Stabilized glycoside hydrolase variants

文档序号：1966733 发布日期：2021-12-14 浏览：17次中文

阅读说明：本技术 稳定化的糖苷水解酶变体 (Stabilized glycoside hydrolase variants ) 是由 L.吉格 F.W.拉斯穆森 S.G.卡斯加德 L.安德森 K.詹森 R.A.帕赫 D.M 于 2020-04-08 设计创作，主要内容包括：披露了例如在蛋白酶的存在下,具有改善的稳定性的糖苷水解酶变体、以及此类变体在洗涤剂应用例如衣物洗涤或餐具洗涤中的用途。(Glycoside hydrolase variants having improved stability, e.g., in the presence of a protease, and the use of such variants in detergent applications, e.g., laundry or dishwashing, are disclosed.)

1. A variant of a parent polypeptide having glycoside hydrolase (EC 3.2.1.-), cellulase or endoglucanase activity, wherein the variant comprises a catalytic domain, a proline-rich linker region and a Carbohydrate Binding Module (CBM), and wherein the variant has glycoside hydrolase, cellulase or endoglucanase activity, wherein the variant has improved stability in an aqueous composition comprising a protease compared to the parent.

2. A variant of a parent polypeptide having glycoside hydrolase (EC 3.2.1.-), cellulase or endoglucanase activity, wherein the variant comprises a catalytic domain, an engineered linker region and a Carbohydrate Binding Module (CBM), and wherein the variant has glycoside hydrolase, cellulase or endoglucanase activity, wherein the variant has improved stability in an aqueous composition comprising a protease compared to the parent.

3. A variant according to any preceding claim which is a family GH45 endoglucanase.

4. The variant according to any of the preceding claims, wherein the CBM is CBM 1.

5. A variant which is a hybrid polypeptide having glycoside hydrolase activity, e.g. endoglucanase activity, preferably GH45 endoglucanase activity, comprising (a) a catalytic domain from a polypeptide having glycoside hydrolase activity, e.g. endoglucanase activity, preferably GH45 endoglucanase activity, (b) a linker selected from the group consisting of: PPPPPPPP (SEQ ID NO:31), PPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) and SPSPSPSPSPG (SEQ ID NO:25), and (c) a Carbohydrate Binding Module (CBM), preferably CBM1, preferably wherein the variant has improved stability in an aqueous composition comprising a protease as compared to the parent.

6. The variant according to any of the preceding claims, wherein improved stability is determined according to the assay described in example 2 and/or example 7.

7. The variant according to any of the preceding claims, wherein the variant comprises an N-terminal catalytic domain and a C-terminal CBM.

8. The variant according to any of the preceding claims, wherein the variant comprises a C-terminal catalytic domain and an N-terminal CBM.

9. The variant according to any of the preceding claims, wherein the variant exhibits improved fabric or textile care and/or improved wash performance relative to the parent, e.g. after storage in the presence of a protease.

10. The variant according to any of the preceding claims, wherein the linker comprises at least 25% proline, e.g., at least 28% proline, at least 30% proline, at least 35% proline, at least 40% proline, at least 50% proline, e.g., at least 60%, at least 66%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% proline.

11. The variant according to any of the preceding claims, wherein the linker has a length of at least four amino acids and comprises one or more of the following optional repeat motifs:

a.[P/S/T/R/K/D/E]P, preferably [ P/S/T]P, most preferably (SP)_aA is 2-10 or P_bB is 4-20, preferably 4-15;

b.P [ S/T/R/K/D/E/N/Q ] P [ S/T/R/K/D/E ] (SEQ ID NO:102), preferably P [ S/E ] PT (SEQ ID NO: 109).

12. The variant according to any of the preceding claims, wherein the linker has a length of at least four amino acids and comprises the following optional repeat motif: [ S/T/R/K/D/E ] P [ S/T/R/K/D/E/N/Q ] [ P/S/T/R/K/D/E ] [ P/S/T/R/K/D/E ] P and/or P [ P/S/T/R/K/D/E ] [ P/S/T/R/K/D/E ].

13. The variant according to any of the preceding claims, wherein the linker comprises:

a.(SP)_a，a＝2-10；

b.(PS)_a，a＝2-10；

c.P_bb is 4-20, preferably 4-15;

d.(PEPT(SEQ ID NO:125))_c，c＝2-5；

e.(PSPT(SEQ ID NO:104))_d，d＝2-5；

f.(P[S/T/R/K/D/E/N/Q]P[S/T/R/K/D/E](SEQ ID NO:102))_e，e＝

2-5；

g.([S/T/R/K/D/E]P)_ff is 2 to 10, preferably 2 to 5;

h.([S/T/R/K/D/E/N/Q]P[S/T/R/K/D/E])_g，g＝2-6；

i.([S/T/R/K/D/E/N/Q][S/T/R/K/D/E/N/Q]P)_h，h＝2-5；

j.(TP)_i，i＝2-10；

k.([S/T/P][S/T/P][S/T/P])_j，j＝2-11；

and/or combinations thereof, wherein the combinations comprise individual monomer units.

14. The variant according to any of the preceding claims, wherein the linker comprises:

a.(SP)_a，a＝2-10；

b.(PS)_a，a＝2-10；

c.P_bb is 4-20, preferably 4-15; or

d.(PEPT(SEQ ID NO:125))_c，c＝2-5。

15. The variant according to any of the preceding claims, wherein the linker has a length of at least 4 amino acids and not more than 30 amino acids, such as 4-28 amino acids, preferably 4-20 amino acids, or even 4-10 amino acids, such as 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids or 10 amino acids.

16. The variant of any preceding claim, wherein the linker comprises SPSP (SEQ ID NO:130), SPSPSP (SEQ ID NO:131), SPSPSPSPSP (SEQ ID NO:132), SPSPSPSPSP (SEQ ID NO:58), SPSPSPSPSPSP (SEQ ID NO:133), SPSPSPSPSPSPSP (SEQ ID NO:134), SPSPSPSPSPSPSPSP (SEQ ID NO:135), PPPP (SEQ ID NO:27), PPPPPPP (SEQ ID NO:28), PPPPPP (SEQ ID NO:29), PPPPPPP (SEQ ID NO:31), PPPPPPP (SEQ ID NO:136), PPPPPPPPP (SEQ ID NO:137), PPPPPPPPPP (SEQ ID NO:138), PPPPPPPPPPP (SEQ ID NO:139), PPPPPPPPPPPP (SEQ ID NO:140), PPPPPPPPPPPPP (SEQ ID NO:141), PPPPPPPPPPPPPP (SEQ ID NO:142), PPPPPPPPPPPPPPP (SEQ ID NO:143), TPEPT (SEQ ID NO:144), PEPTPEPTPEPT (SEQ ID NO:145), PEP NO:145), PEPTPEPTPEPTPEPT (SEQ ID NO:146), PEPTPEPTPEPTPEPTPEPT (SEQ ID NO:79), PSPTPSPT (SEQ ID NO:147), PSPTPSPTPSPT (SEQ ID NO:148), PSPTPSPTPSPTPSPT (SEQ ID NO:149), PSPTPSPTPSPTPSPTPSPT (SEQ ID NO:150), SPSSPS (SEQ ID NO:151), SPSSPSSPS (SEQ ID NO:152), SPSSPSSPSSPS (SEQ ID NO:153), SPSSPSSPSSPSSPS (SEQ ID NO:154), TPTTPT (SEQ ID NO:155), TPTTPTG (SEQ ID NO:96), TPTTPTTPT (SEQ ID NO:156), TPTTPTTPTTPT (SEQ ID NO:157), TPTTPTTPTTPTTPT (SEQ ID NO:158), PEPTPRPTPEPTPRPT (SEQ ID NO:159), PEPTPKPTPEPTPKPT (SEQ ID NO:160), PEPTPQPTPEPTPQPT (SEQ ID NO:161), PRPTPEPTPRPT (SEQ ID NO:162), PKPTPEPTPKPT (SEQ ID NO:163), TPQPT (SEQ ID NO:164), PEPTPQPTPEPT (SEQ ID NO:165), PEPTPRPTPEPTPRPTG (SEQ ID NO: 68685), SEQ ID NO: PEPTPKPTPEPTPKPTG (PEP NO:87), PEPTPQPTPEPTPQPTG (SEQ ID NO:88), PRPTPEPTPRPTG (SEQ ID NO:89), PKPTPEPTPKPTG (SEQ ID NO:90), PEPTPQPTG (SEQ ID NO:91), PEPTPQPTPEPTG (SEQ ID NO:92), PPPGGPGGPGTPTSTAPGSGPTSPGGGSG (SEQ ID NO: 82).

17. The variant according to any of the preceding claims, wherein the linker further comprises a glycine at the C-terminal position.

18. The variant according to any of the preceding claims, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, such as at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, with the amino acid sequence as depicted at positions 1-212 of SEQ ID NO 1, positions 1-211 of SEQ ID NO 2, positions 1-210 of SEQ ID NO 3, positions 1-211 of SEQ ID NO 4.

19. The variant according to any of the preceding claims, wherein the CBM comprises a variant of any of the groups as set forth in SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 180, SEQ ID NO 181, SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 184, SEQ ID NO 185, SEQ ID NO 186, SEQ ID NO 187, SEQ ID NO 188, SEQ ID NO 189, SEQ ID NO 190, SEQ ID NO 191, SEQ ID NO 192, SEQ ID NO 193, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, SEQ ID NO 197, 198, 199, 200, such as, for example, an amino acid sequence having at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity.

20. The variant according to any of the preceding claims, wherein the linker is selected from the group consisting of:

TTPPTPTPTPTPG(SEQ ID NO:12)

TTPTPPTPTPTPTPG(SEQ ID NO:13)

TTPTPTPPTPTPTPTPG(SEQ ID NO:14)

TTPTPTPTPPTPTPTPTPG(SEQ ID NO:15)

TPPTPPTPPTPPTPPTPPTPPTPPTPPTPPTPPG(SEQ ID NO:16)

TPTTPTTPTTPTG(SEQ ID NO:17)

TPTTPTTPTTPTTPTTPTG(SEQ ID NO:18)

SPSSPSSPSSPSG(SEQ ID NO:19)

SPSSPSSPSSPSSPSG(SEQ ID NO:20)

SPPSPPSPPSPPSPPG(SEQ ID NO:21)

SPPSPPSPPSPPSPPSPPSPPSPPSPPSPPG(SEQ ID NO:22)

PPSSPSSPSSPSSPSSPSSPSG(SEQ ID NO:23)

SPSPG(SEQ ID NO:24)

SPSPSPSPSPG(SEQ ID NO:25)

TPTPTPTPTPG(SEQ ID NO:26)

PPPP(SEQ ID NO:27)

PPPPP(SEQ ID NO:28)

PPPPPP(SEQ ID NO:29)

PPPPPPPG(SEQ ID NO:30)

PPPPPPP(SEQ ID NO:31)

PPPPPPPPG(SEQ ID NO:32)

PPPPPPPPPG(SEQ ID NO:33)

PPPPPPPPPPG(SEQ ID NO:34)

PPPPPPPPPPPG(SEQ ID NO:35)

PPPPPPPPPPPPPG(SEQ ID NO:36)

PEPTPEPTG(SEQ ID NO:37)

PEPTPEPTPEPTG(SEQ ID NO:38)

PEPTPEPTPEPTPEPTG(SEQ ID NO:39)

PEPTPEPTPEPTPEPTPEPTG(SEQ ID NO:40)

PSPTPSPTPSPTPSPTG(SEQ ID NO:41)

PSPTPSPTPSPTPSPTPSPTG(SEQ ID NO:42)

PQPTPQPTG(SEQ ID NO:43)

PDPTPDPTG(SEQ ID NO:44)

PRPTPEPTG(SEQ ID NO:45)

PQPTPEPTG(SEQ ID NO:46)

PSPNSPNSPNG(SEQ ID NO:47)

PEPTPRPTG(SEQ ID NO:48)

PQPTPEPTPQPTPEPTPQPTPEPTPQPTG(SEQ ID NO:49)

PDPTPDPTPDPTG(SEQ ID NO:50)

PQPTPQPTPQPTPQPTG(SEQ ID NO:51)

PQPTPEPTPQPTPEPTG(SEQ ID NO:52)

SPSPSPSPPPG(SEQ ID NO:53)

SPSPSPSPDPG(SEQ ID NO:54)

SPSPSPSPKPG(SEQ ID NO:55)

SPSPSPSPAPG(SEQ ID NO:56)

SPSPSPSPSPSG(SEQ ID NO:57)

SPSPSPSPSP(SEQ ID NO:58)

SPSPSPSPSPS(SEQ ID NO:59)

SPSPSPSPSPP(SEQ ID NO:60)

SPSPSPSPSPE(SEQ ID NO:61)

SPSPSPSPSPN(SEQ ID NO:62)

SPSPSPSPSPGG(SEQ ID NO:63)

SPSPSPSPSPK(SEQ ID NO:64)

PEPTPEPTP(SEQ ID NO:65)

PEPTPEPTR(SEQ ID NO:66)

PEPTPEPTPEPTP(SEQ ID NO:67)

PEPTPEPTPEPTPEPTPSPTG(SEQ ID NO:68)

PEPTPEPTPEPTPEPTPTPTG(SEQ ID NO:69)

PEPTPEPTPEPTPEPTPGPTG(SEQ ID NO:70)

PEPTPEPTPEPTPEPTPDPTG(SEQ ID NO:71)

PEPTPEPTPEPTPEPTPETG(SEQ ID NO:72)

PEPTPEPTPEPTPEPTPEPTD(SEQ ID NO:73)

PEPTPEPTE(SEQ ID NO:74)

PEPTPEPTPEPTPEPTPEP(SEQ ID NO:75)

PEPTPEPTPEPTPEPTPSPT(SEQ ID NO:76)

PEPTPEPTPEPTPEPTPRPTT(SEQ ID NO:77)

PEPTPEPTPEPTPEPTPEPTT(SEQ ID NO:78)

PEPTPEPTPEPTPEPTPEPT(SEQ ID NO:79)

PEPTPEPTPEPTPEPTPEPTS(SEQ ID NO:80)

PEPTPEPTPEPTPEPTPEPTR(SEQ ID NO:81)

PPPGGPGGPGTPTSTAPGSGPTSPGGGSG(SEQ ID NO:82)

PPPGGPGGTGTPTSTAPGSGPTSPGGGSG(SEQ ID NO:83)

PPSGGPGGPGTPTSTAPGSGPTSPGGGSG(SEQ ID NO:84)

PEPTPRPTPEPTPRPTG(SEQ ID NO:85)

PKPTPEPTPKPTPEPTG(SEQ ID NO:86)

PEPTPKPTPEPTPKPTG(SEQ ID NO:87)

PEPTPQPTPEPTPQPTG(SEQ ID NO:88)

PRPTPEPTPRPTG(SEQ ID NO:89)

PKPTPEPTPKPTG(SEQ ID NO:90)

PEPTPQPTG(SEQ ID NO:91)

PEPTPQPTPEPTG(SEQ ID NO:92)

TPPTPPG(SEQ ID NO:93)

SPSSPSG(SEQ ID NO:94)

SPSSPSSPSG(SEQ ID NO:95)

TPTTPTG (SEQ ID NO: 96); and

TPTTPTTPTG(SEQ ID NO:97)。

21. the variant according to any of the preceding claims, wherein the linker is PPPPP (SEQ ID NO:31), PPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO: 25).

22. The variant according to any of the preceding claims, wherein the variant is selected from the group consisting of 5:

23. the variant according to any of the preceding claims, wherein the variant is selected from the group consisting of:

variant 401, variant 402, variant 403, variant 404, variant 405, variant 406, variant 407, variant 408, variant 409, variant 410, variant 411, variant 412, variant 413, variant 414, variant 415, variant 416, variant 417, variant 418, variant 419, variant 420, variant 421, variant 422, variant 423, variant 424, variant 425, variant 426, variant 427, variant 428, variant 429, and variant 430.

24. An isolated polynucleotide encoding the variant according to any one of claims 1-23.

25. A nucleic acid construct comprising the polynucleotide of claim 24.

26. An expression vector comprising the polynucleotide according to claim 24.

27. A host cell comprising the polynucleotide of claim 24.

28. A method of producing a variant having glycoside hydrolase (EC3.2.1.), cellulase or endoglucanase activity according to any of claims 1-23, the method comprising

a. Culturing the host cell of claim 27 under conditions suitable for expression of the variant; and

b. recovering the variant.

29. A method for obtaining a variant having glycoside hydrolase (EC3.2.1.-), cellulase or endoglucanase activity, the method comprising introducing a proline-rich linker region into a parent glycoside hydrolase; and recovering the variant.

30. A whole broth formulation or cell culture composition comprising the variant according to any of claims 1-23.

31. A composition comprising the variant according to any one of claims 1-23.

32. The composition of claim 31, further comprising a protease.

33. The composition according to any one of claims 31-32, further comprising one or more additional enzymes selected from the group consisting of: (further) protease, lipase, cutinase, amylase, (further) carbohydrase, (further) cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, nuclease, lichenase, oxidase, e.g., laccase, and/or peroxidase, and combinations thereof.

34. A composition according to any of claims 31-33, which is a detergent composition, preferably a liquid detergent composition.

35. Use of the variant according to any of claims 1-23 or the composition according to any of claims 31-34 for fabric or textile care, such as for pre-treating a stained fabric or for rejuvenating a textile (e.g. by removing fuzz or pills) to restore the visual and sensory properties of the fabric after long term use to match a new textile.

Technical Field

The present invention relates to variants of glycoside hydrolases. These variants may have improved stability, in particular improved stability in the presence of detergents and/or proteases. Furthermore, the present invention relates to liquid detergent compositions comprising the stabilized glycoside hydrolase variants.

Background

Glycoside hydrolases (e.g., cellulases) typically contain a catalytic domain and one or more Carbohydrate Binding Modules (CBMs) linked by a linker region. Linkers are generally flexible linkers that provide connectivity between structured domains, but their functional role is largely unknown.

Cellulases have been used in detergents for many years because of the benefits observed with cellulases in laundry processes, such as color clarification, prevention of redeposition, anti-pilling/lint removal and improved whiteness, and cellulases are characterized by the ability to cleave 1, 4-beta-glucosidic bonds in cellulose molecules into smaller molecules.

In some applications, a complex cellulase composition is used, wherein the composition comprises more than one cellulose degrading enzyme (selected from the endoglucanases used, cellobiohydrolases, and beta-glucosidases), while other applications use an enzyme composition comprising predominantly one or more endoglucanases.

WO 1996/029397 discloses a family 45 endoglucanase for detergent use. Most commercial detergent compositions contain proteases that improve the removal of many common stains. However, proteases also degrade other proteins available in the wash solution, including other enzymes such as cellulases and other glycoside hydrolases. Accordingly, it would be desirable to provide glycoside hydrolases, such as cellulases and variants thereof, with increased stability in the presence of proteases.

Disclosure of Invention

The present invention provides a variant of a parent polypeptide having glycoside hydrolase or (EC 3.2.1.-) activity, wherein the variant comprises a catalytic domain, an engineered linker region (e.g., a proline-rich linker region), and a Carbohydrate Binding Module (CBM). Preferably, the variant has improved linker stability and/or improved CBM stability in an aqueous composition comprising a protease compared to the parent glycoside hydrolase, and wherein the variant has glycoside hydrolase activity.

The invention further relates to polynucleotides and expression constructs comprising the polynucleotides; host cells comprising these polynucleotides or expression constructs, and the use of such host cells for producing variants of the invention.

Compositions, particularly detergent compositions (e.g., liquid detergent compositions), comprising the variants are also disclosed, as well as uses of such compositions for laundering textiles.

Definition of

Detergent component: the term "detergent component" is defined herein to mean the type of chemicals that can be used in a detergent composition. Examples of detergent components are surfactants, hydrotropes, builders, co-builders, chelating agents (chelators) or chelating agents (chelating agents), bleaching systems or bleach components, polymers, fabric hueing agents, fabric conditioners, suds boosters (foam boosters), suds suppressors, dispersants, dye transfer inhibitors, optical brighteners, perfumes, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants and solubilizers. Detergent compositionThe composition may comprise one or more detergent components of any type.

Detergent composition: the term "detergent composition" refers to compositions used to remove undesirable compounds from items to be cleaned (e.g., textiles, dishware, and hard surfaces). The detergent composition may be used, for example, for cleaning textiles, dishes, and hard surfaces, for both household and industrial cleaning, and/or for fabric care. These terms encompass any material/compound selected for the particular type of cleaning composition and form of product desired (e.g., liquid, gel, powder, granule, paste, or spray compositions) and include, but are not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations such as hard surface cleaning formulations for glass, wood, plastic, ceramic, and metal countertops and windows; carpet cleaners; range cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-detergents (pre-spoters), along with dish detergents). In addition to containing the enzyme of the invention, the detergent formulation may contain one or more additional enzymes (e.g., amylases, proteases, peroxidases, cellulases, beta-glucanases, xyloglucanases, hemicellulases, xanthanases, xanthan lyases, lipases, acyltransferases, phospholipases, esterases, laccases, catalases, arylesterases, amylases, alpha-amylases, glucoamylases, cutinases, pectinases, pectin lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenanses, pullulanases, tannases, arabinosidases, hyaluronidase, chondroitinase, xyloglucanases, xylanases, pectin acetyl esterase, polygalacturonases, rhamnogalacturonases, other endo-beta-mannanases, enzymes for example, Exo-beta-mannanase (GH5 and/or GH26), lichenase (lichenase), phosphodiesterase, pectin methylesterase, cellobiohydrolase, transglutaminase, nuclease, combinations thereof, or any mixtures thereof), and/or ingredients such as surfactants, builders, chelating agents (chellators) ) Or chelating agents (chelating agents), bleaching systems or components, polymers, fabric conditioners, suds boosters, suds suppressors, dyes, perfumes, tarnish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, corrosion inhibitors, enzyme inhibitors or stabilizers, enzyme activators, one or more transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

Tableware washing: the term "dishwashing" refers to all forms of washing dishes, such as manual dishwashing (HDW) or Automatic Dishwashing (ADW). Washing dishes includes, but is not limited to, cleaning all forms of dishes, such as plates, cups, glasses, bowls, all forms of cutlery (e.g., spoons, knives, forks), and serving utensils as well as ceramics, plastics, metals, porcelain, glass, and acrylates.

Dishwashing composition: the term "dishwashing composition" refers to all forms of compositions intended for cleaning dishes, table ware, pots, pans, cutlery and for cleaning hard surface areas in the kitchen. The present invention is not limited to any particular type of dishwashing composition or any particular detergent.

Cleaning solution: the term "wash liquor" refers to an aqueous solution containing a detergent composition in diluted form, such as, but not limited to, a detergent solution containing a laundry detergent composition in diluted form, e.g., a wash liquor in a laundry process.

Textile product: the term "textile" means any textile material, including yarns, yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material, fabrics made from these materials, and products made from fabrics (e.g., garments and other articles). The textile or fabric may be in the form of knits, wovens, denims, nonwovens, felts, yarns, and terry cloth. The textile may be cellulose-based, such as natural cellulosics including cotton, flax/linen, jute, flax, or hemp, or mixtures of any of the like,Ramie, sisal or coir, or man-made cellulose (e.g. from wood pulp) including viscose/rayon, cellulose acetate fibers (tricell), lyocell (lyocell) or blends thereof. The textile or fabric may also be not cellulose based, such as natural polyamides including wool, camel hair, cashmere, mohair, rabbit hair and silk, or synthetic polymers such as nylon, aramids, polyesters, acrylates, polypropylene and spandex/elastane (spandex/elastane), or blends thereof and blends of cellulose based fibers and non-cellulose based fibers. Examples of blends are blends of cotton and/or rayon/viscose with one or more companion materials such as wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyester fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers) and/or cellulose-containing fibers (e.g. rayon/viscose, ramie, flax/linen, jute, cellulose acetate fibers, lyocell). The fabric may be a conventional washable garment, such as a stained household garment. When the term fabric or garment is used, it is intended to also include the broad term textile.

Hard surface cleaning: the term "hard surface cleaning" is defined herein as cleaning hard surfaces, wherein hard surfaces may include floors, tables, walls, roofs, etc., as well as surfaces of hard objects such as automobiles (car wash) and dishware (dish wash). Dishwashing includes, but is not limited to, cleaning dishes, cups, glasses, bowls, cutlery (e.g., spoons, knives, forks), serving utensils, ceramics, plastics, metals, porcelain, glass, and acrylates.

Whiteness degree: the term "whiteness" is defined herein as a broad term in different fields and with different meaning for different customers. The loss of whiteness can be attributed, for example, to ashing, yellowing, or removal of optical brightener/toner. Ashing and yellowing can be attributed to soil redeposition, body soils, staining from e.g. iron and copper ions or dye transfer. Whiteness may include one or several issues from the following list: colorant or dye action; incomplete stain removal (example)Such as body soils, sebum, etc.); redeposition (ashing, yellowing or other discoloration of the object) (re-association of removed soil with other parts of the textile (soiled or unsoiled)); chemical changes in the textile during application; and clarification or brightening of the color.

Color clarification: loose or broken fibers can accumulate on the surface of the fabric during washing and wear. One consequence is that the fabric color appears less bright or less intense due to surface contamination. Removing loose or broken fibers from the textile will partially restore the original color and appearance of the textile. The term "color clear" as used herein means a partial restoration of the original color of the textile.

Anti-pilling: the term "anti-pilling" means the removal of pills from the textile surface and/or the prevention of pill formation on the textile surface.

Fabric care: the term fabric care (also referred to as textile care) refers to a treatment that retains or partially restores or fully restores the properties of the textile, for example, by color clarification, anti-pilling or preventing the formation of pills on the textile surface.

Cellulolytic or cellulase enzymes: the term "cellulolytic enzyme" or "cellulase" means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include one or more endoglucanases (e.g., EC 3.2.1.4), one or more cellobiohydrolases, one or more beta-glucosidases, or a combination thereof. Two basic methods for measuring cellulolytic enzyme activity include: (1) measuring total cellulolytic enzyme activity, and (2) measuring individual cellulolytic enzyme activities (endoglucanase, cellobiohydrolase, and beta-glucosidase), as in Zhang et al, 2006, Biotechnology Advances [ Advances in Biotechnology ] ]24:452, 481. Total cellulolytic enzyme activity can be measured using insoluble substrates including Whatman (Whatman) -1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, and the like. The most common measurement of the total cellulolytic activity is carried out using a Whatman No. 1 filter paper as a baseFilter paper assay of substances. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose,1987, Pure appl. chem. [ Pure and applied chemistry ]]59:257-68)。

Cellulosic material: the term "cellulosic material" means any material containing cellulose. The primary polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third most abundant is pectin. The secondary cell wall produced after the cell growth has ceased also contains polysaccharides and is reinforced by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and is therefore a linear beta- (1-4) -D-glucan, whereas hemicellulose comprises a variety of compounds with a range of substituents in complex branched structures, such as xylans, xyloglucans, arabinoxylans, and mannans. Although cellulose is generally polymorphic, it is found in plant tissues to exist primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicellulose is often hydrogen bonded to cellulose and other hemicelluloses, which helps stabilize the cell wall matrix.

Endoglucanase: the term "endoglucanase" means an enzyme that catalyzes the endo-hydrolysis of 1, 4-beta-D-glucosidic linkages in cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethylcellulose), lichenin, mixed beta-1, 3-beta-1, 4 glucans such as cereal beta-D-glucans or xyloglucans, and beta-1, 4 linkages in other plant materials containing a cellulose component. Chem. [ Pure and applied chemistry ] according to Ghose,1987, Pure and appl]59:257-268, endoglucanase activity was determined using carboxymethyl cellulose (CMC) hydrolysis. One unit of endoglucanase activity is defined as producing 1.0 micromole of reducing sugars per minute at 50 ℃, pH 4.8.

A particularly preferred class of endoglucanases are those of "family GH 45", which is classified as glycoside hydrolase family 45 according to the nomenclature of Henrissat et al, biochem.J. [ J.Biochem. ]280:309-316(1991) and the Carbohydrate-Active enzyme database (Carbohydrate Active enZYmes database) found in cam.org. The GH45 enzyme is an endoglucanase of EC 3.2.1.4.

Glycoside hydrolase: the term "glycoside hydrolase" (GH) means an enzyme that catalyzes the hydrolysis of a glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. For more details, see, e.g., Henrissat B., "A classification of sugar based hydrolases based on amino-acid sequences peptides, [ classification of glycosyl hydrolases based on amino acid sequence similarity ] ]"biochem.j. [ journal of biochemistry]280:309-316(1991) and the Carbohydrate Active enzyme database (Carbohydrate Active enZYmes database) found in cam.

Exemplary glycoside hydrolase families with reported cellulase activity useful according to the present invention include those of families GH5, GH6, GH7, GH8, GH9, GH12, GH44, GH45, GH48, GH51, GH124, with family GH45 being particularly preferred.

Carbohydrate binding modules: the term "carbohydrate binding module" means a region within a carbohydrate-active enzyme that provides carbohydrate binding affinity (Boraston et al, 2004, biochem. J. [ J. biochem. ].)]383:769-781). Most known carbohydrate-binding modules (CBMs) are continuous amino acid sequences with discrete folds. Carbohydrate-binding modules (CBM) are typically found at the N-terminal or C-terminal end of the enzyme. Some CBMs are known to have specificity for cellulose.

Exemplary CBM families useful according to the present invention are those of CBM families 1,4, 17, 28, 30, 44, 72 and 79. Again, referring to cazy.org/carbonate-Binding-Modules, CBM family 1 includes a module of approximately 40 residues found almost exclusively in fungi. The cellulose binding function has been demonstrated in many cases and appears to be mediated by three aromatic residues spaced about 10.4 angstroms apart and forming a flat surface. CBM family 4 includes a module of approximately 150 residues found in bacterial enzymes. The binding of these modules has been demonstrated with xylan, beta-1, 3-glucan, beta-1, 3-1, 4-glucan, beta-1, 6-glucan and amorphous cellulose (but not with crystalline cellulose). CBM family 17 includes modules of approximately 200 residues. Binding to amorphous cellulose, cellooligosaccharides and derivatized cellulose has been demonstrated. With respect to CBM family 28, modules of endo-1, 4-glucanases from Bacillus species (Bacillus sp.)1139 bind to non-crystalline cellulose, cellooligosaccharides and β - (1,3) (1,4) -glucans. For CBM family 30, the binding of the N-terminal module of filamentous Bacillus succinogenes (Fibrobacter succinogenes) CelF to cellulose has been demonstrated. It has been demonstrated that the C-terminal CBM44 module of the Clostridium thermocellum (Clostridium thermocellum) enzyme binds cellulose and xyloglucan equally well. CBM family 72 includes a module of 130-180 residues found in C-terminal glycoside hydrolases from multiple families, sometimes as tandem repeats. CBM72 (which was found on endoglucanases from uncultured microorganisms) was found to bind to a broad spectrum of polysaccharides including soluble and insoluble cellulose, β -1,3/1, 4-mixed linked glucans, xylans and β -mannans. CBM family 79 includes modules of approximately 130 residues that have been found only in ruminococcus proteins to date. The binding of ruminococcus flavus (r. flavefaciens) GH9 enzyme to various β -glucans is shown. Most preferred is CBM family 1 (also referred to as "CBM 1").

Catalytic domains: the term "catalytic domain" means the region of an enzyme that contains the catalytic machinery of the enzyme.

Engineered: the term "engineered" means a synthetic construct.

Proline-rich linker: the term "proline-rich linker" means a sequence comprising one or more Pro-Pro, Pro-Xaa (or Xaa-Pro), Xaa-Pro-Xaa, or Xaa-Pro (or Pro-Xaa) units, wherein Pro is a three-letter representation of the amino acid proline and Xaa is a three-letter representation of any amino acid. Preferably, the proline-rich linker comprises the above-described units repeated in combination and/or in succession, e.g., PP, PPP, PPPP (SEQ ID NO:27), PX, PXP, PS, PSP, PXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPX (SEQ ID NO:98), XP, XPX, SP, SPS, XPXP (SEQ ID NO:99), XPXXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPX (SEQ ID NO:101), and the like.

In one aspect, the proline-rich linker comprises one or more of the following optional repeat motifs: [ P/S/T/R/K/D/E ] P and P [ S/T/R/K/D/E/N/Q ] P [ S/T/R/K/D/E ] (SEQ ID NO: 102). In one aspect, the proline-rich linker comprises the following optional repeat motifs: [ S/T/R/K/D/E ] P [ S/T/R/K/D/E/N/Q ], [ P/S/T/R/K/D/E ] [ P/S/T/R/K/D/E ] P and/or P [ P/S/T/R/K/D/E ] [ P/S/T/R/K/D/E ]. In one aspect, the proline-rich linker comprises an optional repeating motif of the same or different amino acids in parentheses as shown: [ P/S/T ] P and P [ S/E ] PT (SEQ ID NO: 109).

In one aspect, the proline-rich linker comprises (a) (SP) a, a ═ 2 to 10; (b) (PS) a, a ═ 2-10; (c) pb, b-4-20, preferably 4-15; (d) (PEPT (SEQ ID NO:125)) c, c ═ 2-5; (e) (PSPT (SEQ ID NO:104)) d, d ═ 2-5; (f) (P [ S/T/R/K/D/E/N/Q ] P [ S/T/R/K/D/E ] (SEQ ID NO:102)) E, E ═ 2-5; (g) ([ S/T/R/K/D/E ] P) f, f is 2-10, preferably 2-5; (h) ([ S/T/R/K/D/E/N/Q ] P [ S/T/R/K/D/E ]) g, g is 2-6; (i) ([ S/T/R/K/D/E/N/Q ] P) h, h ═ 2-5; (j) (TP) i, i ═ 2-10; (k) ([ S/T/P ]) j, j is 2-11; (l) And/or combinations thereof, wherein combinations of individual monomer units are contemplated.

In one aspect, the proline-rich linker comprises a linker in Table A below, such as PPPPPPP (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58), or SPSPSPSPSPG (SEQ ID NO: 25).

Preferably, the proline-rich linker comprises at least 25% proline, like at least 28% proline, at least 30% proline, at least 35% proline, at least 40% proline, at least 50% proline, e.g. at least 60%, at least 66%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% proline. Preferably, the proline-rich linker comprises at least 4 amino acids and not more than 30 amino acids, such as 4-28 amino acids, preferably 4-20 amino acids, or even 4-10 amino acids, such as 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids or 10 amino acids.

Furthermore, a linker region as defined herein may comprise an additional interface of 1-2 amino acids at the junction with the catalytic domain and/or the carbohydrate binding module.

Allelic variants: the term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation and can lead to polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

cDNA: the term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA that is processed through a series of steps, including splicing, and then rendered into mature spliced mRNA.

Coding sequence: the term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequence: the term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding a variant of the invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the variant, or native or foreign with respect to one another. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. Due to introduction of specific restrictions facilitating ligation of control sequences with coding regions of polynucleotides encoding variantsFor purposes of site, the control sequence may be provided with a linker.

Expression of: the term "expression" includes any step involved in the production of a variant, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: the term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a variant and operably linked to control sequences that provide for its expression.

Fragments: the term "fragment" means a polypeptide lacking one or more (e.g., several) amino acids at the amino and/or carboxy terminus of the mature polypeptide; wherein the fragment has cellulolytic activity. In one aspect, the fragment contains at least 260 amino acid residues (e.g., amino acids 1 to 260 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO: 4), at least 240 amino acid residues (e.g., amino acids 1 to 240 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO: 4), or at least residues corresponding to the catalytic domain, for example 210, 211, 212, or 216 amino acid residues (e.g., amino acids 1 to 212 of SEQ ID NO:1 or amino acids 1 to 216 of SEQ ID NO:1, amino acids 1 to 211 of SEQ ID NO:2 or amino acids 1 to 212 of SEQ ID NO:2, amino acids 1 to 211 of SEQ ID NO:3 or amino acids 1 to 210 of SEQ ID NO:3, amino acids 1 to 211 of SEQ ID NO: 4).

Host cell: the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Hybrid polypeptide:the term "hybrid polypeptide" means a polypeptide comprising domains from two or more polypeptides of different origin (origins), e.g., a binding moiety from one polypeptide and a catalytic domain from another polypeptide. The domains may be fused at the N-terminus or C-terminus. Of particular interest herein are polypeptides comprising: from one toA binding moiety of a polypeptide (which may be naturally occurring or further modified), an engineered linker region (e.g., a proline-rich linker region (which is a synthetic construct)), and a catalytic domain from another polypeptide (which may be naturally occurring or further modified).

And (3) hybridization:the term "hybridization" means the pairing of substantially complementary strands of nucleic acids using standard southern blotting procedures. Hybridization can be performed under medium, medium-high, high or very high stringency conditions. Medium stringency conditions mean prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours followed by three washes at 55 ℃ using 0.2X SSC, 0.2% SDS for 15 minutes each. Medium-high stringency conditions mean prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours followed by three washes at 60 ℃ with 0.2X SSC, 0.2% SDS, each for 15 minutes. High stringency conditions mean prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours followed by three washes at 65 ℃ with 0.2X SSC, 0.2% SDS, each for 15 minutes. Very high stringency conditions mean prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours followed by three washes at 70 ℃ with 0.2X SSC, 0.2% SDS, each for 15 minutes.

Improved properties: the term "improved property" means a characteristic associated with an improved variant compared to a reference/parent enzyme. Some aspects of the invention relate to variants having an improvement factor of higher than 1 when the variant is tested for a property of interest in a related assay, wherein the property of the reference/parent enzyme is given a value of 1.

Improved stability: the term "improved stability" means that the enzyme has better stability in the presence of protease relative to the stability of the reference/parent enzyme and includes, for example, proteolytic stability, storage stability in detergent, stability in detergentImproved stability during production of the composition and in-wash stability. The improvement in stability was quantified by: stability was determined according to the assays described in example 2 (linker stability assay-in the presence of protease) and/or example 7 (in-wash linker stability assay using protease) herein.

Improved cleaning performance: the term "improved wash performance" is defined herein as an enzyme exhibiting increased wash performance in a detergent composition, e.g., by increased color clarification and/or anti-pilling effect, relative to the wash performance of a reference enzyme/parent enzyme when evaluated on a new sample and/or after storage of the sample under the same conditions. The term "improved wash performance" includes wash performance in laundry washing and also in e.g. hard surface cleaning such as Automated Dish Wash (ADW).

Separated from each other: the term "isolated" means a substance in a form or environment not found in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or more or all of the naturally occurring components associated with its property; (3) any substance that is modified by man relative to substances found in nature; or (4) any substance that is modified by increasing the amount of the substance relative to other components that are intrinsically associated with the substance (e.g., multiple copies of a gene encoding the substance; using a promoter that is stronger than the promoter intrinsically associated with the gene encoding the substance). The isolated substance may be present in a sample of fermentation broth.

Mature polypeptide:the term "mature polypeptide" means a polypeptide that is in its mature form following N-terminal processing (e.g., removal of a signal peptide).

Mature polypeptide coding sequence: the term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having cellulase (e.g., endoglucanase) activity.

Mutants: the term "mutant"Meaning a polynucleotide encoding a variant.

Nucleic acid constructs: the term "nucleic acid construct" means a nucleic acid molecule, either single-or double-stranded, that is isolated from a naturally occurring gene or that has been modified to contain segments of nucleic acids in a manner not otherwise found in nature, or that is synthetic, that contains one or more control sequences.

Operatively connected to:the term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

Parent or parent cellulase: the term "parent" or "parent cellulase" means any polypeptide having a glycosidic hydrolase activity (particularly cellulolytic activity or even endoglucanase activity) to which changes are made to produce the enzyme variants of the invention.

Purified: the term "purified" means a nucleic acid or polypeptide that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., the purified polypeptide or nucleic acid can form discrete bands in an electrophoretic gel, a chromatographic eluate, and/or media subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or more pure (e.g., weight percent on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" means that a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component is present in a composition at a relative or absolute concentration that is higher than the starting composition.

Recombination: when used in reference to cells, nucleic acids, proteins or vectorsIn vivo, the term "recombinant" means that it has been modified from its native state. Thus, for example, a recombinant cell expresses a gene that is not found within the native (non-recombinant) form of the cell, or expresses the native gene at a different level or under different conditions than found in nature. Recombinant nucleic acids differ from the native sequence by one or more nucleotides and/or are operably linked to a heterologous sequence (e.g., a heterologous promoter in an expression vector). The recombinant protein may differ from the native sequence by one or more amino acids and/or be fused to a heterologous sequence. The vector comprising the nucleic acid encoding the polypeptide is a recombinant vector. The term "recombinant" is synonymous with "genetically modified" and "transgenic".

Sequence identity: the degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For The purposes of The present invention, The sequence identity between two amino acid sequences is determined using The Needman-Wunsch algorithm (Needleman and Wunsch,1970, J.Mol.biol. [ J.M.Biol ]48: 443-. The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrix. The output of niedel labeled "longest identity" (obtained using non-simplified options) is used as the percent identity and is calculated as follows:

(same residue x 100)/(alignment Length-total number of vacancies in alignment)

For The purposes of The present invention, The sequence identity between two deoxyribonucleotide sequences is determined using The Needman-Wusch algorithm (Needleman and Wunsch,1970, supra) as implemented in The Nidel program of The EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, supra) (preferably version 5.0.0 or later). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC 4.4) substitution matrix. The output of niedel labeled "longest identity" (obtained using non-simplified options) is used as the percent identity and is calculated as follows:

(identical deoxyribonucleotides x 100)/(alignment length-total number of vacancies in alignment)

Variants: the term "variant" means a polypeptide having cellulolytic activity comprising a substitution at one or more (e.g., several) positions. Substitution means the substitution of an amino acid occupying a position with a different amino acid. These variants of the invention have at least 20%, like at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the cellulolytic activity of the parent (e.g. the mature polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO: 4). "variants" as used herein may also include hybrid polypeptides.

Wild type: the term "wild-type" when referring to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a naturally or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., protein, amino acid, or nucleic acid sequence) found in nature. In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in the laboratory, or modifications of wild-type sequences).

Variant naming conventions

For the purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO 1 is used to identify the corresponding amino acid residues in another cellulase. The amino acid sequence of another cellulase is aligned with The mature polypeptide disclosed in SEQ ID NO:1 and based on this alignment The amino acid position numbering corresponding to any amino acid residue in The mature polypeptide disclosed in SEQ ID NO:1 is determined using The Needman-Weng algorithm (Needleman and Wunsch, 1970, J.mol.biol. [ J.M. 48: 443. 453) as implemented in The Nidel program of The EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite), Rice et al 2000, Trends Genet. [ genetic Trends ]16: 276. 277), preferably version 5.0.0 or later. The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrix.

The identification of corresponding amino acid residues in another cellulase can be determined by one alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (by log-expected multiple sequence comparison; version 3.5 or later; Edgar,2004, Nucleic Acids Research [ Nucleic acid Research ] using its corresponding default parameters]1792-1797), MAFFT (6.857 version or updated version; katoh and Kuma,2002, Nucleic Acids Research [ Nucleic Acids Research ]]3059-3066; katoh et al, 2005, Nucleic Acids Research [ Nucleic Acids Research ]]33: 511-518; katoh and Toh,2007, Bioinformatics]23: 372-374; katoh et al, 2009,Methods in Molecular Biology[ method of molecular biology]537:39-64(ii) a The results of Katoh and Toh, 2010,Bioinformatics[ bioinformatics]26:1899-1900) And with ClustalW (1.83 or later; thompson et al, 1994, Nucleic Acids Research [ Nucleic Acids Research]22: 4673-4680).

Other pairwise sequence comparison algorithms can be used when other enzymes deviate from the mature polypeptide of SEQ ID NO:1, such that conventional sequence-based comparison methods cannot detect their relationship (Lindahl and Elofsson,2000, J.Mol.biol. [ J.Mol.M.295: 613-) -615). Higher sensitivity in sequence-based searches can be obtained using search programs that utilize probabilistic representations (profiles) of polypeptide families to search databases. For example, the PSI-BLAST program generates multiple spectra by iterative database search procedures and is capable of detecting distant homologues (Atschul et al, 1997, Nucleic Acids Res. [ Nucleic Acids research ]25: 3389-. Even greater sensitivity can be achieved if a family or superfamily of polypeptides has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones,1999, J.mol.biol. [ journal of molecular biology ]287: 797-. Similarly, the method of Gough et al, 2000, J.mol.biol. [ J. Mol. ]313: 903-. These alignments can in turn be used to generate homology models for polypeptides, and the accuracy of such models can be assessed using a variety of tools developed for this purpose.

For proteins of known structure, several tools and resources are available to retrieve and generate structural alignments. For example, the SCOP superfamily of proteins has been aligned structurally, and those alignments are accessible and downloadable. Two or more Protein structures may be aligned using a variety of algorithms such as distance alignment matrices (Holm and Sander,1998, Proteins [ Protein ]33:88-96) or combinatorial extensions (Shindyalov and Bourne,1998, Protein Engineering [ Protein Engineering ]11: 739-.

For example, the parent polypeptide may comprise any of SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 or mature polypeptides thereof.

In describing variations of the invention, the nomenclature described below is adapted for ease of reference. Accepted IUPAC single letter or three letter amino acid abbreviations are used.

Substitution. For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, substitution of threonine at position 226 with alanine is denoted as "Thr 226 Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), e.g., "Gly 205Arg + Ser411 Phe" or "G205R + S411F" representing glycine (G) and serine at positions 205 and 411 (S) is substituted with arginine (R) and phenylalanine (F), respectively.

Absence of. For amino acid deletions, the following nomenclature is used: original amino acids, positions. Accordingly, the deletion of glycine at position 195 is denoted as "Gly 195" or "G195". Multiple deletions are separated by a plus sign ("+"), e.g., "Gly 195 + Ser 411" or "G195 + S411".

Insert into. For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Accordingly, insertion of a lysine after the glycine at position 195 is denoted as "Gly 195 GlyLys" or "G195 GK". The insertion of multiple amino acids is denoted as [ original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid # 2; etc. of]. For example, the insertion of lysine and alanine after glycine at position 195 is denoted as "Gly 195 GlyLysAla" or "G195 GKA".

In such cases, the inserted one or more amino acid residues are numbered by adding a lower case letter to the position number of the amino acid residue preceding the inserted one or more amino acid residues. In the above example, the sequence would thus be:

Parent strain:	variants:
		195	195 195a 195b
G	G-K-A

multiple changes. Variants comprising multiple alterations are separated by a plus sign ("+"), e.g., "Arg 170Tyr + Gly195 Glu" or "R170Y + G195E" representing substitutions of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

Different changes. Where different changes can be introduced at one position, the different changes are separated by a comma, e.g., "Arg 170Tyr, Glu" represents the substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Tyr 167Gly, Ala + Arg170Gly, Ala" denotes the following variants:

"Tyr 167Gly + Arg170 Gly", "Tyr 167Gly + Arg170 Ala", "Tyr 167Ala + Arg170 Gly", and "Tyr 167Ala + Arg170 Ala".

Nomenclature

For the purposes of the present invention, brackets are used to indicate alternative amino acids at specific positions in the sequence (using their one-letter codes). For example, the nomenclature [ S/E ] means that the amino acid at this position can be serine (Ser, S) or glutamic acid (Glu, E). Likewise, the nomenclature [ P/S/T ] means that the amino acid at this position can be proline (Pro, P), serine (Ser, S), or threonine (Thr, T), and so on for other combinations as described herein. Amino acids indicated in parentheses using this nomenclature may be separated by a vertical line or, in some cases, not a line, e.g., [ P/S/T ] may also be designated [ PST ].

In certain instances, a sequence motif includes more than one set of parentheses, each set of parentheses independently representing a position in the sequence. Thus, P [ S/T/R/K/D/E/N/Q ] P [ S/T/R/K/D/E ] (SEQ ID NO:102) means: p (conserved amino acid) at the first position; s, T, R, K, D, E, N, or Q is at a second position; p (conserved amino acid) at the third position; and either S, T, R, K, D, or E is at the fourth position. The motif represented by this nomenclature can thus be any of PSPS (SEQ ID NO:103), PSPT (SEQ ID NO:104), PSPR (SEQ ID NO:105), PSPK (SEQ ID NO:106), PSPD (SEQ ID NO:107), PSPE (SEQ ID NO:108), and the like.

Unless further limited otherwise, amino acid X (or Xaa) is used herein to represent any of the 20 natural amino acids.

Detailed Description

Many proteins are composed of structural domains joined by linkers. For example, cellulases and other Glycoside Hydrolases (GH) are often found as modular enzymes having one or more catalytic domains linked to one or more CBMs via a peptide called a linker, which is sometimes partially glycosylated. The catalytic domain is responsible for the hydrolytic degradation of cellulose, and when present, the CBM acts by increasing the effective concentration of the enzyme near the substrate surface. In contrast, linkers are generally flexible linkers that provide connectivity between structured domains, but their functional role is largely unknown.

The present invention relates to variants of glycoside hydrolases having a three-domain structure, wherein the catalytic domain is linked to one or more carbohydrate-binding modules via a linker. The present invention relates to variants having a peptide stretch that makes the natural linker more stable, i.e., less susceptible to proteolytic cleavage.

Especially cellulases, are frequently cleaved (nicked) by proteases or partially or completely degraded in exposed areas in liquid detergents. Most commonly, proteases cleave in the unstructured linker region of cellulases and thereby reduce the ability of cellulases to remove fuzz and pills and to maintain or restore the color of textiles by reducing the ability of cellulases to bind to insoluble cellulosic substrates. The loss of binding affinity severely affects the performance of cellulases and therefore protease stable linkers are of great value in the liquid laundry/dish washing detergent field as well as in softeners.

Variants

The present invention provides a variant of a parent polypeptide having glycoside hydrolase (EC 3.2.1.-) activity, wherein the variant comprises a catalytic domain, an engineered linker region (which may be, for example, a proline-rich linker region, such as a non-naturally occurring proline-rich linker region), and a Carbohydrate Binding Module (CBM), wherein the variant has improved linker stability and/or improved CBM stability in an aqueous detergent composition containing a protease compared to the parent glycoside hydrolase, and wherein the variant has glycoside hydrolase activity.

In embodiments, the parent polypeptide is a cellulase, and even more preferably, an endoglucanase, and even more preferably, a GH45 endoglucanase.

Polypeptides having N-terminal and/or C-terminal CBM's are contemplated.

Joint

The variants according to the invention comprise a proline-rich amino acid sequence linking the catalytic core to the CBM (linker region), e.g. a proline-rich linker region.

These proline-rich linkers as described herein comprise one or more Pro-Pro, Pro-Xaa (or Xaa-Pro), Xaa-Pro-Xaa, or Xaa-Xaa-Pro (or Pro-Xaa-Xaa) units, e.g., PPPP (SEQ ID NO:27), PXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPX (SEQ ID NO:98), XP (SEQ ID NO:99), XPXXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPX (SEQ ID NO:100), XXPXXP (SEQ ID NO:101), and the like, optionally further combined and/or repeated.

For example, the linker region may comprise at least 25% proline, e.g., at least 28% proline, at least 30% proline, at least 40% proline, at least 50% proline, e.g., at least 60%, at least 66%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% proline. In other embodiments, the linker comprises at least 50% proline, e.g., at least 60%, at least 66%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and has an overall negative charge. For example, the linker region comprises an acidic amino acid.

Preferred linker regions have a length of at least 4 amino acids and not more than 30 amino acids, such as 4-28 amino acids, preferably 4-20 amino acids, or even 4-10 amino acids, such as 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids.

Exemplary linker regions comprise one or more of the following optional repeat motifs:

[ P/S/T/R/K/D/E ] P and

P[S/T/R/K/D/E/N/Q]P[S/T/R/K/D/E](SEQ ID NO:102)

other preferred linker regions include the following optional repeat motifs:

[S/T/R/K/D/E]P[S/T/R/K/D/E/N/Q]

[ P/S/T/R/K/D/E ] [ P/S/T/R/K/D/E ] P and/or

P[P/S/T/R/K/D/E][P/S/T/R/K/D/E]。

Particularly preferred linkers include optional repeat motifs of the same or different amino acids in parentheses as shown:

[ P/S/T ] P and

P[S/E]PT(SEQ ID NO:109)。

or more specifically, the optional repeating motifs represented by [ P/S/T ] P include PPPPPPPP (SEQ ID NO:29) and PPSPTP (SEQ ID NO:110), PPTPPTP (SEQ ID NO:111), PPSPSP (SEQ ID NO:112), SPPPTP (SEQ ID NO:113), SPTPPP (SEQ ID NO:114), SPPPPP (SEQ ID NO:115), SPTPTP (SEQ ID NO:116), TPPPSP (SEQ ID NO:117), TPSPPP (SEQ ID NO:118), TPPPPP (SEQ ID NO:119), TPSPSP (SEQ ID NO:120), and the optional repeating motifs represented by P [ S/E PT ] P (SEQ ID NO:109) include PSPTPEPT (SEQ ID NO:121), PSPTPEPTPSPTPEPT (SEQ ID NO:122), PEPTSPT (SEQ ID NO:123), PEPTPSPTPSPT (SEQ ID NO:124), and the like.

The exemplary fitting further comprises

(a)(SP)_a，a＝2-10；

(b)(PS)_a，a＝2-10；

(c)P_bB is 4-20, preferably 4-15;

(d)(PEPT(SEQ ID NO:125))_c，c＝2-5；

(e)(PSPT(SEQ ID NO:104))_d，d＝2-5；

(f)(P[S/T/R/K/D/E/N/Q]P[S/T/R/K/D/E](SEQ ID NO:102))_e，e＝2-5；

(g)([S/T/R/K/D/E]P)_ff is 2 to 10, preferably 2 to 5;

(h)([S/T/R/K/D/E/N/Q]P[S/T/R/K/D/E])_g，g＝2-6；

(i)([S/T/R/K/D/E/N/Q][S/T/R/K/D/E/N/Q]P)_h，h＝2-5；

(j)(TP)_i，i＝2-10；

(k)([S/T/P][S/T/P][S/T/P])_j，j＝2-11；

(l) And/or combinations thereof, wherein combinations of individual monomer units are contemplated.

When combinations of these motifs are included, the minimal repeating unit is a monomeric unit. For example, linkers include SPPEPT (SEQ ID NO:126), SPPSPT (SEQ ID NO:127), PSPEPT (SEQ ID NO:128), PSPSPSPT (SEQ ID NO: 129).

Additional exemplary joints include:

SPSP(SEQ ID NO:130)、SPSPSP(SEQ ID NO:131)、SPSPSPSP(SEQ ID NO:132)、SPSPSPSPSP(SEQ ID NO:58)、SPSPSPSPSPSP(SEQ ID NO:133)、SPSPSPSPSPSPSP(SEQ ID NO:134)、SPSPSPSPSPSPSPSP(SEQ ID NO:135)、PPPP(SEQ ID NO:27)、PPPPP(SEQ ID NO:28)、PPPPPP(SEQ ID NO:29)、PPPPPPP(SEQ ID NO:31)、PPPPPPPP(SEQ ID NO:136)、PPPPPPPPP(SEQS ID NO:137)、PPPPPPPPPP(SEQ ID NO:138)、PPPPPPPPPPP(SEQ ID NO:139)、PPPPPPPPPPPP(SEQ ID NO:140)、PPPPPPPPPPPPP(SEQ ID NO:141)、PPPPPPPPPPPPPP(SEQ ID NO:142)、PPPPPPPPPPPPPPP(SEQ ID NO:143)、PEPTPEPT(SEQ ID NO:144)、PEPTPEPTPEPT(SEQ ID NO:145)、PEPTPEPTPEPTPEPT(SEQ ID NO:146)、PEPTPEPTPEPTPEPTPEPT(SEQ ID NO:79)、PSPTPSPT(SEQ ID NO:147)、PSPTPSPTPSPT(SEQ ID NO:148)、PSPTPSPTPSPTPSPT(SEQ ID NO:149)、PSPTPSPTPSPTPSPTPSPT(SEQ ID NO:150)、SPSSPS(SEQ ID NO:151)、SPSSPSSPS(SEQ ID NO:152)、SPSSPSSPSSPS(SEQ ID NO:153)、SPSSPSSPSSPSSPS(SEQ ID NO:154)、TPTTPT(SEQ ID NO:155)、TPTTPTTPT(SEQ ID NO:156)、TPTTPTTPTTPT(SEQ ID NO:157)、TPTTPTTPTTPTTPT(SEQ ID NO:158)、PEPTPRPTPEPTPRPT(SEQ ID NO:159)、PEPTPKPTPEPTPKPT(SEQ ID NO:160)、PEPTPQPTPEPTPQPT(SEQ ID NO:161)、PRPTPEPTPRPT(SEQ ID NO:162)、PKPTPEPTPKPT(SEQ ID NO:163)、PEPTPQPT(SEQ ID NO:164)、PEPTPQPTPEPT(SEQ ID NO:165)、PEPTPRPTPEPTPRPTG(SEQ ID NO:85)、PEPTPKPTPEPTPKPTG(SEQ ID NO:87)、PEPTPQPTPEPTPQPTG(SEQ ID NO:88)、PRPTPEPTPRPTG(SEQ ID NO:89)、PKPTPEPTPKPTG(SEQ ID NO:90)、PEPTPQPTG(SEQ ID NO:91)、PEPTPQPTPEPTG(SEQ ID NO:92)、PPPGGPGGPGTPTSTAPGSGPTSPGGGSG(SEQ ID NO:82)、TTPPTPTPTPTP(SEQ ID NO:166)；TTPTPPTPTPTPTP(SEQ ID NO:167)、TTPTPTPPTPTPTPTP(SEQ ID NO:168)、TPPTPPTPPTPPTPPTPPTPPTPPTPPTPPTPP(SEQ ID NO:169)。

additional exemplary linkers include those described above as well as C-terminal glycines, e.g., SPSPG (SEQ ID NO:24), SPSPSPSPG, SPSPSPSPSPSPSPG (SEQ ID NO:25), SPSPSPSPSPSPG, SPSPSPSPSPSPSPG, SPSPSPSPSPSPSPSPG, PPPPG, PPPPPG, PPPPPPG, PPPPPPPG (SEQ ID NO:30), PPPPPPPPG (SEQ ID NO:32), PPPPPPPPPG (SEQ ID NO:33), PPPPPPPPPPG (SEQ ID NO:34), PPPPPPPPPPPG (SEQ ID NO:35), PPPPPPPPPPPPG (SEQ ID NO:170), PPPPPPPPPPPPPG (SEQ ID NO:36), PPPPPPPPPPPPPPG (SEQ ID NO:171), PPPPPPPPPPPPPPPG (SEQ ID NO:172), PEPTPEPTG (SEQ ID NO:37), PEPTPEPTPEPTG (SEQ ID NO:38), PEPTPEPTPEPTPEPTG (SEQ ID NO:39), PEPTPEPTPEPTPEPTPEPTG (SEQ ID NO:40), PSPTPSPTG, PSPTPSPTPSPTG, PSPTPSPTPSPTPSPTG (SEQ ID NO:41), PSPTPSPTPSPTPSPTPSPTG (SEQ ID NO:42), SPSSPSG (SEQ ID NO:94), SPSSPSSPSG (SEQ ID NO:95), SPSSPSSPSSPSG (SEQ ID NO:19), SPSSPSSPSSPSSPSG (SEQ ID NO:20), TPTTPTG (SEQ ID NO:96), TPTTPTTPTG (SEQ ID NO:97), TPTTPTTPTTPTG (SEQ ID NO:17), TPTTPTTPTTPTTPTG, PEPTPRPTPEPTPRPTG (SEQ ID NO:85), PEPTPKPTPEPTPKPTG (SEQ ID NO:87), PEPTPQPTPEPTPQPTG (SEQ ID NO:88), PRPTPEPTPRPTG (SEQ ID NO:89), PKPTPEPTPKPTG (SEQ ID NO:90), PEPTPQPTG (SEQ ID NO:91), PEPTPQPTPEPTG (SEQ ID NO:92), PPPGGPGGPGTPTSTAPGSGPTSPGGGSG (SEQ ID NO:82), TTPPTPTPTPTPG (SEQ ID NO: 12); TTPTPPTPTPTPTPG (SEQ ID NO:13), TTPTPTPPTPTPTPTPG (SEQ ID NO:14), TTPTPTPTPPTPTPTPTPG (SEQ ID NO:15), TPPTPPTPPTPPTPPTPPTPPTPPTPPTPPTPPG (SEQ ID NO: 16).

Particularly preferred linkers are those comprising mainly or only proline, e.g., PPPP (SEQ ID NO:27), PPPPP (SEQ ID NO:28), PPPPPP (SEQ ID NO:29), PPPPP (SEQ ID NO:31), PPPPPPPP (SEQ ID NO:136), PPPPPPPPP (SEQ ID NO:137), PPPPPPPPPP (SEQ ID NO:138), PPPPPPPPPPP (SEQ ID NO:139), PPPPPPPPPPPP (SEQ ID NO:140), PPPPPPPPPPPPP (SEQ ID NO:141), PPPPPPPPPPPPPP (SEQ ID NO:142), PPPPPPPPPPPPPPP (SEQ ID NO:143), PPPPPPPPPG, PPPPPPPG (SEQ ID NO:30), PPPPPPPPG (SEQ ID NO:32), PPPPPPPPPG (SEQ ID NO:33), PPPPPPPPPPG (SEQ ID NO:34), PPPPPPPPPPPG (SEQ ID NO:35), PPPPPPPPPPPPG (SEQ ID NO:170), PPG PPPPPPPPPPPPPG (SEQ ID NO:36), PPPPPPPPPPPPPPG (SEQ ID NO:171), PPPPPPPPPPPPPPPG (SEQ ID NO: 172).

For the examples considered above, one skilled in the art will recognize that the goal is to replace the linker of the parent of interest with the proline-rich linker herein to provide additional stability.

In an alternative embodiment, the linker may be considered a variant of the linker of the parent molecule, which variant has stable point mutations, including a mutation to proline.

Accordingly, in some embodiments, the linker can comprise an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence depicted at position 213-241 of SEQ ID NO: 1.

In embodiments, the linker comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence depicted at position 211-246 of SEQ ID NO 2.

In particularly preferred embodiments, the linker is selected from any one of the following linkers in table a:

TABLE A. preferred linkers

TTPPTPTPTPTPG(SEQ ID NO:12)

TTPTPPTPTPTPTPG(SEQ ID NO:13)

TTPTPTPPTPTPTPTPG(SEQ ID NO:14)

TTPTPTPTPPTPTPTPTPG(SEQ ID NO:15)

TPPTPPTPPTPPTPPTPPTPPTPPTPPTPPTPPG(SEQ ID NO:16)

TPTTPTTPTTPTG(SEQ ID NO:17)

TPTTPTTPTTPTTPTTPTG(SEQ ID NO:18)

SPSSPSSPSSPSG(SEQ ID NO:19)

SPSSPSSPSSPSSPSG(SEQ ID NO:20)

SPPSPPSPPSPPSPPG(SEQ ID NO:21)

SPPSPPSPPSPPSPPSPPSPPSPPSPPSPPG(SEQ ID NO:22)

PPSSPSSPSSPSSPSSPSSPSG(SEQ ID NO:23)

SPSPG(SEQ ID NO:24)

SPSPSPSPSPG(SEQ ID NO:25)

TPTPTPTPTPG(SEQ ID NO:26)

PPPP(SEQ ID NO:27)

PPPPP(SEQ ID NO:28)

PPPPPP(SEQ ID NO:29)

PPPPPPPG(SEQ ID NO:30)

PPPPPPP(SEQ ID NO:31)

PPPPPPPPG(SEQ ID NO:32)

PPPPPPPPPG(SEQ ID NO:33)

PPPPPPPPPPG(SEQ ID NO:34)

PPPPPPPPPPPG(SEQ ID NO:35)

PPPPPPPPPPPPPG(SEQ ID NO:36)

PEPTPEPTG(SEQ ID NO:37)

PEPTPEPTPEPTG(SEQ ID NO:38)

PEPTPEPTPEPTPEPTG(SEQ ID NO:39)

PEPTPEPTPEPTPEPTPEPTG(SEQ ID NO:40)

PSPTPSPTPSPTPSPTG(SEQ ID NO:41)

PSPTPSPTPSPTPSPTPSPTG(SEQ ID NO:42)

PQPTPQPTG(SEQ ID NO:43)

PDPTPDPTG(SEQ ID NO:44)

PRPTPEPTG(SEQ ID NO:45)

PQPTPEPTG(SEQ ID NO:46)

PSPNSPNSPNG(SEQ ID NO:47)

PEPTPRPTG(SEQ ID NO:48)

PQPTPEPTPQPTPEPTPQPTPEPTPQPTG(SEQ ID NO:49)

PDPTPDPTPDPTG(SEQ ID NO:50)

PQPTPQPTPQPTPQPTG(SEQ ID NO:51)

PQPTPEPTPQPTPEPTG(SEQ ID NO:52)

SPSPSPSPPPG(SEQ ID NO:53)

SPSPSPSPDPG(SEQ ID NO:54)

SPSPSPSPKPG(SEQ ID NO:55)

SPSPSPSPAPG(SEQ ID NO:56)

SPSPSPSPSPSG(SEQ ID NO:57)

SPSPSPSPSP(SEQ ID NO:58)

SPSPSPSPSPS(SEQ ID NO:59)

SPSPSPSPSPP(SEQ ID NO:60)

SPSPSPSPSPE(SEQ ID NO:61)

SPSPSPSPSPN(SEQ ID NO:62)

SPSPSPSPSPGG(SEQ ID NO:63)

SPSPSPSPSPK(SEQ ID NO:64)

PEPTPEPTP(SEQ ID NO:65)

PEPTPEPTR(SEQ ID NO:66)

PEPTPEPTPEPTP(SEQ ID NO:67)

PEPTPEPTPEPTPEPTPSPTG(SEQ ID NO:68)

PEPTPEPTPEPTPEPTPTPTG(SEQ ID NO:69)

PEPTPEPTPEPTPEPTPGPTG(SEQ ID NO:70)

PEPTPEPTPEPTPEPTPDPTG(SEQ ID NO:71)

PEPTPEPTPEPTPEPTPETG(SEQ ID NO:72)

PEPTPEPTPEPTPEPTPEPTD(SEQ ID NO:73)

PEPTPEPTE(SEQ ID NO:74)

PEPTPEPTPEPTPEPTPEP(SEQ ID NO:75)

PEPTPEPTPEPTPEPTPSPT(SEQ ID NO:76)

PEPTPEPTPEPTPEPTPRPTT(SEQ ID NO:77)

PEPTPEPTPEPTPEPTPEPTT(SEQ ID NO:78)

PEPTPEPTPEPTPEPTPEPT(SEQ ID NO:79)

PEPTPEPTPEPTPEPTPEPTS(SEQ ID NO:80)

PEPTPEPTPEPTPEPTPEPTR(SEQ ID NO:81)

PPPGGPGGPGTPTSTAPGSGPTSPGGGSG(SEQ ID NO:82)

PPPGGPGGTGTPTSTAPGSGPTSPGGGSG(SEQ ID NO:83)

PPSGGPGGPGTPTSTAPGSGPTSPGGGSG(SEQ ID NO:84)

PEPTPRPTPEPTPRPTG(SEQ ID NO:85)

PKPTPEPTPKPTPEPTG(SEQ ID NO:86)

PEPTPKPTPEPTPKPTG(SEQ ID NO:87)

PEPTPQPTPEPTPQPTG(SEQ ID NO:88)

PRPTPEPTPRPTG(SEQ ID NO:89)

PKPTPEPTPKPTG(SEQ ID NO:90)

PEPTPQPTG(SEQ ID NO:91)

PEPTPQPTPEPTG(SEQ ID NO:92)

TPPTPPG(SEQ ID NO:93)

SPSSPSG(SEQ ID NO:94)

SPSSPSSPSG(SEQ ID NO:95)

TPTTPTG(SEQ ID NO:96)

TPTTPTTPTG(SEQ ID NO:97)

In particular embodiments, the linker is PPPPP (SEQ ID NO:31), PPPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58), or SPSPSPSPSPG (SEQ ID NO: 25).

In embodiments, the variant comprises a catalytic domain having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58), or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 6.

In embodiments, the variant comprises a catalytic domain having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58), or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 7.

In embodiments, the variant comprises a catalytic domain having an amino acid sequence with at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58), or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 173.

In embodiments, the variant comprises a catalytic domain having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58), or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 178.

In embodiments, the variant comprises a catalytic domain having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58), or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 182.

In embodiments, the variant comprises a catalytic domain having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58), or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 186.

In embodiments, the variant comprises a catalytic domain having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58), or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 188.

In embodiments, the variant comprises a catalytic domain having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58), or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 194.

In embodiments, the variant comprises a catalytic domain having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58), or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 199.

In some aspects, the variants of the invention have improved properties relative to a reference/parent enzyme.

In one aspect, the improved property is increased stability, e.g., improved proteolytic stability, improved detergent stability, improved in-wash stability, or improved thermal stability. In another aspect, the improved property is increased stability during production of the detergent composition or increased performance after storage in the detergent composition relative to the performance of the parent molecule stored under similar conditions. Some aspects of the invention relate to cellulase variants having an improvement factor of higher than 1 when the cellulase variant is tested for a property of interest in a related assay, wherein the property of the reference/parent enzyme is given a value of 1. In some aspects, the property is stability, e.g., improved proteolytic stability. Some aspects of the invention relate to cellulase variants having an improvement factor of greater than 1 when the cellulase variant is tested for a property of interest in the assay described in example 2, wherein the property of the reference/parent enzyme is given a value of 1. In some aspects, the property is stability, e.g., proteolytic stability.

In some aspects, the improved characteristic is increased stability, such as improved detergent stability, improved wash stability and improved thermal stability. Some aspects of the invention relate to cellulase variants having an improvement factor of greater than 1 when the cellulase variant is tested for a property of interest in a related assay (e.g., when the cellulase variant is tested for a property of interest in an assay described in example 7), wherein the property of the reference/parent enzyme is given a value of 1.

In some aspects, the improved characteristic is improved thermal stability.

In some aspects, the improved characteristic is improved stability in a detergent.

In some aspects, the improved property is improved proteolytic stability.

In some aspects, the improved property is one or more or even all of improved thermal stability, improved detergent stability, improved proteolytic stability.

Under the measurement conditions, when the residual activity ratio is defined as

Residual Activity Ratio (RAR) ═ (RA of variant)/(RA of reference)

The variants according to the invention are improved when compared to the reference cellulase above 1.0.

In a particularly preferred aspect, the variants according to the invention result in improved stability (e.g., thermostability, detergent stability, proteolytic stability, or more than one or even all of these), wherein RAR > 1.0. In some aspects, the variant according to the invention has a Residual Activity Ratio (RAR) compared to the parent or reference enzyme and in particular to the cellulase of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 of at least: 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7, 2.8; 2.9; 3.0, 3.1; 3.2; 3.3; 3.4; 3.5, 3.6, 3.7, 3.8, 3.9; 4.0, 4.1; 4.2; 4.3; 4.4; 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1; 5.2; 5.3; 5.4; 5.5, 5.6, 5.7, 5.8, 5.9; 3.0 and 6.1; 6.2; 6.3; 6.4; 6.5, 6.6, 6.7, 6.8, 6.9; 7.0, 7.1; 7.2; 7.3; 7.4 of the total weight of the mixture; 7.5, 7.6, 7.7, 7.8, 7.9; 8.0, 8.1; 8.2; 8.3; 8.4 of the total weight of the mixture; 8.5, 8.6, 8.7, 8.8, 8.9; 9.0, 9.1; 9.2; 9.3; 9.4 of the total weight of the mixture; 9.5, 9.6, 9.7, 9.8, 9.9; 10.0, 10.1; 10.2; 10.3; 10.4; 10.5, 10.6, 10.7, 10.8, 10.9; 12. 15, 16, 20, 25 or 30.

A preferred embodiment relates to cellulase variants with improved stability wherein RAR >1.0 compared to SEQ ID NO: 1. One preferred embodiment relates to cellulase variants with improved stability wherein the Residual Activity Ratio (RAR) is at least 1.5 compared to SEQ ID NO:1 when measured as described in example 2.

Catalytic domains

Particularly preferred enzymes are those having cellulase, e.g. endoglucanase, activity. In particular, the relevant catalytic domain is an enzyme from glycoside hydrolase family 45(GH45), using the nomenclature of Henrissat et al, described in the CAZY database of CAZY.

The catalytic domain may comprise a wild-type or a variant thereof.

In embodiments, the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence depicted at positions 1-212 of SEQ ID No. 1.

In one aspect, the catalytic domain further comprises some substitutions in the variants of the invention, which are 2-20, e.g., 2-10 and 2-5, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.

In another aspect, the variant comprises or consists of two substitutions at positions selected from the group consisting of positions corresponding to: 25, 32, 41, 44, 56, 77, 104, 132, 146, 147, 156, 162, 169, 183, 186, 194 or 201 of SEQ ID NO. 1. In another aspect, the amino acid at this position is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

In one aspect, the variant comprises or consists of one or more substitutions in: X25G; X32S; X41T; X44D; X56A; X77N; X85I; X103A; X104K; X114W or X114F; X134D; X137K or X137R; X146D or X146S; X147R; X152K; X156E; X159D or X159E; X162E; X169Y; X179T; X183V; X186R; X194L or X194S; and/or X201K.

In some embodiments, these variants comprise the substitution X32S and one or more substitutions corresponding to: substitution A25G of a polypeptide having the sequence of SEQ ID NO. 1 or SEQ ID NO. 5; S41T; S56A; S77N; T104K; N134D; a146D or a 146S; Q147R; Q156E; A162E; Q169Y; F183V; Q186R; I194L; K201R and G219W, wherein the variant has cellulolytic activity.

In some embodiments, these variants comprise the substitution X56A and one or more substitutions corresponding to: substitution A25G of a polypeptide having the sequence of SEQ ID NO. 1 or SEQ ID NO. 5; a 32S; S41T; S77N; T104K; N134D; a146D or a 146S; Q147R; Q156E; A162E; Q169Y; F183V; Q186R; I194L; K201R and G219W, wherein the variant has cellulolytic activity.

In some embodiments, these variants comprise the substitution X134D and one or more substitutions corresponding to: substitution A25G of a polypeptide having the sequence of SEQ ID NO. 1 or SEQ ID NO. 5; a 32S; S41T; S56A; S77N; S85I; T104K; G114F; G114W; S137E; S137R; S137D; S137K; a146D or a 146S; Q147R; S152K; Q156E; S159E; S159D; A162E; Q169Y; D179T; F183V; Q186R; I194L; I194S; K201R and G219W, wherein the variant has cellulolytic activity.

In some embodiments, the variants comprise substitution a146D, and further comprise a substitution selected from the group consisting of substitutions corresponding to the following substitutions in SEQ ID NO: 1: A25G of a polypeptide having the sequence of SEQ ID NO. 1 or SEQ ID NO. 5; a 32S; S41T; S56A; S77N; K103A; T104K; G114F; G114W; N134D; S137R; S152K; Q156E; S159D; S159E; A162E; Q169Y; D179T; F183V; Q186R; I194L; K201R and G219W, wherein the variant has cellulolytic activity.

In some embodiments, these variants comprise the substitution X147R and one or more substitutions corresponding to: substitution A25G of a polypeptide having the sequence of SEQ ID NO. 1 or SEQ ID NO. 5; a 32S; S41T; S56A; S77N; T104K; N134D; a146D or a 146S; Q156E; A162E; Q169Y; F183V; Q186R; I194L; K201R and G219W, wherein the variant has cellulolytic activity.

In some embodiments, these variants comprise the substitution S159D, and further comprise a substitution selected from the group consisting of substitutions corresponding to the following substitutions in SEQ ID NO: 1: A25G of a polypeptide having the sequence of SEQ ID NO. 1 or SEQ ID NO. 5; a 32S; S41T; S56A; S77N; K103A; T104K; G114F; G114W; N134D; S137R; a 146D; S152K; Q156E; A162E; Q169Y; D179T; F183V; Q186R; I194L; K201R and G219W, wherein the variant has cellulolytic activity.

In some embodiments, these variants comprise the substitution X169Y and one or more substitutions corresponding to: substitution A25G of a polypeptide having the sequence of SEQ ID NO. 1 or SEQ ID NO. 5; a 32S; S41T; S56A; S77N; T104K; N134D; a146D or a 146S; Q147R; Q156E; A162E; F183V; Q186R; I194L; K201R and G219W, wherein the variant has cellulolytic activity.

In embodiments, the variant comprises one or more of the following combinations: 25G +56A, 25G +114W, 25G +134D, 25G +146D, 25G +147R, 25G +156E, 25G +162E, 25G +169Y, 25G +183V, 56A +114W, 56A +134D, 56A +146D, 56A +147R, 56A +156E, 56A +162E, 56A +169Y, 56A +183V, 114W +134D, 114W +146D, 114W +147R, 114W +156E, 114W +162E, 114W +169Y, 114W +183V, 134D +146D, 134D +147R, 134D +156E, 134D +169Y, 134D +183V, 146D +147R, 146D + 147E, 147Y, 147R +156E, 183Y +162E, 183E +162E, 183Y, 183E + 169E, 169Y + 169E, 169E +156E, 183E +162E, 183Y +169Y, 169E + 169E, 169Y, and 183E +156E, 162E +183V, 169Y +183V, wherein numbering is performed using SEQ ID NO. 1 or SEQ ID NO. 5.

In embodiments, the variant comprises one or more of the following combinations: 25G +56A +114W, 25G +56A +134D, 25G +56A +146D, 25G +56A +147R, 25G +56A +156E, 25G +56A +162E, 25G +56A +169Y, 25G +56A +183V, 25G +114W +134D, 25G +114W +146D, 25G +114W +147R, 25G +114W +156E, 25G +114W +162E, 25G +114W +169Y, 25G +114W +183V, 25G +134D +146D, 25G +134D +147R, 25G +134D +156E, 25G +134D +162E, 25G +134D +169Y, 25G +134D +183V, 25G +146D +147R, 25G +146D +156E, 25G +146D +162E, 25G +146D + 146Y, 25G +134D +169Y, 25G +134D + 147Y, 147R + 147V, 25G + 134E, 25G +183V +147R, 25G + 134E, 25G +147R +183V, 25G + 134E, 25G +147, 25G +147R +183V, 25G +156E +162E, 25G +156E +169Y, 25G +156E +183V, 25G +162E +169Y, 25G +162E +183V, 25G +169Y +183V, 56A +114W +134D, 56A +114W +146D, 56A +114W +147R, 56A +114W +156E, 56A +114W +162E, 56A +114W +169Y, 56A +114W +183V, 56A +134D +146D, 56A +134D +147R, 56A +134D +156E, 56A +134D +162E, 56A +134D +169Y, 56A +134D +183V, 56A +146D +147R, 56A +146D +156E, 56A +146D +162E, 56A +146D + 146Y, 183A +146D + 147V, 56A +147R + 147V, 56A + 169R +147R, 147V, 56A +147R + 156A + 147V, 56A +147R, 56A +156E, 56A +147R + 147V, 56A +147R, 56A +156E +162E, 56A +156E +169Y, 56A +156E +183V, 56A +162E +169Y, 56A +162E +183V, 56A +169Y +183V, 114W +134D +146D, 114W +134D +147R, 114W +134D +156E, 114W +134D +162E, 114W +134D +169Y, 114W +134D +183V, 114W +146D +147R, 114W +146D +156E, 114W +146D +162E, 114W +146D +169Y, 114W +146D +183V, 114W +147R +156E, 114W +147R +169Y, 114W + R +183V, 147W +156E + 147E, 114W +147R +169Y, 114W + R +183V, 147W +156E +162E, 114W +156E +169Y, 114W +156E +183V, 183W +156E + 183Y, 183W + 146E +134D + 134V, 114W +134D, 134D +146D +162E, 134D +146D +169Y, 134D +146D +183V, 134D +147R +156E, 134D +147R +162E, 134D +147R +169Y, 134D +147R +183V, 134D +156E +162E, 134D +156E +169Y, 134D +156E +183V, 134D +162E +169Y, 134D +162E +183V, 134D +169Y +183V, 146D +147R +156E, 146D +147R +162E, 146D +147R + Y, 146D +147R +183V, 146D +156E +162E, 146D +156E +169Y, 146D +156E +183V, 146D +162E +169Y, 146D +162E +183V, 146D + 183Y +183V, 147R +156E +162E, 183Y +147R + 147Y, 183Y +162E + 147Y, 183Y +147R +162E + R + 147Y, 147Y +147R +156E + 183Y +162E, and 147Y +162E, 156E +162E +169Y, 156E +162E +183V, 156E +169Y +183V, 162E +169Y +183V, wherein numbering is performed using SEQ ID NO. 1 or SEQ ID NO. 5.

In embodiments, the variant comprises one or more of the following combinations: 25G +56A +114W +134D, 25G +56A +114W +146D, 25G +56A +114W +147R, 25G +56A +114W +156E, 25G +56A +114W +162E, 25G +56A +114W +169Y, 25G +56A +114W +183V, 25G +56A +134D +146D, 25G +56A +134D +147R, 25G +56A +134D +156E, 25G +56A +134D +162E, 25G +56A +134D + 162Y, 25G +56A +134D +183V, 25G +56A +146D +147R, 25G +56A + 156D +156E, 25G +56A +146D +162E, 25G +56A +146D +169Y, 25G +56A + 146V, 183G +146D +147R, 25G +56A +147R + 147E, 25G +56A +147R + 147V, 25G +56A +147R + 162A +147R, 25G +56A + 147V, 25G +147R +56A +147R, 25A +162E, 25G + V + 147A +56A +147R, 25G +56A +147R, 25A + X, 25G +56A +156E +162E, 25G +56A +156E +169Y, 25G +56A +156E +183V, 25G +56A +162E +169Y, 25G +56A +162E +183V, 25G +56A +169Y +183V, 25G +114W +134D +146D, 25G +114W +134D +147R, 25G +114W +134D +156E, 25G +114W +134D +162E, 25G +114W +134D +169Y, 25G +114W +134D +183V, 25G +114W +146D +147R, 25G +114W +146D +156E, 25G +114W + 162D +162E, 25G +114W +146D +169Y, 25G +114W +146D +183V, 25G +114W +147R + 147E, 25G + 156W + 147E, 25G + 162W +162E, 25G +114W +162E, 25G +114W + 147V, 25G +114W +147R +162E, 25G +114W +162E, 25G + X, 25G +114W +156E +169Y, 25G +114W +156E +183V, 25G +114W +162E +169Y, 25G +114W +162E +183V, 25G +114W +169Y +183V, 25G +134D +146D +147R, 25G +134D +146D +156E, 25G +134D +162E, 25G +134D +146D +169Y, 25G +134D +146D +183V, 25G +134D +147R +156E, 25G +134D +147R +162E, 25G +134D +147R +169Y, 25G +134D +147R + 147V, 25G +134D +156E +162E, 25G +134D +156E + 156Y, 25G +134D + 134E +183V, 25G +134D +162E + 183Y, 25G +134D +156E + 162Y, 25G +134D +156E + 147V, 25G +134D + 134E +169Y, 25G +134D + 134E +156E +169Y, 25G + 147V, 25G +134D + 147E + 156Y, 25G +134D + 134E +156E, 25G +146D +147R +169Y, 25G +146D +147R +183V, 25G +146D +156E +162E, 25G +146D +156E +169Y, 25G +146D +156E +183V, 25G +146D +162E +169Y, 25G +146D +162E +183V, 25G +146D +169Y +183V, 25G +147R +156E +162E, 25G +147R +156E +169Y, 25G +147R +156E +183V, 25G +147R +162E +169Y, 25G +147R +162E +183V, 25G +147R +169Y +183V, 25G +156E +169Y, 25G +156E + 169E +183V, 25G +156E +169Y +183V, 25G +162E + 183Y + 56Y, 56W +56A + 134W + 156D + 134W +134D + 134W + 134A +134D + 134W +134D + 134A + 134W, 56A +114W +134D +169Y, 56A +114W +134D +183V, 56A +114W +146D +147R, 56A +114W +146D +156E, 56A +114W +146D +162E, 56A +114W +146D +169Y, 56A +114W +146D +183V, 56A +114W +147R +156E, 56A +114W +147R +162E, 56A +114W +147R +169Y, 56A +114W +147R +183V, 56A +114W +156E +162E, 56A +114W +156E +169Y, 56A +114W +156E +183V, 56A +114W +162E +169Y, 56A +114W +162E +183V, 56A +114W +169Y +183V, 56A +134D + 146R, 56A +146D +162E, 56A +134D, 56A +134D, 56A +134D + 134E, 56A +134D + 134E, 56A +134D + 134E, 56A + 134E, and 56A +134D + 134E, 56A +134D +147R +156E, 56A +134D +147R +162E, 56A +134D +147R +169Y, 56A +134D +147R +183V, 56A +134D +156E +162E, 56A +134D +156E +169Y, 56A +134D +156E +183V, 56A +134D +169Y, 56A +134D +162E +183V, 56A +134D +169Y +183V, 56A +146D +147R +156E, 56A +146D +147R +162E, 56A +146D +147R +169Y, 56A +146D +147R + 147D +147R + 183Y, 56A +146D +147R +183V, 56A +146D +156E +162E, 56A +146D +156E +169Y, 56A +146D +183V, 56A +146D +162E + 183Y, 56A + 183A + 162D + 156Y, 56A +156E +169Y, and 147E +169Y, 56A +147R +156E +183V, 56A +147R +162E +169Y, 56A +147R +162E +183V, 56A +147R +169Y +183V, 56A +156E +162E +169Y, 56A +156E +162E +183V, 56A +156E +169Y +183V, 56A +162E +169Y +183V, 114W +134D +146D +147R, 114W +134D +146D +156E, 114W +134D +146D + 162D, 114W +134D +146D +169Y, 114W +134D +146D +183V, 114W +134D +147R +156E, 114W +134D +147R + 162Y, 114W +134D +147R +183V, 114W +134D +162E, 183Y +134D +162E, 114W +134D + 162Y, 114W +134D +162E, 114W +134D +169Y +183V, 114W +146D +147R +156E, 114W +146D +147R +162E, 114W +146D +147R +169Y, 114W +146D +147R +183V, 114W +146D +156E +162E, 114W +146D +156E +169Y, 114W +146D +156E +183V, 114W +146D +162E +169Y, 114W +146D +162E +183V, 114W +146D +169Y +183V, 114W +147R +156E +162E, 114W +147R +156E +169Y, 114W +147R +156E +183V, 114W + 162R +162E +169Y, 114W + R + 147V, 114W +147R +169Y +183V, 114W +156E + 162Y + 183Y, 183W +156E +183V, 114W +156E + 162Y, 183W +162E +169Y, 114W +169Y + 169V, 114W + 147V, and 134E +156E +162E +183V, 134D +146D +147R +162E, 134D +146D +147R +169Y, 134D +146D +147R +183V, 134D +146D +156E +162E, 134D +146D +156E +169Y, 134D +146D +156E +183V, 134D +146D +162E +169Y, 134D +146D +162E +183V, 134D +146D +169Y +183V, 134D +147R +156E +162E, 134D +147R +156E +169Y, 134D +147R +156E +183V, 134D +147R +162E +169Y, 134D +147R +162E + 183Y, 134D +156E + 147V, 134D +147R + 183Y +183V, 183D + 147E + 147Y + 169E +156E +169Y, 134D + 147E +156E + 147V, 134D + 147Y +156E +169Y, 134D +156E +169Y, 134D + 147E +156E +169Y + E +169Y, 146D +147R +162E +169Y, 146D +147R +162E +183V, 146D +147R +169Y +183V, 146D +156E +162E +169Y, 146D +156E +162E +183V, 146D +156E +169Y +183V, 146D +162E +169Y +183V, 147R +156E +169Y, 147R +156E +162E +183V, 147R +156E +169Y +183V, 147R +162E +169Y +183V, 156E +162E +169Y +183V, wherein numbering is performed using SEQ ID NO 1 or SEQ ID NO 5.

Particularly preferred variants in the catalytic domain include variants comprising substitutions selected from the group consisting of:

X147R+X156E；

X147R+X169Y；

X56A+X147R；

X147R+X162E；

X147R+X156E+X162E；

X25G+X56A+X147R；

X134D+X156E+X162E；

X56A+X134D+X156E+X162E；

X25G+X56A+X156E+X162E；

X25G+X134D+X156E+X162E；

X25G+X56A+X134D+X169Y；

X56A+X134D+X162E；

X56A+X147R+X169Y；

X134D+X147R；

X156E+X169Y；

X56A+X134D+X147R；

X56A+X134D+X156E+X169Y；

X56A+X146D+X147R+X169Y；

X56A+X134D+X147R+X169Y；

X56A+X147R+X162E+X169Y；

X2*+X56A+X147R+X169Y；

X41T+X56A+X147R+X169Y；

X56A+X77N+X147R+X169Y；

X56A+X104K+X147R+X169Y；

X56A+X147R+X165Q+X169Y；

X56A+X147R+X169Y+X194L；

X56A+X147R+X169Y+X201R；

X56A+X147R+X169Y+X219W；

X44D+X56A+X147R+X169Y；

X50E+X56A+X147R+X169Y；

X32S+X56A+X147R+X169Y；

X44D+X56A+X147R+X169Y；

X56A+X147R+X169Y+X186R；

X56A+X147R+X169Y+X183V；

X56A+X146S+X147R+X162E+X169Y；

X56A+X134D+X147R；

X56A+X134D+X147R+X162E；

X32S+X56A+X134D+X147R+X169Y+X183V；

X56A+X134D+X147R+X162E+X169Y+X183V；

X32S+X56A+X77N+X134D+X147R+X162E+X169Y；

X32S+X56A+X134D+X146D+X147R+X169Y+X183V；

X32S+X56A+X134D+X147R+X169Y；

X56A+X134D+X147R+X162E+X169Y；

X32S+X56A+X134D+X146S+X147R+X169Y；

X32S+X56A+X134D+X146D+X147R+X169Y；

X32S+X56A+X134D+X147R+X169Y+X183V；

X32S+X56A+X134D+X147R+X169Y+X201R；

X56A+X134D+X146D+X147R+X169Y+X183V；

X56A+X134D+X146D+X147R+X162E+X169Y；

X56A+X134D+X146D+X147R+X169Y+X201R；

X56A+X134D+X147R+X162E+X169Y+X183V；

X56A+X134D+X147R+X169Y+X183V+X201R；

X32S+X56A+X77N+X134D+X147R+X169Y+X183V；

X32S+X56A+X77N+X134D+X147R+X162E+X169Y；

X32S+X56A+X134D+X146S+X147R+X169Y+X183V；

X32S + X56A + X134D + X146D + X147R + X169Y + X183V; or

X32S+X56A+X134D+X146D+X147R+X162E+X169Y，

Wherein numbering is performed using SEQ ID NO 1 or SEQ ID NO 5.

In particularly preferred embodiments, the catalytic domain comprises variants of SEQ ID No. 5 comprising or consisting of one or more of: A25G; a 32S; S41T; N44D; S56A; S77N; S85I; K103A; T104K; G114W or G114F; N134D; S137K or S137R; a146D or a 146S; Q147R; S152K; Q156E; S159D or S159E; A162E; Q169Y; D179T; F183V; Q186R; I194L; K201R; and combinations thereof.

In embodiments, the parent cellulase is a cellulase having SEQ ID No. 1 or SEQ ID No. 5 and the variant comprises one or more of the following combinations: a25G + S56A, a25G + G114W, a25G + N134D, a25G + a146D, a25G + Q147R, a 25R + Q156R, a 25R + a 162R, a 25R + Q169R, a 25R + F183R, S56R + G114R, S56R + N134R, S56R + a 146R, S56R + Q147R, S56R + Q36156R, S56R + Q R, S56R + F183R, G114R + N134R, G R + a R, G114 + Q R, G R + Q R, a 36183 + F R, a R + N R, a R + F183 + R, a R + F R, a R + N R + F R, a R + F R + R, a R + F R + R, a R + F R, a R + F R + R, a R + F R + R, a R + F R + F R, a R + F R + R, a R + F R + F R + F R + F R + R, G R + F R + F R + F R + F R + F R + F R + F R + 36.

In embodiments, the parent cellulase is a cellulase having SEQ ID No. 1 or SEQ ID No. 5 and the variant comprises one or more of the following combinations: a25 + S56 + G114, A25 + S56 + N134, A25 + S56 + A146, A25 + S56 + Q147, A25 + S56 + Q156, A25 + S56 + A162, A25 + S56 + Q169, A25 + S56 + F183, A25 + G114 + N134, A25 + G114 + A146, A25 + G114 + Q147, A25 + G114 + Q156, A25 + G114 + A162, A25 + G114 + Q169, A25 + G114 + F183, A25 + N134 + A146, A25 + N134 + Q147, A25 + N134 + Q156, A25 + N134 + A162, A25 + N134 + Q169, A25 + N134 + F183, A25 + A146 + Q147, A25 + A146 + Q156, A25 + N146 + Q183, A183 + Q183 + F156, A25 + Q147, A25 + F56 + Q + F156, A25 + Q183 + F156, A + Q + F156, A25 + Q147, A25 + Q + F156, A25 + Q + F169, A25 + F156, A + Q + F183, A24 + F183, A + F183, A + F156 + Q147, A + Q147, A25 + Q + F156, A56 + Q147, A + Q + F156, A25 + F156, A + Q + F156, A + F169, A25 + F156, A25 + Q + F156, A + Q147, A25 + Q + F156, A24 + Q147, A + Q147, A56 + Q147, A + Q147, A56 + Q + F156, A + Q + F156, A56 + Q147, A + Q + F156, A24 + F156, A + F169, A + F156, A + Q + F156, A25 + Q + F156, A25 + F156, A24 + Q147, A + Q + F156, A24 + F156, A25 + Q147, A24 + F156, A + Q147, A56 + Q + F156, A24 + F156, A + Q + F156, A + Q + F156, A + Q + F169, A + F156, A24 + F156, A25 + F156, A, S56 + N134 + A146, S56 + N134 + Q147, S56 + N134 + Q156, S56 + N134 + A162, S56 + N134 + Q169, S56 + N134 + F183, S56 + A146 + Q147, S56 + A146 + Q156, S56 + A146 + A162, S56 + A146 + Q169, S56 + A146 + F183, S56 + Q147 + Q156, S56 + Q147 + A162, S56 + Q147 + Q169, S56 + Q147 + F183, S56 + Q156 + A162, S56 + Q156 + Q169, S56 + Q156 + F183, S56 + A183, S183 + Q183, S56 + A162 + F183, S56 + Q169 + F183, S56 + Q183, G114 + N134 + A146, G114 + N134 + Q147, G114 + G134 + Q183, G + N134 + Q183, G156 + Q183, S56 + A183 + Q183 + F183, S114 + G156 + G + F147, G114 + G + F147, G + G114 + F147, G + G114 + Q + G + F147, G114 + G114 + F147, G + G114 + F147, G + Q + F + G114 + G + F147, G + F147 + G114 + F147, G114 + F183 + F147, G + F147 + F183 + F147, G114 + F183 + F147, G + F, N134 + a146 + Q156, N134 + a146 + a162, N134 + a146 + Q169, N134 + a146 + F183, N134 + Q147 + Q156, N134 + Q147 + a162, N134 + Q147 + Q169, N134 + Q147 + F183, N134 + Q156 + a162, N134 + Q156 + Q169, N134 + Q156 + F183, N134 + a162 + Q169, N134 + a162 + F183, N134 + Q169 + F183, a146 + Q147, a146 + Q147 + a162, a146 + Q147 + Q169, a146 + Q147 + F183, a146 + Q183, a 183 + Q156 + Q169, a146 + Q156 + F183, a146 + a162 + Q169, a146 + a162 + F169, a 183 + Q147 + Q183 + Q156 + Q169, a 183 + Q156 + F183, 183 + Q156, a 183 + Q147 + Q156 + Q147, a 183 + Q147, a162 + Q147, a 183 + Q147 + Q169, a162, a 183 + Q147, a162 + Q147, a 169, a 183 + Q147, a 156, a162, a 183 + Q147, a 156, a162, a 169, a162, a 183 + Q147 + Q169, a, and Q147 + Q169, a 156, a162 + Q147 + Q169, a 156, a162 + Q147 + Q169, a162 + Q147 + Q169, a 156, and Q147 + Q169, a 134 + Q147 + Q169, a 156, a162 + Q147 + Q169, a 156, a162 + Q169, a162, a 156, a162 + Q169, and Q169, a162 + Q147 + Q169, a 162.

In embodiments, the parent cellulase is a cellulase having SEQ ID No. 1 or SEQ ID No. 5 and the variant comprises one or more of the following combinations: 25 + S56 + G114 + N134, A25 + S56 + G114 + A146, A25 + S56 + G114 + Q147, A25 + S56 + G114 + Q156, A25 + S56 + G114 + A162, A25 + S56 + G114 + Q169, A25 + S56 + G114 + F183, A25 + S56 + N134 + A146, A25 + S56 + N134 + Q147, A25 + S56 + N134 + Q156, A25 + S56 + N134 + A162, A25 + S56 + N134 + Q169, A25 + S56 + N134 + F183, A25 + S56 + A183 + Q147, A25 + S56 + A156 + Q156, A25 + S56 + A146 + A162, A25 + S56 + A146 + Q169, A25 + S56 + Q147, A183 + S183 + Q147, A25 + S56 + Q + A156 + Q147, A25 + S56 + Q147, A25 + A24 + S56 + Q147, A24 + A + Q147, A25 + S56 + Q134 + Q147, A134 + Q147, A + Q134 + A147, A134 + Q183, A147, A25 + Q183, A + Q147, A + Q134 + Q183, A147, A25 + Q183 + Q134 + Q147, A25 + A156, A + A25 + S156 + Q134 + Q147, A24 + Q134 + A24 + Q147, A56 + A + Q147, A25 + Q147, A24 + Q134 + Q147, A24 + Q134 + A + Q147, A24 + Q134 + Q147, A25 + G114 + N134 + F183, A25 + G114 + A146 + Q147, A25 + G114 + A146 + Q156, A25 + G114 + A146 + A162, A25 + G114 + A146 + Q169, A25 + G114 + A146 + F183, A25 + G114 + Q147 + Q156, A25 + G114 + Q147 + A162, A25 + G114 + Q147 + Q169, A25 + G114 + Q147 + Q183, A25 + G114 + Q156 + A162, A25 + G114 + Q156 + Q169, A25 + G114 + Q183, A25 + G183 + A183 + Q183, A25 + G114 + G183, A25 + G114 + F183, A25 + N134 + Q147, A25 + N134 + Q147, A134 + N134 + A183 + N134 + A134 + Q147, A183 + N134 + A183, A183 + N134 + Q147 + A134 + N134 + Q183, A134 + N147, A134 + N134 + Q147, A25 + N134 + Q183, A134 + N183, A + N134 + Q134 + N183, A134 + Q183, A134 + N147 + Q134 + Q183, A134 + N183, A134 + N147 + N183, A25 + N134 + N147 + N134 + F183, A, A25 + A146 + Q147 + Q169, A25 + A146 + Q147 + F183, A25 + A146 + Q156 + A162, A25 + A146 + Q156 + Q169, A25 + A146 + Q156 + F183, A25 + A146 + A162 + Q169, A25 + A146 + A162 + F183, A25 + A146 + Q169 + F183, A25 + Q147 + Q156 + A162, A25 + Q147 + Q156 + Q169, A25 + Q147 + Q156 + F183, A25 + Q147 + A147 + Q147 + F183, A25 + Q147 + Q183, A25 + Q147 + Q169 + Q183, A183 + Q183, A25 + Q183, A156 + Q147 + G147, A25 + Q156 + Q169, A25 + Q156 + Q183, A25 + Q147 + G56 + G147, A56 + G114 + G147 + G114 + G + S114 + G147, A56 + G + S114 + G147, A134 + G + S + G147 + G + Q147 + Q134 + G + S134 + G + S134 + S114 + G + S134, A134 + G147, A134 + G147 + G147 + G134 + G134 + G + F183, A134 + G147, A134 + G147 + G134 + G147 + G134 + G147 + G134 + G, S56 + G114 + Q156 + A162, S56 + G114 + Q156 + Q169, S56 + G114 + Q156 + F183, S56 + G114 + A162 + Q169, S56 + G114 + A162 + F183, S56 + G114 + Q169 + F183, S56 + N134 + A146 + Q147, S56 + N134 + Q156, S56 + N134 + A146 + A162, S56 + N134 + A146 + Q169, S56 + N134 + A146 + F183, S56 + N134 + Q147 + Q156, S56 + N134 + Q147 + A162, S56 + N134 + Q183 + Q147 + Q183, S56 + N134 + F183, S56 + N147 + Q147, S56 + N134 + Q147, S56 + Q183 + Q147, S56 + Q183 + Q146 + Q147 + Q146 + Q147, S56 + A146 + F146 + Q147, S56 + F146 + Q147 + Q146 + Q147, S56 + Q183 + Q147, S56 + F146 + Q147, S56 + Q183 + F146 + Q147, S56 + Q147 + Q183 + Q147, S56 + F146 + Q147, S56 + Q147 + F146 + Q147, S56 + F146 + F, S56 + F146 + F183 + F, S56 + F146 + Q147 + F, S56 + F146 + F, S56 + F, S56 + Q147 + Q156 + F183, S56 + Q147 + A162 + Q169, S56 + Q147 + A162 + F183, S56 + Q147 + Q169 + F183, S56 + Q156 + A162 + Q169, S56 + Q156 + A162 + F183, S56 + Q156 + Q169 + F183, S56 + A162 + Q169 + F183, G114 + N134 + A146 + Q147, G114 + N134 + A146 + Q156, G114 + N134 + A146 + A162, G114 + N134 + A146 + Q169, G114 + N134 + A146 + F183, G114 + N134 + Q147 + Q183, G114 + N183 + Q183, G114 + N134 + Q147 + G114 + F147 + Q183, G114 + Q183 + Q156 + Q183, G114 + N183 + Q183, G156 + A183 + Q183 + A146 + F146 + Q146 + G114 + F146 + F147, G114 + F146 + Q + G146 + G114 + Q146 + Q + F146 + G114 + G146 + G114 + F, G114 + Q147 + Q156 + A162, G114 + Q147 + Q156 + Q169, G114 + Q147 + Q156 + F183, G114 + Q147 + A162 + Q169, G114 + Q147 + A162 + F183, G114 + Q147 + Q169 + F183, G114 + Q156 + A162 + Q169, G114 + Q156 + A183, G114 + Q156 + Q169 + F183, G114 + A162 + Q169 + F183, N134 + A146 + Q147 + Q156, N134 + A146 + Q147 + Q169, N134 + A183 + Q183, N134 + A183 + Q183, N134 + Q156 + Q147, N134 + Q147, N134 + A183 + Q183, N183 + Q147, N134 + F183 + Q183 + F183 + Q147, N134 + Q156 + Q134 + Q147, N134 + F183 + Q147, N134 + Q147, N134 + F183 + Q134 + Q147, N134 + F183 + Q147, N134 + F183 + Q134 + F183, N134 + F183 + Q134 + F147, N134 + F183 + F147, N134 + F183 + Q134 + Q147, N134 + F183 + F, A146D + Q147R + a162E + Q169Y, a 146Y + Q147Y + a 162Y + F183Y, a 146Y + Q147Y + Q169Y + F183Y, a 146Y + Q156Y + a 162Y + Q169Y, a 146Y + Q156Y + a 162Y + F183Y, a 146Y + Q156Y + Q169Y + F183Y, a 146Y + a Y + Q169Y + F183Y, Q147Y + Q Y + a 162Y + Q169Y, Q147Y + Q156Y + a 162Y + F Y, Q Y + Q36147 + Q Y + F Y, Q147 + Q Y + F183 + F Y.

Further preferred variants comprise substitutions in the catalytic domain, e.g., SEQ ID NO:5, selected from the group consisting of:

Q147R+Q156E；

Q147R+Q169Y；

S56A+Q147R；

Q147R+A162E；

Q147R+Q156E+A162E；

A25G+S56A+Q147R；

N134D+Q156E+A162E；

S56A+N134D+Q156E+A162E；

A25G+S56A+Q156E+A162E；

A25G+N134D+Q156E+A162E；

A25G+S56A+N134D+Q169Y；

S56A+N134D+A162E；

S56A+Q147R+Q169Y；

N134D+Q147R；

Q156E+Q169Y；

S56A+N134D+Q147R；

S56A+N134D+Q156E+Q169Y；

S56A+A146D+Q147R+Q169Y；

S56A+N134D+Q147R+Q169Y；

S56A+Q147R+A162E+Q169Y；

S2*+S56A+Q147R+Q169Y；

S41T+S56A+Q147R+Q169Y；

S56A+S77N+Q147R+Q169Y；

S56A+T104K+Q147R+Q169Y；

S56A+Q147R+K165Q+Q169Y；

S56A+Q147R+Q169Y+I194L；

S56A+Q147R+Q169Y+K201R；

S56A+Q147R+Q169Y+G219W；

N44D+S56A+Q147R+Q169Y；

N50E+S56A+Q147R+Q169Y；

A32S+S56A+Q147R+Q169Y；

N44D+S56A+Q147R+Q169Y；

S56A+Q147R+Q169Y+Q186R；

S56A+Q147R+Q169Y+F183V；

S56A+A146S+Q147R+A162E+Q169Y；

S56A+N134D+Q147R；

S56A+N134D+Q147R+A162E；

A32S+S56A+N134D+Q147R+Q169Y+F183V；

S56A+N134D+Q147R+A162E+Q169Y+F183V；

A32S+S56A+S77N+N134D+Q147R+A162E+Q169Y；

A32S+S56A+N134D+A146D+Q147R+Q169Y+F183V；

A32S+S56A+N134D+Q147R+Q169Y；

S56A+N134D+Q147R+A162E+Q169Y；

A32S+S56A+N134D+A146S+Q147R+Q169Y；

A32S+S56A +N134D+A146D+Q147R+Q169Y；

A32S+S56A +N134D+Q147R+Q169Y+F183V；

A32S+S56A +N134D+Q147R+Q169Y+K201R；

S56A+N134D+A146D+Q147R+Q169Y+F183V；

S56A+N134D+A146D+Q147R+A162E+Q169Y；

S56A+N134D+A146D+Q147R+Q169Y+K201R；

S56A+N134D+Q147R+A162E+Q169Y+F183V；

S56A+N134D+Q147R+Q169Y+F183V+K201R；

A32S+S56A+S77N+N134D+Q147R+Q169Y+F183V；

A32S+S56A+S77N+N134D+Q147R+A162E+Q169Y；

A32S+S56A+N134D+A146S+Q147R+Q169Y+F183V；

a32S + S56A + N134D + a146D + Q147R + Q169Y + F183V; or

A32S+S56A+N134D+A146D+Q147R+A162E+Q169Y。

Further preferred variants comprise substitutions in the catalytic domain, e.g., SEQ ID NO:5, selected from the group consisting of:

a variant may further comprise one or more additional changes at one or more (e.g., several) other positions.

Amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; typically a small deletion of 1-30 amino acids; small amino-terminal or carboxy-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by altering the net charge or another function (such as a polyhistidine segment, an epitope, or a binding domain).

Examples of conservative substitutions are within these groups as follows: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions which do not normally alter specific activity are known in The art and are described, for example, by H.Neurath and R.L.Hill,1979, in The Proteins, Academic Press, N.Y.. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.

Alternatively, the amino acid changes have the property of altering the physicochemical properties of the polypeptide. For example, amino acid changes can improve the thermostability of the polypeptide, change substrate specificity, change the pH optimum, and the like.

Essential amino acids in polypeptides can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,1989, Science 244: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the cellulolytic activity of the resulting mutant molecule is tested to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al, 1996, J.biol.chem. [ J.Biol ]271: 4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, as determined by the following technique: nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, as well as mutating putative contact site amino acids. See, e.g., de Vos et al, 1992, Science [ Science ]255: 306-); smith et al, 1992, J.mol.biol. [ J.Mol.224: 899-); wlodaver et al, 1992, FEBS Lett. [ Provisions of the European Association of biochemistry ]309: 59-64. The identity of the essential amino acids can also be inferred from alignment with the relevant polypeptide.

For example, the catalytic residues of a cellulase having the amino acid sequence of SEQ ID NO. 1 are identified as Asp 12 and Asp 122.

Carbohydrate Binding Module (CBM)

The Carbohydrate Binding Module (CBM) may comprise a wild-type or variant thereof, and it is also contemplated that these variants herein may comprise a wild-type catalytic domain of a first microorganism (which is wild-type or a variant thereof), and a carbohydrate binding module from a second microorganism (which is wild-type or a variant thereof) linked by a linker region.

For example, the variant may comprise the catalytic domain of SEQ ID NO. 1 or a variant thereof, and a carbohydrate binding module from SEQ ID NO. 2 linked by a linker region.

Preferably, the CBM is CBM 1.

In embodiments, the carbohydrate binding module/domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% or 100% sequence identity, to the amino acid sequence set forth in SEQ ID No. 6.

In embodiments, the carbohydrate binding module comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 7.

In embodiments, the carbohydrate binding module comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% or 100% sequence identity, to the amino acid sequence set forth in SEQ ID No. 197.

In embodiments, the variant comprises SEQ ID No. 5 or a variant thereof, and SEQ ID No. 6. In a further embodiment, the variant comprises, in order from N-terminus to C-terminus, SEQ ID NO:5 or a variant thereof, a linker, and SEQ ID NO: 6.

In embodiments, the variant comprises SEQ ID No. 5 or a variant thereof, and SEQ ID No. 7. In a further embodiment, the variant comprises, in order from N-terminus to C-terminus, SEQ ID NO:5 or a variant thereof, a linker, and SEQ ID NO: 7.

In embodiments, the variant comprises SEQ ID No. 5 or a variant thereof, and SEQ ID No. 8. In a further embodiment, the variant comprises, in order from N-terminus to C-terminus, SEQ ID NO:5 or a variant thereof, a linker, and SEQ ID NO: 8.

In embodiments, the variant comprises SEQ ID No. 5 or a variant thereof, and SEQ ID No. 9. In a further embodiment, the variant comprises, in order from N-terminus to C-terminus, SEQ ID NO:5 or a variant thereof, a linker, and SEQ ID NO: 9.

In embodiments, the variant comprises SEQ ID No. 5 or a variant thereof, and SEQ ID No. 173. In a further embodiment, the variant comprises, in order from N-terminus to C-terminus, SEQ ID NO. 5 or a variant thereof, a linker, and SEQ ID NO. 173.

In embodiments, the variant comprises SEQ ID No. 5 or a variant thereof, and SEQ ID No. 174. In a further embodiment, the variant comprises, in order from N-terminus to C-terminus, SEQ ID NO:5 or a variant thereof, a linker, and SEQ ID NO: 174.

Tables B1-B2, C1-C2 and table D provide exemplary preferred variants according to the present invention, which are provided in tabular form for ease of comparative reference. As used in the tables herein, these variants are represented in their entirety in order from N-terminus to C-terminus, without additional linkers or further modifications between the named sequences in the respective columns. Thus, represented in the table as follows

Variant 1 of (a) may equally be represented as:

Table B1.

Table B2.

Table C1.

Table C2.

Table D.

Stability in the Presence of proteases

In embodiments, the variant has improved stability in the presence of a protease compared to the parent enzyme. Preferably, the variant has improved stability in the presence of a protease and a surfactant (e.g., a detergent composition) as compared to the parent cellulase.

Stability in the presence of a protease is beneficial for use of, for example, a cellulase in the presence of a protease, because it extends the time for the cellulase to function and be active and perform its intended function.

A preferred use of the variants of the invention is in detergents, where proteases are typically included to improve washing. The improved stability of the variants of the invention means that the variants can exert a longer cellulolytic activity during the laundry process as compared to the parent cellulase and thus provide improved wash performance benefits as compared to the parent cellulase.

For liquid detergent compositions, these variants of the invention further have the benefit of improved stability in the presence of a protease, which means that liquid detergent compositions comprising a protease and further comprising the variant of the invention have a longer shelf life compared to the same liquid detergent composition comprising the parent cellulase.

Stability in the presence of protease can be determined by: a given cellulase is incubated under defined conditions in the presence of a protease, the cellulolytic activity after incubation is measured and compared to a cellulase sample that has not been incubated with a protease.

Another method for determining stability in the presence of a protease is: two identical tubes containing a given cellulase to be tested in a defined solution containing a protease are prepared, one tube being incubated at a high temperature, for example in the range of 30-90 ℃ (stressed), and the other tube being incubated at a low temperature, for example in the range of 0-5 ℃ (unstressed). The tubes are incubated for a predetermined time, for example between 1 and 24 hours, typically 16 hours. After incubation, both samples were analyzed for cellulolytic activity and the residual activity was determined as follows:

residual activity (%) - (activity, stress/activity, non-stress) × 100.

For example, the residual activity in 50% liquid detergent a containing 0.166 v/v-% protease can be determined, wherein the samples are incubated for 16 hours at high temperature (stressed) and 5 ℃ (non-stressed) before the activity is determined. The temperature should be chosen such that the residual activity of the parent molecule is in the range of 10% -50%.

The core stabilization method is illustrated in more detail in example 1.

The variants of the invention have higher residual activity than the parent cellulase. In one embodiment, the variant of the invention has at least 10% higher residual activity compared to the parent cellulase, e.g. at least 20% higher residual activity, e.g. at least 30% higher residual activity, e.g. at least 40% higher residual activity, e.g. at least 50% higher residual activity, e.g. at least 60% higher residual activity, e.g. at least 70% higher residual activity, e.g. at least 80% higher residual activity, e.g. at least 90% higher residual activity, or at least 100% higher residual activity compared to the parent cellulase.

However, in conventional enzyme stability assays for testing thermostability, the measurement of activity of stressed and unstressed samples typically focuses on measuring changes affecting the catalytic site of the enzyme molecule, for example by using small synthetic substrates such as 4-methylumbelliferone- β -cellopentaglucoside or soluble carboxymethylcellulose (CMC).

Importantly, however, it is not necessarily possible to detect changes in other properties of the enzyme of interest due to stress in these assays, which properties are important for the enzyme to function in use but do not directly affect the active site of the enzyme. One such example is a glycosyl hydrolase having a separate catalytic domain and CBM linked by a linker, as in cellulases used, for example, in laundry detergents and textile care products for the removal of fuzz and pills. If stress affects only the linker and/or CBM portion of the molecule and not the catalytic domain portion, then these changes will not be detected by conventional assays as described above and/or in example 1. When using simple substrates such as CMC or 4-methylumbelliferone- β -pentoside, the activity will appear to be maintained during stress but the performance is significantly affected, as the CBM portion of the enzyme molecule plays an important role in directing the enzyme to the proper location of the textile to be treated.

In addition, the importance of a CBM for performance can be tested by comparing the performance of the catalytic domain with that of a catalytic domain with an intact linker and CBM.

To detect changes in the linker and/or CBM after storage under stress conditions, specific measurements must be taken when testing whether stress affects the performance of the enzyme. This can be done by comparing the performance of the enzyme before and after stress. Alternatively, it can be tested by: ensuring that the enzyme binds to its natural insoluble substrate (e.g. cotton linters) is included as part of an assay for testing stability, and/or the binding of the enzyme to microcrystalline cellulose or cotton linters is first probed and then the activity of the enzyme that loses its ability to bind to cellulose compared to the total activity is measured.

Thus, linker and/or CBM stability is measured by: the method comprises incubating cellulase in a detergent comprising a protease, and then determining the ability of the incubated cellulase to bind to cellulose fibers. If the linker or cellulose binding domain is affected by a protease, the binding affinity of the cellulase to the cellulose fiber will be reduced.

The conditions described in example 2 illustrate this linker and CBM specific assay.

Parent cellulase

The parent cellulase may be a polypeptide having cellulolytic activity and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a polypeptide having the sequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or SEQ ID No. 4. In one aspect, the amino acid sequence of the parent differs from the mature polypeptide of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4 by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or SEQ ID NO 4.

The parent cellulase may be a polypeptide having cellulolytic activity and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the catalytic domain of a mature polypeptide having the sequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or SEQ ID No. 4. In one aspect, the amino acid sequence of the parent differs from the catalytic domain of the mature polypeptide of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4 by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In another aspect, the parent comprises the catalytic domain of SEQ ID NO. 1, e.g., amino acids 1 to 212 or amino acids 1 to 216 of SEQ ID NO. 1. In another aspect, the parent comprises SEQ ID NO 5.

In another aspect, the parent comprises the catalytic domain of SEQ ID NO. 2, e.g., amino acids 1 to 211 or amino acids 1 to 213 of SEQ ID NO. 2.

In another aspect, the parent comprises the catalytic domain of SEQ ID NO. 3, e.g., amino acids 1 to 210 of SEQ ID NO. 3.

In another aspect, the parent comprises the catalytic domain of SEQ ID NO. 4, e.g., amino acids 1 to 211 of SEQ ID NO. 4.

In another embodiment, the parent is an allelic variant of the mature polypeptide or catalytic domain of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or SEQ ID NO 4.

The polypeptides may be hybrid polypeptides in which a region of one polypeptide is fused at the N-terminus or C-terminus of a region of another polypeptide.

The parent may be a fusion polypeptide or a cleavable fusion polypeptide wherein another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the invention. Fusion polypeptides are produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides such that they are in frame and expression of the fusion polypeptide is under the control of one or more of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, where the fusion polypeptide is produced post-translationally (Cooper et al, 1993, EMBO J. [ J. European society of molecular biology ]12: 2575-.

The fusion polypeptide may further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in the following documents: martin et al, 2003, J.Ind.Microbiol.Biotechnol. [ journal of Industrial microorganism Biotechnology ]3: 568-576; svetina et al 2000, J.Biotechnol. [ J.Biotechnology ]76: 245-; Rasmussen-Wilson et al 1997, appl. environ. Microbiol. [ application and environmental microbiology ]63: 3488-; ward et al, 1995, Biotechnology [ Biotechnology ]13: 498-503; and Contreras et al, 1991, Biotechnology [ Biotechnology ]9: 378-; eaton et al, 1986, Biochemistry [ Biochemistry ]25: 505-512; Collins-Racie et al, 1995, Biotechnology [ Biotechnology ]13: 982-; carter et al, 1989, Proteins: Structure, Function, and Genetics [ Proteins: structure, function, and genetics ]6: 240-; and Stevens,2003, Drug Discovery World 4: 35-48.

The parent may be obtained from a microorganism of any genus. For the purposes of the present invention, the term "obtained from … …" as used herein in connection with a given source shall mean that the parent encoded by the polynucleotide is produced by that source or by a strain into which a polynucleotide from that source has been inserted. In one aspect, the parent is secreted extracellularly. The parent may be a bacterial cellulase. For example, the parent may be a gram-positive bacterial polypeptide, such as a Bacillus (Bacillus), Clostridium (Clostridium), Enterococcus (Enterococcus), Geobacillus (Geobacillus), Lactobacillus (Lactobacillus), Lactococcus (Lactococcus), marine Bacillus (Oceanobacillus), Staphylococcus (Staphylococcus), Streptococcus (Streptococcus) or Streptomyces (Streptomyces) cellulase; or a gram-negative bacterial polypeptide, such as Campylobacter (Campylobacter), Escherichia coli (E.coli), Flavobacterium (Flavobacterium), Clostridium (Fusobacterium), Helicobacter (Helicobacter), Clavibacterium (Ilyobacter), Neisseria (Neisseria), Pseudomonas (Pseudomonas), Salmonella (Salmonella) or Ureabasma (Ureabasma) cellulases.

In one aspect, the parent is an alkalophilic Bacillus (Bacillus alkalophilus), a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), a Bacillus brevis (Bacillus brevis), a Bacillus circulans (Bacillus circulans), a Bacillus clausii (Bacillus clausii), a Bacillus coagulans (Bacillus coagulosus), a Bacillus firmus (Bacillus fimbriae), a Bacillus lautus (Bacillus lautus), a Bacillus lentus (Bacillus lentus), a Bacillus subtilis (Bacillus licheniformis), a Bacillus megaterium (Bacillus megaterium), a Bacillus pumilus (Bacillus pumilus), a Bacillus stearothermophilus (Bacillus thermophilus), a Bacillus subtilis (Bacillus subtilis), or a Bacillus thuringiensis (Bacillus thuringiensis) cellulase.

In another aspect, the parent is a Streptococcus equisimilis (Streptococcus equisimilis), Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus uberis (Streptococcus uberis), or Streptococcus equi subsp.

In another aspect, the parent is a Streptomyces achromogenicus (Streptomyces achromogens), Streptomyces avermitilis (Streptomyces avermitilis), Streptomyces coelicolor (Streptomyces coelicolor), Streptomyces griseus (Streptomyces griseus), or Streptomyces lividans (Streptomyces lividans) cellulase.

The parent may be a fungal cellulase. For example, the parent may be a yeast cellulase, such as a Candida (Candida), Kluyveromyces (Kluyveromyces), Pichia (Pichia), Saccharomyces (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), or Yarrowia (Yarrowia) cellulase; or filamentous fungal cellulases, for example Acremonium (Acremonium), Agaricus (Agaric), Alternaria (Alternaria), Aspergillus, Aureobasidium (Aureobasidium), Staphylocodiophora (Botryospora), Ceriporiopsis (Ceriporiopsis), Chaetomium (Chaetomium), Chrysosporium (Chrysosporium), Claviceps (Claviceps), Cochlosporium (Cochliobolus), Coprinus (Coprinopsis), Coprinoides (Coptotermes), Coprinus (Coptotermes), Corynococcus (Corynascus), Cochlosporium (Cryptoterria), Cryptococcus (Cryptocococcus), Micrococcus (Cryptococcus), Chromospora (Diplodia), Umbilica (Exidiella), Filipendum (Filisium), Fusarium (Fusarium), Rhodosporium (Leptosporium), Fusarium (Leptosporium), Leptosporium (Leptosporium), Leptosporium (Mucor), etc.), Leptosporium), etc.) Neocallimastix (Neocallimastix), Neurospora (Neurospora), Paecilomyces (Paecilomyces), Penicillium, Phanerochaete (Phanerochaete), Ruminochytrium (Piromyces), Poitrasia, Pseudoplectania (Pseudoplectania), Pseudotrichomonas (Pseudotrichomonas), Rhizomucor (Rhizomucor), Schizophyllum (Schizophyllum), Scytalidium (Scytalidium), Talaromyces (Talaromyces), Thermoascus (Thermoascus), Thielavia (Thielavia), Tolypocladium (Tolypocladium), Trichoderma (Trichoderma), Trichosporoides (Trichosporoidea), Verticillium (Verticillium), Pediobolus (Volvillaria), or Xylaria (Xylaria).

In another aspect, the parent is a Saccharomyces carlsbergensis (Saccharomyces cerevisiae), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces diastaticus (Saccharomyces diastaticus), Saccharomyces douglasii (Saccharomyces douglasii), Saccharomyces kluyveri (Saccharomyces kluyveri), Saccharomyces norbensis (Saccharomyces norbensis), or Saccharomyces oviformis (Saccharomyces oviformis) cellulase.

In another aspect, the parents are Acremonium Chrysosporium (Acremonium cellulolyticus), Aspergillus aculeatus (Aspergillus aculeatus), Aspergillus awamori (Aspergillus awamori), Aspergillus foetidus (Aspergillus foetidus), Aspergillus fumigatus (Aspergillus fumigatus), Aspergillus japonicus (Aspergillus japonicus), Aspergillus nidulans (Aspergillus nidulans), Aspergillus niger (Aspergillus niger), Aspergillus oryzae (Aspergillus oryzae), Chrysosporium angustifolia (Chrysosporium inops), Chrysosporium cuticulosum (Chrysosporium keratophilum), Chrysosporium lucknowenum (Chrysosporium lucknowense), Chrysosporium coprinum (Chrysosporium Chrysosporium), Chrysosporium Chrysosporium (Chrysosporium kejinophilum), Chrysosporium lucknowenum (Fusarium), Chrysosporium cucumerinum (Fusarium Chrysosporium), Fusarium gramineum (Fusarium Chrysosporium), Fusarium graminearum (Fusarium solanum), Fusarium graminearum fulvum (Fusarium fulvum), Fusarium graminearum (Fusarium fulvum), Fusarium fulvum), Fusarium solanum fulvum (Fusarium solanum falcatum), Fusarium graminum (Fusarium graminum), Fusarium solanum fulvum (Fusarium solanum) and Fusarium graminum (Fusarium graminum) in a (Fusarium solanum) in a. sp Fusarium heterosporum (Fusarium heterosporum), Fusarium albizium (Fusarium negundi), Fusarium oxysporum (Fusarium oxysporum), Fusarium reticulatum (Fusarium reticulatum), Fusarium roseum (Fusarium roseum), Fusarium sambucinum (Fusarium sambucinum), Fusarium sarcochroum (Fusarium sarcochroum), Fusarium sporotrichioides (Fusarium sporotrichioides), Fusarium sulphureum (Fusarium supherum), Fusarium torulosum (Fusarium torulosum), Fusarium trichothecioides (Fusarium trichothecioides), Fusarium venenatum (Fusarium trichothecosum), Fusarium griseum (Fusarium trichothecioides), Fusarium griseum, special-putrescens (Humicola), Fusarium trichothecioides (Fusarium trichothecorum), Fusarium trichothecorum (trichothecorum), Fusarium trichothecorum (trichothecorum), Fusarium trichothecorum (trichothecorum), Fusarium trichothecorum (trichothecorum), Fusarium trichothecorum (trichothecorum), and Fusarium trichothecorum (trichothecorum), Fusarium trichothecorum), or trichothecorum (trichothecorum), or trichothecorum), or trichothecorum (trichothecorum), or trichothecorum (trichothecorum), or trichothecorum, trichothecorum, trichothecum, trichothecorum, or trichothecum trichothecorum, trichothecum, trichothecorum, or trichothecorum trichothecum, trichothecorum, or trichothecorum, trichothecum, or trichothecum, or trichothecum, trichothecorum, trichothecum, or trichothecum, or trichothecorum, trichothecum, or trichothecorum, trichothecum, or trichothecum, or trichothecum, or trichothecellum, or trichothecum, or trichothecium roseum, or trichothecum, or trichothec, Thielavia australis (Thielavia australis), Thielavia faecalis (Thielavia fimeti), Thielavia microspora (Thielavia microspora), Thielavia ovata (Thielavia ovira ovispora), Clostridium peruvii (Thielavia peruviana), Thielavia trichotheca (Thielavia setosa), Thielavia glabrata (Thielavia spidonium), Thielavia nodosa (Thielavia spidonium), Thielavia thermotolerans (Thielavia sublmophilum), Thielavia terrestris (Thielavia terrestris), Trichoderma harzianum (Trichoderma harzianum), Trichoderma koningii (Trichoderma koningii), Trichoderma longibrachiatum (Trichoderma longibrachiatum), Trichoderma reesei (Trichoderma reesei), or Trichoderma viride (Trichoderma viride) cellulase.

In another aspect, the parent is a Thielavia terrestris cellulase, e.g., the cellulase of SEQ ID NO:1 or a mature polypeptide thereof.

It is to be understood that for the aforementioned species, the invention encompasses complete and incomplete stages (perfect and perfect states), and other taxonomic equivalents (equivalents), such as anamorphs, regardless of their known species names. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily available to the public at many Culture collections, such as the American Type Culture Collection (ATCC), the German Culture Collection of microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), the Dutch cultures Collection (CBS), and the Northern Regional Research Center of the American Agricultural Research Service Culture Collection (NRRL).

The parents can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.), using the above probes. Techniques for the direct isolation of microorganisms and DNA from natural habitats are well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening a genomic DNA or cDNA library or mixed DNA sample of another microorganism. Once a polynucleotide encoding a parent has been detected using one or more probes, the polynucleotide can be isolated or cloned by utilizing techniques known to those of ordinary skill in the art (see, e.g., Sambrook et al, 1989, supra).

Preparation of variants

The present invention also relates to methods for obtaining variants having glycoside hydrolase activity, comprising: (a) introducing one or more substitutions of the mature polypeptide of the parent polypeptide into the parent glycoside hydrolase; and (b) recovering the variant.

Variants can be prepared using any mutagenesis method known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, and the like.

Site-directed mutagenesis is a technique in which one or more (e.g., several) mutations are introduced at one or more defined sites in a polynucleotide encoding a parent.

Site-directed mutagenesis can be achieved in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. In vitro site-directed mutagenesis may also be performed by cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid comprising a polynucleotide encoding a parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Typically, the restriction enzymes that digest the plasmid and oligonucleotide are the same, allowing the sticky ends of the plasmid and insert to ligate to each other. See, e.g., Scherer and Davis,1979, Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. ]76: 4949-; and Barton et al, 1990, Nucleic Acids Res. [ Nucleic Acids research ]18: 7349-.

Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., U.S. patent application publication nos. 2004/0171154; storici et al, 2001, Nature Biotechnol [ natural biotechnology ]19: 773-; kren et al, 1998, Nat. Med. [ Nature medicine ]4: 285-; and Calissano and Macino,1996, Fungal Genet.Newslett. [ Fungal genetics newslett. ]43: 15-16.

Any site-directed mutagenesis procedure can be used in the present invention. There are many commercially available kits that can be used to prepare variants.

Synthetic gene construction requires in vitro synthesis of designed polynucleotide molecules to encode the polypeptide of interest. Gene synthesis can be performed using a variety of techniques, such as the multiplex microchip-based technique described by Tian et al (2004, Nature 432: 1050-.

Single or multiple amino acid substitutions, deletions and/or insertions can be made and tested using known mutagenesis, recombination and/or shuffling methods, followed by relevant screening procedures such as those described by Reidhaar-Olson and Sauer,1988, Science [ Science ]241: 53-57; bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. ]86: 2152-2156; WO 95/17413; or those disclosed in WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al, 1991, Biochemistry [ Biochemistry ]30: 10832-.

The mutagenesis/shuffling approach can be combined with high throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al, 1999, Nature Biotechnology [ Nature Biotechnology ]17: 893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from the host cells and rapidly sequenced using methods standard in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

Semi-synthetic gene construction is achieved by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction typically utilizes a process of synthesizing polynucleotide fragments in combination with PCR techniques. Thus, defined regions of a gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, while still other regions can be subjected to error-prone PCR or non-error-prone PCR amplification. The polynucleotide subsequences may then be shuffled.

Polynucleotide

The invention also relates to polynucleotides encoding the variants of the invention.

Nucleic acid constructs

The invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

The polynucleotide can be manipulated in a variety of ways to provide for expression of the variant. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

The control sequence may be a promoter, i.e., a polynucleotide, which is recognized by a host cell for expression of the polynucleotide. The promoter contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of the nucleic acid construct of the invention in a bacterial host cell are promoters obtained from the following genes: bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levan sucrase gene (sacB), bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA Gene (Agaisse and Lereclus,1994, Molecular Microbiology [ Molecular Microbiology ]13:97-107), Escherichia coli lac operon, Escherichia coli trc promoter (Egon et al, 1988, Gene [ Gene ]69: 301-. Other promoters are described in Gilbert et al, 1980, Scientific American [ Scientific Americans ]242:74-94, "Useful proteins from recombinant bacteria ]; and Sambrook et al, 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.

Examples of suitable promoters for directing the transcription of the nucleic acid construct of the invention in a filamentous fungal host cell are promoters obtained from the genes for the following enzymes: aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Aspergillus niger dextranase I, Aspergillus niger endoglucanase II, Aspergillus niger endoglucanase III, Aspergillus niger, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, and NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene wherein the untranslated leader sequence is replaced by the untranslated leader sequence of an Aspergillus triose phosphate isomerase gene; non-limiting examples include a modified promoter from an Aspergillus niger neutral alpha-amylase gene wherein the untranslated leader sequence has been replaced with the untranslated leader sequence from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof.

In yeast hosts, useful promoters are obtained from the following genes: saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for Yeast host cells are described by Romanos et al, 1992, Yeast [ Yeast ]8: 423-488.

The control sequence may also be a transcription terminator which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' -terminus of the polynucleotide encoding the variant. Any terminator which is functional in the host cell may be used.

Preferred terminators for bacterial host cells are obtained from the following genes: bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

Preferred terminators for filamentous fungal host cells are obtained from the genes: aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the following genes: saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al (1992, supra).

The control sequence may also be an mRNA stability region downstream of the promoter and upstream of the coding sequence of the gene, which increases the expression of the gene.

Examples of suitable mRNA stability regions are obtained from: bacillus thuringiensis cryIIIA gene (WO 94/25612) and Bacillus subtilis SP82 gene (Hue et al, 1995, Journal of Bacteriology 177: 3465-.

The control sequence may also be a leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' -terminus of the polynucleotide encoding the variant. Any leader sequence that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

Suitable leader sequences for yeast host cells are obtained from the following genes: saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH 2/GAP).

The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3' -terminus of the variant coding sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for the following enzymes: aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman,1995, mol.Cellular Biol. [ molecular cell biology ]15: 5983-.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the variant and directs the variant into the cell's secretory pathway. The 5' -end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence encoding the variant. Alternatively, the 5' -end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence in order to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for use in bacterial host cells are those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Other signal peptides are described by Simonen and Palva,1993, Microbiological Reviews [ Microbiological review ]57:109- & 137.

An effective signal peptide coding sequence for use in a filamentous fungal host cell is a signal peptide coding sequence obtained from the genes for the following enzymes: aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase and Rhizomucor miehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al (1992, supra).

The control sequence may also be a propeptide coding sequence that codes for a propeptide positioned at the N-terminus of a variant. The resulting polypeptide is called a pro-enzyme (proenzyme) or propolypeptide (or zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the following genes: bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei (Rhizomucor miehei) aspartic protease, and Saccharomyces cerevisiae alpha-factor.

In the case where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is positioned next to the N-terminus of the variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulate the expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter may be used. Other examples of regulatory sequences are those which allow gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene amplified in the presence of methotrexate, and the metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the variant will be operably linked to the regulatory sequence.

Expression vector

The invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector such that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome or chromosomes into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell may be used, or a transposon may be used.

The vector preferably contains one or more selectable markers that allow for convenient selection of transformed cells, transfected cells, transduced cells, and the like. A selectable marker is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance (e.g., ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance). Suitable markers for yeast host cells include, but are not limited to: ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA 3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5' -phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and the Streptomyces hygroscopicus (Streptomyces hygroscopicus) bar gene.

The vector preferably contains one or more elements that allow the vector to integrate into the genome of the host cell or the vector to replicate autonomously in the cell, independently of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide sequence encoding the variant or any other vector element for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the host cell genome at one or more precise locations in one or more chromosomes. To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, e.g., 100 to 10000 base pairs, 400 to 10000 base pairs, and 800 to 10000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. Alternatively, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicon mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in E.coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAM β 1, which allow replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN 6.

Examples of origins of replication useful in filamentous fungal cells are AMA1 and ANS1(Gems et al, 1991, Gene [ 98: 61-67; Cullen et al, 1987, Nucleic Acids Res. [ Nucleic Acids research ]15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of a plasmid or vector containing the gene can be accomplished according to the method disclosed in WO 00/24883.

More than one copy of a polynucleotide of the invention may be inserted into a host cell to increase production of the variant. An increased copy number of the polynucleotide may be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, wherein cells comprising amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, may be selected for by culturing the cells in the presence of the appropriate selectable agent.

Procedures for ligating the elements described above to construct the recombinant expression vectors of the invention are well known to those of ordinary skill in the art (see, e.g., Sambrook et al, 1989, supra).

Host cell

The present invention also relates to recombinant host cells comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the production of the variant of the present invention. The construct or vector comprising the polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extra-chromosomal vector, as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will depend to a large extent on the gene encoding the variant and its source.

The host cell may be any cell useful in the recombinant production of variants, such as a prokaryotic cell or a eukaryotic cell.

The prokaryotic host cell may be any gram-positive or gram-negative bacterium. Gram-positive bacteria include, but are not limited to: bacillus, Clostridium, enterococcus, Geobacillus, Lactobacillus, lactococcus, Paenibacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include, but are not limited to: campylobacter (Campylobacter), Escherichia coli, Flavobacterium (Flavobacterium), Clostridium (Fusobacterium), Helicobacter (Helicobacter), Clavibacterium (Ilyobacter), Neisseria (Neisseria), Pseudomonas (Pseudomonas), Salmonella (Salmonella), and Ureabasma (Ureapasma).

The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell, including but not limited to Streptococcus equisimilis (Streptococcus equisimilis), Streptococcus pyogenes (Streptococcus pyogenenes), Streptococcus uberis (Streptococcus uberis) and Streptococcus equi subsp.

The bacterial host cell may also be any streptomyces cell, including but not limited to: streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

Introduction of DNA into bacillus cells can be achieved by: protoplast transformation (see, e.g., Chang and Cohen,1979, mol.Gen. Genet. [ molecular and general genetics ]168: 111-. The introduction of DNA into E.coli cells can be achieved by: protoplast transformation (see, e.g., Hanahan,1983, J.mol.biol. [ J.Biol. ]166: 557-. The introduction of DNA into Streptomyces cells can be achieved by: protoplast transformation, electroporation (see, e.g., Gong et al, 2004, Folia Microbiol. (Praha) [ leaf-line microbiology (Bragg) ]49: 399-. The introduction of DNA into a Pseudomonas cell can be achieved by: electroporation (see, e.g., Choi et al, 2006, J.Microbiol. methods [ journal of microbiological methods ]64: 391-. The introduction of DNA into Streptococcus cells can be achieved by: natural competence (natural competence) (see, e.g., Perry and Kuramitsu,1981, infection. Immun. [ infection and immunity ]32: 1295-. However, any method known in the art for introducing DNA into a host cell may be used.

The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

The host cell may be a fungal cell. "Fungi" as used herein include Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, Oomycota and all mitosporic Fungi (as defined by Hawksworth et al in The literature: Ainsworth and Bisby's Dictionary of The Fungi [ Anschofsis and Bessebi Dictionary ], 8 th edition, 1995, CAB International [ International centre of applied bioscience ], University Press [ University Press ], Cambridge, UK [ Cambridge ]).

The fungal host cell may be a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Ascomoogenous yeast) (Endomycetales), basidiogenous yeast (basidiogenous yeast) and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeasts may vary in the future, for the purposes of the present invention, yeasts should be defined as described in Biology and Activities of Yeast [ Biology and Activity of Yeast ] (Skinner, Passmore and Davenport, ed., Soc.App.bacteriol.Symphosis Series No.9[ application society for bacteriology monograph Series 9], 1980).

The yeast host cell may be a Candida cell, a Hansenula cell, a Kluyveromyces cell, a Pichia cell, a Saccharomyces cell, a Schizosaccharomyces cell, a Yarrowia cell, such as a Kluyveromyces lactis cell, a Saccharomyces carlsbergensis cell, a Saccharomyces cerevisiae cell, a Saccharomyces diastaticus cell, a Saccharomyces dowrayanus cell, a Saccharomyces cerevisiae cell, a Yarrowia cell, or a Yarrowia cell.

The fungal host cell may be a filamentous fungal cell. "filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al, 1995 (supra)). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, while carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding (budding) of unicellular thallus and carbon catabolism may be fermentative.

The filamentous fungal host cell may be a member of the genera Acremonium (Acremonium), Aspergillus (Aspergillus), Aureobasidium (Aureobasidium), Cladosporium (Bjerkandra), Ceriporiopsis (Ceriporiopsis), Chrysosporium (Chrysosporium), Coprinus (Coprinus), Coriolus (Coriolus), Cryptococcus (Cryptococcus), Filibasidiaceae (Filibasidium), Fusarium (Fusarium), Humicola (Humicola), Magnaporthe (Magnaporthe), Mucor (Mucor), Myceliophthora (Myceliophthora), Neocallimastix (Neocallimastix), Neurospora (Neurospora), Paecilomyces (Paecilomyces), Penicillium (Penicillium), Phanerium (Thermobacterium), Thermobacteroid (Trichoderma), Trichosporoides (Trichoderma), Trichoderma (Trichoderma), Trichoderma (Trichoderma).

For example, the filamentous fungal host cell may be Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus niger (Bjerkandra adusta), Ceriporiopsis xerophila (Ceriporiopsis anerina), Ceriporiopsis carinatus (Ceriporiopsis caregiea), Ceriporiopsis superficialis (Ceriporiopsis gilviscus), Ceriporiopsis pannicus (Ceriporiopsis panocicola), Ceriporiopsis annulata (Ceriporiopsis rivulosa), Ceriporiopsis micus (Ceriporiopsis subrufa), Ceripopsis pomicus (Ceriporiopsis subspecies), Ceriporiopsis crispa (Ceriporiopsis subrufimbriatus), Ceriporiopsis cuticola (Ceriporiosa), Ceriporiosa flavivirus (Ceriporiosa), Ceriporiopsis fulvescens (Ceriporiopsis fulvellus), Ceriporiopsis (Chrysosporium), Chrysosporium (Chrysosporium lucorum, Chrysosporium (Chrysosporium), Chrysosporium) Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium heterosporum, Fusarium albizium, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola lanuginosa, Mucor miehei, myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii (Pleurous eryngii), Thielavia terrestris, Trametes villosa (Trastomyces villosa), Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichoderma viride cells.

Fungal cells may be transformed by methods involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transforming aspergillus and trichoderma host cells are described in the following documents: EP 238023, Yelton et al, 1984, Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. ]81: 1470-. Suitable methods for transforming Fusarium species are described by Malardier et al, 1989, Gene [ Gene ]78:147-156 and WO 96/00787. Yeast can be transformed using procedures described by the following references: becker and guard, edited in Abelson, j.n. and Simon, m.i., Guide to Yeast Genetics and Molecular Biology [ Guide to Molecular Biology ], Methods in Enzymology [ Methods in Enzymology ], volume 194, page 182-; ito et al, 1983, j. bacteriol [ journal of bacteriology ]153: 163; and Hinnen et al, 1978, Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. ]75: 1920.

Generation method

The invention also relates to methods of producing variants, the methods comprising: (a) culturing the host cell of the invention under conditions suitable for expression of the variant; and (b) recovering the variant.

The host cell is cultured in a nutrient medium suitable for producing the variant using methods known in the art. For example, the cell may be cultured by shake flask culture, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing expression and/or isolation of the variant. Culturing occurs in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions, for example, in catalogues of the American Type Culture Collection. If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from the cell lysate.

Variants can be detected using methods known in the art that are specific for the variant. These detection methods include, but are not limited to: the use of specific antibodies, the formation of enzyme products or the disappearance of enzyme substrates. For example, enzymatic assays can be used to determine the activity of a variant.

Variants can be recovered using methods known in the art. For example, the variant may be recovered from the nutrient medium by a variety of conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

Variants can be purified by a variety of procedures known in the art to obtain substantially pure variants, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden editors, VCH Publishers, new york, VCH Publishers, 1989).

In an alternative aspect, the variant is not recovered, but rather a host cell of the invention expressing the variant is used as a source of the variant.

Detergent composition

In one embodiment, the present invention relates to detergent compositions comprising the enzyme of the present invention in combination with one or more additional cleaning composition components. The selection of additional components is within the ability of the skilled artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.

For textile care, the selection of components may include the following considerations: the type of textile to be cleaned, the type and/or degree of soil, the temperature at which cleaning is carried out, and the formulation of the detergent product. Although the components mentioned below are classified by general headings according to specific functionality, this is not to be construed as a limitation, as the components may comprise additional functionality as will be appreciated by the skilled person.

In one embodiment, the present invention relates to a liquid laundry detergent composition comprising an enzyme of the present invention in combination with one or more additional laundry detergent composition components (in particular, a protease). In another embodiment, the invention comprises an adjunct product for use in laundry washing, such as a pre-detergent or a stain release enhancer. The invention also relates to ADW (automatic dishwashing) compositions comprising an enzyme of the invention in combination with one or more additional ADW composition components. The selection of additional components is within the ability of the skilled artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.

The enzyme of the present invention

In one embodiment of the invention, the polypeptide of the invention may be added to a detergent composition in an amount corresponding to: from 0.001 to 200mg of protein per liter of wash liquor, e.g.from 0.005 to 100mg of protein, preferably from 0.01 to 50mg of protein, more preferably from 0.05 to 20mg of protein, even more preferably from 0.1 to 10mg of protein.

One or more enzymes of the detergent compositions of the invention may be stabilised using conventional stabilisers, for example polyols, such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives, for example aromatic borate esters, or phenyl boronic acid derivatives, for example 4-formylphenyl boronic acid, and the compositions may be formulated as described in, for example, WO 92/19709 and WO 92/19708.

The polypeptides of the invention may also be incorporated into detergent formulations as disclosed in WO 97/07202, which is hereby incorporated by reference.

Surface active agent

The detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or nonionic and/or semi-polar and/or zwitterionic, or mixtures thereof. In particular embodiments, the detergent composition comprises a mixture of one or more nonionic surfactants and one or more anionic surfactants. The one or more surfactants are typically present at a level of from about 5% to 60% (e.g., about 5% to about 50%, or about 10% to about 50%, or about 20% to about 50%) by weight. The one or more surfactants are selected based on the desired cleaning application, and may include any one or more conventional surfactants known in the art.

When included therein, the detergent will typically contain from about 5% to about 60% (such as from about 5% to about 40%, including from about 10% to about 25%) by weight of one or more anionic surfactants. Non-limiting examples of anionic surfactants include sulfates and sulfonates, specifically Linear Alkylbenzene Sulfonate (LAS), isomers of LAS, branched alkylbenzene sulfonate (BABS), phenylalkane sulfonate, alpha-olefin sulfonate (AOS), olefin sulfonate, alkene sulfonate, alkane-2, 3-diylbis (sulfate), hydroxyalkane sulfonate and disulfonate, Alkyl Sulfate (AS) such AS Sodium Dodecyl Sulfate (SDS), Fatty Alcohol Sulfate (FAS), Primary Alcohol Sulfate (PAS), alcohol ether sulfate (AES or AEOS or FES, also known AS alcohol ethoxy sulfate or fatty alcohol ether sulfate), Secondary Alkane Sulfonate (SAS), Paraffin Sulfonate (PS), ester sulfonate, sulfonated fatty acid glycerides, alpha-sulfonated fatty acid methyl esters (alpha-SFMe or SES) including Methyl Ester Sulfonate (MES), Alkyl or alkenyl succinic acids, dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acid or fatty acid salts (soaps) or fatty acids, and combinations thereof.

When included therein, the detergent will typically contain from about 0.1% to about 10% (e.g., from about 0.1% to about 5%) by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary amine (ADMEAQ), Cetyl Trimethyl Ammonium Bromide (CTAB), dimethyl distearyl ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, Alkoxylated Quaternary Ammonium (AQA) compounds, ester quaternary ammonium, and combinations thereof.

When included therein, the detergent will typically contain from about 0.2% to about 60% (e.g., from about 1% to about 40%, particularly from about 5% to about 20%, from about 3% to about 15%) by weight of nonionic surfactant. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, Propoxylated Fatty Alcohols (PFA), alkoxylated fatty acid alkyl esters (e.g., ethoxylated and/or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), Alkylpolyglycosides (APG), alkoxylated amines, Fatty Acid Monoethanolamides (FAM), Fatty Acid Diethanolamides (FADA), Ethoxylated Fatty Acid Monoethanolamides (EFAM), Propoxylated Fatty Acid Monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamide (GA), or Fatty Acid Glucamides (FAGA)), Methyl Ester Ethoxylates (MEE), as well as products available under the trade names SPAN and TWEEN And combinations thereof.

When included therein, the detergent will typically contain from about 0, 1% to about 10% by weight of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include Amine Oxides (AO), such as alkyl dimethylamine oxide, N- (cocoalkyl) -N, N-dimethylamine oxide, and N- (tallow-alkyl) -N, N-bis (2-hydroxyethyl) amine oxide, and combinations thereof.

When included therein, the detergent will typically contain from about 0, 1% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines, such as alkyl dimethyl betaines, sulfobetaines, and combinations thereof.

Solvent system: to dissolve surfactants and other detergent ingredients, a solvent system is required. The solvent is typically water, an alcohol, a polyol, a sugar and/or mixtures thereof. Preferred solvents are water, glycerol, sorbitol, propylene glycol (MPG, 1, 2-or 1, 3-propanediol), dipropylene glycol (DPG), the polyethylene glycol family (PEG300-600), hexylene glycol, inositol, mannitol, ethanol, isopropanol, n-butoxypropropanol, ethanolamine (monoethanolamine, diethanolamine and triethanolamine), sucrose, dextrose, glucose, ribose, xylose and related mono-and dipyryranosides and furanosides.

The solvent system is typically present in a total of 5% -90%, 5% -60%, 5% -40%, 10% -30% by weight.

The water content of the unit dose encapsulated in the PVA film is typically in the range of 1% -15%, 2% -12%, 3% -10%, 5% -10%.

The polyol content of the unit dose contained in the PVA film is typically in the range of 5% to 50%, 10% to 40%, or 20% to 30%.

In embodiments, the surfactant is a non-naturally occurring surfactant.

Hydrotropic agent

Hydrotropes are compounds that dissolve hydrophobic compounds in aqueous solutions (or conversely, polar materials in a non-polar environment). Typically, hydrotropes have both hydrophilic and hydrophobic characteristics (as known from surfactants as so-called amphiphilic properties), however the molecular structure of hydrotropes generally does not favour spontaneous self-aggregation, see for example the reviews by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science [ colloidal and Interface Science new ]12: 121-. Hydrotropes do not exhibit the critical concentrations found in surfactants and lipids that form micelles, lamellae, or other well-defined mesophases (meso-phases), above which self-aggregation occurs. In contrast, many hydrotropes show a continuous type of aggregation process in which the aggregate size grows with increasing concentration. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing materials of both polar and non-polar character, including mixtures of water, oils, surfactants, and polymers. Hydrotropes are routinely used in a variety of industries ranging from pharmaceutical, personal care, food to technical applications. The use of hydrotropes in detergent compositions allows, for example, for more concentrated surfactant formulations (such as during the compaction of liquid detergents by removal of water) without causing undesirable phenomena such as phase separation or high viscosity.

The detergent may contain 0-10% by weight, such as 0-5% by weight, for example from about 0.5% to about 5%, or from about 3% to about 5% of a hydrotrope. Any hydrotrope known in the art for use in detergents can be utilized. Non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate (STS), Sodium Xylene Sulfonate (SXS), Sodium Cumene Sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthalene formate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfonate, and combinations thereof.

Builders and co-builders

The detergent composition may contain from about 0% to about 65%, from 0% to about 20%, or from about 0.5% to about 5% of a detergent builder or co-builder, or mixtures thereof. In dishwashing detergents, the level of builder is typically from 10% to 65%, especially from 20% to 40%. The builder and/or co-builder may in particular be a chelating agent forming a water-soluble complex with Ca and Mg. Any builder and/or co-builder known in the art for use in laundry detergents may be utilized. Non-limiting examples are citrate, sodium carbonate, sodium bicarbonate, and sodium citrate. Examples of the phosphate include 1-hydroxyethylidene-1, 1-diphosphonic acid (HEDP, etidronic acid), diethylenetriaminepenta (methylenephosphoric acid) (DTPMP), ethylenediaminetetra (methylenephosphoric acid) (EDTMPA), aminotri (methylenephosphoric Acid) (ATMP), nitrilotrimethylene phosphoric acid (NTMP), 2-aminoethylphosphoric acid (AEPn), Dimethyl Methylphosphonate (DMPP), tetramethylenediaminetetra (methylenephosphoric acid) (TDTMP), hexamethylenediaminetetra (methylenephosphoric acid) (HDTMP), butanetricarboxylic acid Phosphate (PBTC), N- (phosphonomethyl) iminodiacetic acid (PMIDA), 2-carboxyethylphosphoric acid (CEPA), 2-hydroxyphosphorylcarboxylic acid (HPAA), and amino-tris- (methylene-phosphoric Acid) (AMP). L-glutamic acid N, N-diacetic acid tetrasodium salt (GLDA), methylglycinediacetic acid (MGDA). Non-limiting examples of builders include homopolymers of polyacrylates or copolymers thereof, such as poly (acrylic acid) (PAA) or copoly (acrylic acid/maleic acid) (PAA/PMA). Further non-limiting examples include citrates, chelating agents (such as aminocarboxylates, aminopolycarboxylates, and phosphonates), and alkyl succinic acids, or alkenyl succinic acids. Additional specific examples include 2,2 ', 2 "-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N, N' -disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N, N-diacetic acid (GLDA), 1-hydroxyethane-1, 1-diphosphonic acid (HEDP), ethylenediaminetetra (methylenephosphonic acid) (EDTMPA), diethylenetriaminepenta (methylenephosphonic acid) (DTMPA or DTPMPA), N- (2-hydroxyethyl) iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N, N-diacetic acid (ASDA), aspartic acid-N-monopropionic Acid (ASMP), iminodisuccinic acid (IDA), N- (2-sulfomethyl) -aspartic acid (SMAS), N- (2-sulfoethyl) -aspartic acid (SEAS), N- (2-sulfomethyl) -glutamic acid (SMGL), N- (2-sulfoethyl) -glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), alpha-alanine-N, N-diacetic acid (alpha-ALDA), serine-N, N-diacetic acid (SEDA), isoserine-N, N-diacetic acid (ISDA), phenylalanine-N, N-diacetic acid (PHDA), anthranilic acid-N, N-diacetic acid (ANDA), sulfanilic acid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA), and sulfomethyl-N, n-diacetic acid (SMDA), N- (2-hydroxyethyl) -ethylenediamine-N, N', N "-triacetate (HEDTA), Diethanolglycine (DEG), diethylenetriamine penta (methylene phosphonic acid) (DTPMP), aminotri (methylene phosphonic Acid) (ATMP), and combinations and salts thereof. Further exemplary builders and/or co-builders are described in e.g. WO 09/102854, US 5977053

In embodiments, the builder or co-builder is a non-naturally occurring builder or co-builder.

Bleaching system

The detergent may contain 0-30% by weight, such as from about 1% to about 20%, of a bleaching system. Any bleaching system known in the art for use in laundry detergents may be utilized. Suitable bleach system components include bleach catalysts, photobleaches, bleach activators, sources of hydrogen peroxide such as sodium percarbonate, sodium perborate and hydrogen peroxide-urea (1:1), preformed peracids and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, diperoxydicarboxylic acids, perimidic acids and salts, peroxymonosulfuric acids and salts (e.g., oxone (R)) and mixtures thereof non-limiting examples of bleach systems include peroxide-based bleach systems combined with peracid-forming bleach activators, which can include, for example, inorganic salts, including alkali metal salts such as the sodium salt of perborate (usually monohydrate or tetrahydrate), percarbonate, persulfate, perphosphate, persilicate salts 4- [ (3,5, 5-trimethylhexanoyl) oxy ] benzene-1-sulfonic acid sodium salt (ISONOBS), 4- (dodecanoyloxy) benzene-1-sulfonate (LOBS), 4- (decanoyloxy) benzene-1-sulfonate, 4- (decanoyloxy) benzoate (DOBS or DOBA), 4- (nonanoyloxy) benzene-1-sulfonate (NOBS), and/or those disclosed in WO 98/17767. A particular family of bleach activators of interest is disclosed in EP 624154 and particularly preferred in this family is Acetyl Triethyl Citrate (ATC). ATC or short chain triglycerides like triacetin have the advantage that it is environmentally friendly. In addition, acetyl triethyl citrate and triacetin have good hydrolytic stability in the product upon storage and are effective bleach activators. Finally, ATC is multifunctional in that citrate released in the perhydrolysis reaction may act as a builder. Alternatively, the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type. The bleaching system may also comprise peracids such as 6- (phthalimido) Perhexanoic Acid (PAP). The bleaching system may also include a bleach catalyst. In some embodiments, the bleaching component may be an organic catalyst selected from the group consisting of: an organic catalyst having the formula:

(iii) And mixtures thereof,

wherein each R¹Independently a branched alkyl group containing from 9 to 24 carbons or a straight alkyl group containing from 11 to 24 carbons, preferably each R¹Independently a branched alkyl group containing from 9 to 18 carbons or a straight alkyl group containing from 11 to 18 carbons, more preferably each R¹Independently selected from the group consisting of: 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl and isotentadecyl. Other exemplary bleaching systems are described in, for example, WO 2007/087258, WO 2007/087244, WO 2007/087259, EP 1867708 (vitamin K), and WO 2007/087242. Suitable photobleaches may for example be sulfonated zinc or aluminium phthalocyanines.

Preferably, the bleach component comprises a source of peracid in addition to the bleach catalyst, particularly an organic bleach catalyst. The peracid source may be selected from (a) preformed peracids; (b) percarbonate, perborate or persulfate salts (sources of hydrogen peroxide), preferably in combination with bleach activators; and (c) a perhydrolase enzyme and an ester for forming a peracid in situ in the presence of water in the textile or hard surface treatment step.

In an embodiment, the bleaching system is a non-naturally occurring bleaching system.

Polymer and method of making same

The detergent may contain 0-10% (such as 0.5% -5%, 2% -5%, 0.5% -2%, or 0.2% -1%) by weight of the polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning, and/or anti-foam properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs. Exemplary polymers include (carboxymethyl) cellulose (CMC), poly (vinyl alcohol) (PVA), poly (vinylpyrrolidone) (PVP), poly (ethylene glycol) or poly (ethylene oxide) (PEG), ethoxylated poly (ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA, poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligoethylene glycol, copolymers of poly (ethylene terephthalate) and poly (oxyethylene terephthalate) (PET-POET), PVP, poly (vinylimidazole) (PVI), poly (vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternary ammonium ethoxysulfate. Other exemplary polymers are disclosed in, for example, WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.

In embodiments, the polymer is a non-naturally occurring polymer.

Fabric toner

The detergent composition of the present invention may also comprise a fabric hueing agent, such as a dye or pigment, which when formulated in a detergent composition, may deposit on the fabric when said fabric is contacted with a wash liquor which comprises said detergent composition and which therefore changes the colour of said fabric by absorption/reflection of visible light. Optical brighteners emit at least some visible light. Conversely, when fabric toners absorb at least a portion of the visible spectrum, they change the color of the surface. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include those selected from the group consisting of the following dyes falling into the color Index (color Index, c.i.) classification: direct blue, direct red, direct violet, acid blue, acid red, acid violet, basic blue, basic violet and basic red, or mixtures thereof, for example as described in WO 2005/03274, WO 2005/03275, WO 2005/03276 and EP 1876226 (which are incorporated herein by reference). The detergent composition preferably comprises from about 0.00003 wt% to about 0.2 wt%, from about 0.00008 wt% to about 0.05 wt%, or even from about 0.0001 wt% to about 0.04 wt% fabric hueing agent. The composition may comprise from 0.0001 wt% to 0.2 wt% of a fabric hueing agent, which may be particularly preferred when the composition is in the form of a unit dose pouch. Suitable toners are also disclosed in, for example, WO 2007/087257 and WO 2007/087243.

Additional enzymes

The detergent additive together with the detergent composition may comprise one or more [ further ] enzymes, such as a hydrolase (EC 3. -), such as a hydrolase (EC 3.1. -), a glycosidase (EC 3.2. -), and a hydrolase (EC 3.4. -), an oxidoreductase (EC 1. -), such as a laccase (EC 1.10. -) or a peroxidase (EC 1.11. -), or a lyase (EC 4. -), such as a carbon-oxygen lyase (EC 4.2. -). In particular embodiments, the detergent composition may comprise one or more [ additional ] enzymes, such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase (e.g., a laccase), and/or a peroxidase.

Generally, the properties of the selected enzyme or enzymes should be compatible with the selected detergent (i.e., pH optimum, compatibility with other enzymatic or non-enzymatic ingredients, etc.), and the enzyme or enzymes should be present in effective amounts.

Cellulase enzymes

Suitable cellulases include those of bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Suitable cellulases include cellulases from bacillus, pseudomonas, humicola, fusarium, thielavia, cladosporium, e.g., fungal cellulases produced by humicola insolens, myceliophthora thermophila and fusarium oxysporum as disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259.

Particularly suitable cellulases are the alkaline cellulases or neutral cellulases providing or maintaining whiteness and preventing redeposition or having colour care benefits. Examples of such cellulases are the cellulases described in EP 0495257, EP 0531372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0531315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and WO 99/001544.

Other cellulases are endo-beta-1, 4-glucanases having a sequence with at least 97% identity to the amino acid sequence from position 1 to position 773 of SEQ ID No. 2 of WO 2002/099091 or a family 44 xyloglucanase having a sequence with at least 60% identity to positions 40-559 of SEQ ID No. 2 of WO 2001/062903.

Commercially available cellulases include Celluzyme^TMAnd Carezyme^TM(Novoxin Co.), Carezyme Premium^TM(Novoxil Co., Ltd.) Celluclean^TM(Novoxil Co., Ltd.) Celluclean Classic^TM(Novoxin Co., Ltd.) Cellusoft^TM(Novoxin Co.), Whitezyme^TM(Novoxil, Inc.), Clazinase ^TMAnd Puradax HA^TM(Jencology International Inc.) and KAC-500(B)^TM(Kao Corporation )).

Mannanase

Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. The mannanase may be an alkaline mannanase of family 5 or 26. It may be a wild type from the genus bacillus or humicola, in particular from bacillus autohesis (b.agaradhhaerens), bacillus licheniformis (b.licheniformis), bacillus alcalophilus (b.halodurans), bacillus clausii (b.clausii), or humicola insolens. Suitable mannanases are described in WO 1999/064619. The commercially available mannanase is Mannaway (novicent).

Protease enzyme

Suitable proteases include those of bacterial, fungal, plant, viral or animal origin, for example of plant or microbial origin. Preferably of microbial origin. Chemically modified mutants or protein engineered mutants are included. It may be an alkaline protease, such as a serine protease or a metalloprotease. The serine protease may for example be a serine protease of the S1 family (such as trypsin) or the S8 family (such as subtilisin). The metalloprotease may for example be a thermolysin from e.g. the M4 family or other metalloprotease such as those from the M5, M7 or M8 families.

The term "subtilase" refers to the serine protease subgroup according to Siezen et al, Protein Eng. [ Protein engineering ]4(1991)719-737 and Siezen et al Protein Science [ Protein Science ]6(1997) 501-523. Serine proteases are a subset of proteases characterized by a serine at the active site that forms a covalent adduct with a substrate. Subtilases can be divided into 6 subclasses, namely, the subtilisin family, the thermolysin family, the proteinase K family, the lanthionine antibiotic peptidase family, the Kexin family and the Pyrrolysin family.

Examples of subtilases are those derived from Bacillus, e.g., Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii as described in US 7262042 and WO 09/021867; and subtilisin lenus, subtilisin Novo, subtilisin Carlsberg, bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO 89/06279 and protease PD138 described in (WO 93/18140). Other useful proteases may be those described in WO 92/175177, WO 01/016285, WO 02/026024 and WO 02/016547. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and fusarium protease (described in WO 89/06270, WO 94/25583 and WO 05/040372), as well as chymotrypsin derived from cellulomonas (Cellumonas) (described in WO 05/052161 and WO 05/052146).

Further preferred proteases are alkaline proteases from B.lentus DSM 5483 (as described e.g.in WO 95/23221) and variants thereof (described in WO 92/21760, WO 95/23221, EP 1921147 and EP 1921148).

Examples of metalloproteases are neutral metalloproteases as described in WO 07/044993 (Jencology International Inc. (Genencor Int.)), e.g.those derived from Bacillus amyloliquefaciens.

Examples of useful proteases are the variants described in: WO 92/19729, WO 96/034946, WO 98/20115, WO 98/20116, WO 99/011768, WO 01/44452, WO 03/006602, WO 04/03186, WO 04/041979, WO 07/006305, WO 11/036263, WO 11/036264, in particular variants with substitutions at one or more of the following positions: 3. 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252, and 274, numbered with BPN'. More preferably, the subtilase variant may comprise the following mutations: S3T, V4I, S9R, a15T, K27R, 36D, V68A, N76D, N87S, R, 97E, A98S, S99G, D, G, S101G, M, G103G, V104G, Y, G106, G118G, G120G, G123G, S128G, P129G, S130G, G160G, Y167G, R170G, a 194G, G195G, V199G, V205G, L217G, N218G, M222G, a 232G, K G, Q236G, Q245G, N252G, T274G (numbering is done using BPN'.

Suitable commercially available proteases include those sold under the following trade names: Duralase^Tm、Durazym^Tm、Ultra、 Ultra、Ultra、Ultra、 Progress and Progress(novifin corporation), those sold under the following trade names:PurafectPreferenz^Tm、Purafect Purafect Purafect Effectenz^Tm、and(Danisco/DuPont ), Axappem^TM(Gistedbury Broards, Inc. (Gist-Brocases N.V.)), BLAP (sequence shown in FIG. 29 of US 5352604) and variants thereof (Henkel AG) and KAP (Bacillus alcalophilus subtilisin) from Kao.

Lipase and cutinase:

suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipases from the genus Thermomyces, e.g., from Thermomyces lanuginosus (earlier named Humicola lanuginosa) as described in EP 258068 and EP 305216; cutinases from the genus Humicola, such as Humicola insolens (WO 96/13580); lipases from strains of the genus pseudomonas (some of these are now renamed to burkholderia), such as pseudomonas alcaligenes or pseudoalcaligenes alcaligenes (EP 218272), pseudomonas cepacia (EP 331376), pseudomonas strain SD705(WO 95/06720 and WO 96/27002), pseudomonas wisconsiensis (p.wisconsinensis) (WO 96/12012); GDSL-type Streptomyces lipases (WO 10/065455); cutinases from Pyricularia oryzae (WO 10/107560); cutinases from pseudomonas mendocina (US 5,389,536); a lipase from Thermobifida fusca (WO 11/084412); geobacillus stearothermophilus lipase (WO 11/084417); lipases from Bacillus subtilis (WO 11/084599); and lipases (WO 12/137147) from Streptomyces griseus (WO 11/150157) and Streptomyces pristinaespiralis (S.pristinaespiralis).

Further examples are lipase variants such as those described in EP 407225, WO 92/05249, WO 94/01541, WO 94/25578, WO 95/14783, WO 95/30744, WO 95/35381, WO 95/22615, WO 96/00292, WO 97/04079, WO 97/07202, WO 00/34450, WO 00/60063, WO 01/92502, WO 07/87508 and WO 09/109500.

Preferred commercial productsThe lipase product comprises Lipolase^TM、Lipex^TM；Lipolex^TMAnd Lipoclean^TM(Novozymes A/S)), Lumafast (from Jennoniaceae (Genencor)), and Lipomax (from Giste Brocads, Inc. (Gist-Brocades)).

Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, such as acyltransferase with homology to Candida antarctica lipase A (WO 10/111143), acyltransferase from Mycobacterium smegmatis (WO 05/56782), perhydrolase from the CE 7 family (WO 09/67279) and variants of Mycobacterium smegmatis perhydrolase (in particular the S54V variant used in commercial product title Power Bleach from Huntington Textile dyeing, Inc. (Huntsman Textile Effects Pte Ltd)) (WO 10/100028).

Amylase:

suitable amylases which can be used with the variants of the invention may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a specific strain of Bacillus licheniformis (described in more detail in GB 1,296,839).

Suitable amylases include those having SEQ ID NO 2 of WO 95/10603 or variants thereof having 90% sequence identity to SEQ ID NO 3. Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and in SEQ ID NO4 of WO 99/019467, such as variants having substitutions at one or more of the following positions: 15. 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444.

Different suitable amylases include the amylase having SEQ ID NO 6 of WO 02/010355 or a variant thereof having 90% sequence identity to SEQ ID NO 6. Preferred variants of SEQ ID NO 6 are those having deletions in positions 181 and 182 and substitutions in position 193.

Other suitable amylases are hybrid alpha-amylases comprising residues 1-33 of the Bacillus amyloliquefaciens derived alpha-amylase shown in SEQ ID NO 6 of WO 2006/066594 and residues 36-483 of the Bacillus licheniformis alpha-amylase shown in SEQ ID NO4 of WO 2006/066594 or variants thereof having 90% sequence identity. Preferred variants of this hybrid alpha-amylase are those having a substitution, deletion, or insertion in one or more of the following positions: g48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from Bacillus amyloliquefaciens shown in SEQ ID NO. 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO. 4 are those having the following substitutions:

M197T；

H156Y + a181T + N190F + a209V + Q264S; or

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S。

Further suitable amylases are those having SEQ ID NO 6 of WO 99/019467 or variants thereof having 90% sequence identity to SEQ ID NO 6. Preferred variants of SEQ ID NO 6 are those having a substitution, deletion, or insertion in one or more of the following positions: r181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having a deletion in positions R181 and G182, or positions H183 and G184.

Further amylases which may be used are those having SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 2 or SEQ ID NO 7 of WO 96/023873 or variants thereof having 90% sequence identity to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 7. Preferred variants of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or SEQ ID NO 7 are those having substitutions, deletions, or insertions at one or more of the following positions: 140. 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476 (numbered using SEQ ID 2 of WO 96/023873). More preferred variants are those having a deletion in two positions selected from 181, 182, 183, and 184, such as 181 and 182, 182 and 183, or positions 183 and 184. The most preferred amylase variants of SEQ ID NO 1, SEQ ID NO 2, or SEQ ID NO 7 are those having deletions in positions 183 and 184 and substitutions in one or more of positions 140, 195, 206, 243, 260, 304, and 476.

Other amylases which may be used are those having SEQ ID NO 2 of WO 08/153815, SEQ ID NO 10 of WO 01/66712, or a variant thereof having 90% sequence identity to SEQ ID NO 2 of WO 08/153815, or a variant thereof having 90% sequence identity to SEQ ID NO 10 of WO 01/66712. Preferred variants of SEQ ID No. 10 in WO 01/66712 are those having a substitution, deletion, or insertion in one or more of the following positions: 176. 177, 178, 179, 190, 201, 207, 211, and 264.

Further suitable amylases are those of SEQ ID NO. 2 of WO 09/061380 or variants thereof having 90% sequence identity to SEQ ID NO. 2. Preferred variants of SEQ ID NO 2 are those having a C-terminal truncation, and/or substitution, deletion, or insertion in one or more of the following positions: q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID No. 2 are those having a substitution in one or more of the following positions: Q87E, R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E, R, N272E, R, S243Q, a, E, D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and/or those having deletions in positions R180 and/or S181 or T182 and/or G183. The most preferred amylase variants of SEQ ID NO 2 are those having the following substitutions:

N128C+K178L+T182G+Y305R+G475K；

N128C+K178L+T182G+F202Y+Y305R+D319T+G475K；

S125A + N128C + K178L + T182G + Y305R + G475K; or

S125A + N128C + T131I + T165I + K178L + T182G + Y305R + G475K, wherein the variants are C-terminally truncated and optionally further comprise a substitution at position 243 and/or a deletion at position 180 and/or position 181.

Other suitable amylases are alpha-amylases with SEQ ID NO 12 in WO 01/66712 or variants having at least 90% sequence identity with SEQ ID NO 12. Preferred amylase variants are those having a substitution, deletion or insertion in one or more of the following positions of SEQ ID NO:12 in WO 01/66712: r28, R118, N174; r181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; r320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and having substitutions R118K, N195F, R320K and R458K, and additionally having substitutions at one or more positions selected from the group consisting of: m9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and a339, most preferred are variants additionally having substitutions in all these positions.

Other examples are amylase variants such as those described in WO 2011/098531, WO 2013/001078 and WO 2013/001087.

A commercially available amylase is Duramyl^TM、Termamyl^TM、Fungamyl^TM、Stainzyme^TM、Stainzyme Plus^TM、Natalase^TMAnd BAN^TM(from Novozymes A/S), and Rapidase^TM、Purastar^TM/Effectenz^TM、Powerase^TM、Preferenz S1000^TM Preferenz S110^TMAnd Preferenz S100^TM(from Jencology International Inc./DuPont).

Peroxidase/oxidase:

peroxidase is a peroxidase comprised by the enzyme classification EC 1.11.1.7 described by the International Commission on the Nomenclature of the International Union of Biochemistry and Molecular Biology (IUBMB), or any fragment derived therefrom which exhibits peroxidase activity.

Suitable peroxidases include those of plant, bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, for example Coprinus cinereus (C.cinerea) (EP 179,486), and variants thereof, such as those described in WO 93/24618, WO 95/10602 and WO 98/15257.

Peroxidases may also include haloperoxidases, such as chloroperoxidase, bromoperoxidase, and compounds exhibiting chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidase (e.c.1.11.1.10) catalyzes the formation of hypochlorite from chloride ions.

In embodiments, the haloperoxidase is a chloroperoxidase. Preferably, the haloperoxidase is a vanadium haloperoxidase, i.e. a vanadate-containing haloperoxidase. In a preferred method of the invention, the vanadate-containing haloperoxidase is combined with a source of chloride ions.

Haloperoxidases have been isolated from a number of different fungi, in particular from the group of the fungi hyphomycetes, such as the genera Caldariomyces (e.g. Hemeromyces coaliphora), Alternaria, Curvularia (e.g. Curvularia verruculosa) and Curvularia inequality (C.inaegus), Helminthosporium, Geobacillus and Botrytis.

Haloperoxidases have also been isolated from bacteria such as the genera Pseudomonas, e.g., P.pyrrocinia, and Streptomyces, e.g., S.aureofaciens.

In a preferred embodiment, the haloperoxidase may be derived from Curvularia, in particular Curvularia verruculosa or Curvularia inequality, as described in WO 95/27046 for Curvularia inequality CBS 102.42; or Curvularia verruculosa CBS 147.63 or Curvularia verruculosa CBS 444.70 as described in WO 97/04102; or from Drechslera hartlebii as described in WO 01/79459, from Tryphialla crassa as described in WO 01/79458, from Phaeotrichonica crotalarie as described in WO 01/79461, or from the genus Genichosporium as described in WO 01/79460.

The oxidase may in particular comprise any laccase comprised by the enzyme classification EC 1.10.3.2 or any fragment derived therefrom exhibiting laccase activity, or a compound exhibiting similar activity, such as catechol oxidase (EC 1.10.3.1), o-aminophenol oxidase (EC 1.10.3.4) or bilirubin oxidase (EC 1.3.3.5).

Preferred laccases are enzymes of microbial origin. These enzymes may be derived from plants, bacteria or fungi (including filamentous fungi and yeasts).

Suitable examples from fungi include laccases that may be derived from the following strains: aspergillus (Aspergillus), Neurospora (Neurospora) (e.g., Neurospora crassa), coprospora (Podospora), botrytis, lysimachia (collectinia), Fomes (Fomes), Lentinus (Lentinus), Pleurotus (Pleurotus), Trametes (Trametes), e.g., Trametes hirsuta (t.villosa) and Trametes versicolor (t.versicolor)), Rhizoctonia (Rhizoctonia) (e.g., Rhizoctonia solani), coprinus (e.g., coprinus cinerea), coprinus (e.g., coprinus), coprinus (c. crispatus) (e.g., Polyporus fulvus), Polyporus crispatus (e.g., Polyporus culus), Polyporus crispatus (papuloides), Polyporus pinus (e.g., Polyporus thermophilus), Polyporus pinus (papuloides), Polyporus pinus (e.g., Polyporus pinus) Phlebia (Phlebia) (e.g., P.radiata)) or Coriolus (Coriolus) (e.g., Coriolus hirsutus (C.hirsutus)) (JP 2238885).

Suitable examples from bacteria include laccases which may be derived from strains of bacillus.

Preferred are laccases derived from Coprinus or myceliophthora; in particular laccase derived from Coprinus cinereus, as disclosed in WO 97/08325; or from myceliophthora thermophila, as disclosed in WO 95/33836.

Nuclease enzymes

Suitable nucleases include deoxyribonuclease (dnase) and ribonuclease (rnase), which are any enzymes that catalyze the hydrolytic cleavage of phosphodiester bonds in the DNA or RNA backbone, respectively, thereby degrading DNA and RNA. There are two main classifications based on the site of activity. Exonucleases digest nucleic acids from the ends. Endonucleases act on the region in the middle of the target molecule. The nuclease is preferably a dnase, which is preferably obtainable from a microorganism, preferably a fungus or a bacterium. In particular, dnases obtainable from species of bacillus are preferred; in particular, dnases obtainable from Bacillus foodborne (Bacillus cibi), Bacillus subtilis or Bacillus licheniformis are preferred. Examples of such dnases are described in WO 2011/098579, WO 2014/087011 and WO 2017/060475. Also particularly preferred are dnases obtainable from Aspergillus (Aspergillus) species; in particular a DNase obtainable from Aspergillus oryzae (Aspergillus oryzae), such as the DNase described in WO 2015/155350.

Lichenase

Suitable lichenases (lichenases) include enzymes which catalyze the hydrolysis of beta-1, 4-glucosidic bonds to produce beta-glucans. Lichenase (or lichenase) (e.g. EC 3.2.1.73) hydrolyses the (1,4) - β -D-glycosidic linkages in β -D-glucans containing (1,3) -and (1,4) -linkages and may act on lichen starch and cereal β -D-glucans but not on β -D-glucans containing only 1, 3-linkages or 1, 4-linkages. Examples of such lichenases are described in patent applications WO 2017/097866 and WO 2017/129754.

One or more detergent enzymes may be included in the detergent composition by adding a separate additive containing one or more enzymes, or by adding a combined additive containing all of these enzymes. The detergent additives of the present invention, either alone or in combination, may be formulated, for example, as granules, liquids, slurries, and the like. Preferred detergent additive formulations are granules, in particular non-dusting granules; liquids, in particular stabilizing liquids; or a slurry.

Non-dusting granulates may for example be produced as disclosed in US 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly (ethylene oxide) products (polyethylene glycol, PEG) having an average molecular weight of 1000 to 20000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; an ethoxylated fatty alcohol, wherein the alcohol contains from 12 to 20 carbon atoms, and wherein 15 to 80 ethylene oxide units are present; a fatty alcohol; a fatty acid; and mono-and diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. The liquid enzyme preparation may be stabilized, for example, by adding a polyol (such as propylene glycol), a sugar or sugar alcohol, lactic acid or boric acid according to established methods. The protected enzymes may be prepared according to the methods disclosed in EP 238,216.

Auxiliary materials

Any detergent component known in the art for use in laundry detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrinkage agents, anti-soil redeposition agents, anti-wrinkle agents, bactericides, binders, corrosion inhibitors, disintegrants/disintegrating agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and/or polyols such as propylene glycol), fabric conditioners (including clays), fillers/processing aids, optical brighteners/optical brighteners, suds boosters, suds (foam) regulators, perfumes, soil suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, alone or in combination. Any ingredient known in the art for use in laundry detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.

Dispersants-the detergent compositions of the present invention may also contain dispersants. In particular, the powder detergent may contain a dispersant. Suitable water-soluble organic materials include homo-or co-polymeric acids or salts thereof, wherein the polycarboxylic acid comprises at least two carboxyl groups separated from each other by not more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant science series, volume 71, Marcel Dekker, Inc.

Dye transfer inhibiting agents-the detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone and polyvinylimidazole, or mixtures thereof. When present in the subject compositions, the dye transfer inhibiting agents may be present at a level of from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3%, by weight of the composition.

Optical brighteners-the detergent compositions of the present invention will preferably also contain additional components which can colour the articles being cleaned, such as optical brighteners or optical brighteners. When present, the level of brightener is preferably from about 0.01% to about 0.5%. Any fluorescent whitening agent suitable for use in laundry detergent compositions may be used in the compositions of the present invention. The most commonly used fluorescent whitening agents are those belonging to the following classes: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives and diphenyl-distyryl derivatives. Examples of diaminostilbene-sulphonic acid derivative types of optical brighteners include the following sodium salts: 4,4' -bis- (2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2, 2' -disulfonate, 4' -bis- (2, 4-dianilino-s-triazin-6-ylamino) stilbene-2, 2' -disulfonate, 4' -bis- (2-anilino-4- (N-methyl-N-2-hydroxy-ethylamino) -s-triazin-6-ylamino) stilbene-2, 2' -disulfonate, 4' -bis- (4-phenyl-1, 2, 3-triazol-2-yl) stilbene-2, 2' -disulfonate and sodium 5- (2H-naphtho [1,2-d ] [1,2,3] triazol-2-yl) -2- [ (E) -2-phenylethenyl ] benzenesulfonate. Preferred optical brighteners are Tianlibao (Tinopal) DMS and Tianlibao CBS available from Ciba-Geigy AG (Basel, Switzerland). Heliotrope DMS is the disodium salt of 4,4 '-bis- (2-morpholino-4-anilino-s-triazin-6-ylamino) stilbene-2, 2' -disulfonate. Celecoxib CBS is the disodium salt of 2,2' -bis- (phenyl-styryl) -disulfonate. It is also preferred that the optical brightener is commercially available as Parawhite KX, supplied by Palamon Minerals and Chemicals, Inc., of Monmony, India. Other fluorescers suitable for use in the present invention include 1-3-diarylpyrazolines and 7-aminoalkylcoumarins.

Suitable levels of fluorescent brightener include lower levels from about 0.01 wt%, from 0.05 wt%, from about 0.1 wt%, or even from about 0.2 wt% to higher levels of 0.5 wt% or even 0.75 wt%.

Soil release polymers-the detergent compositions of the present invention may also comprise one or more soil release polymers which aid in the removal of soil from fabrics, such as cotton or polyester based fabrics, especially hydrophobic soil from polyester based fabrics. Soil release polymers can be, for example, nonionic or anionic terephthalic acid based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see, for example, Powdered Detergents, Surfactant science series, volume 71, chapter 7, massel Dekker (Marcel Dekker, Inc). Another type of soil release polymer is an amphiphilic alkoxylated greasy cleaning polymer comprising a core structure and a plurality of alkoxylated groups attached to the core structure. The core structure may comprise a polyalkyleneimine structure or a polyalkanolamine structure as described in detail in WO 2009/087523 (incorporated herein by reference). In addition, random graft copolymers are suitable soil release polymers. Suitable graft copolymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (incorporated herein by reference). Other soil release polymers are substituted polysaccharide structures, especially substituted cellulose structures, such as modified cellulose derivatives, such as those described in EP 1867808 or WO 2003/040279 (both incorporated herein by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, non-ionically modified cellulose, cationically modified cellulose, zwitterionic modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, ester carboxymethyl cellulose, and mixtures thereof.

Antiredeposition agents-the detergent compositions of the present invention may also include one or more antiredeposition agents, such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene oxide and/or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose-based polymers described above under soil release polymers may also function as anti-redeposition agents.

Rheology modifiers-are structurants or thickeners, distinct from viscosity reducers. The rheology modifier is selected from the group consisting of: non-polymeric crystalline, hydroxyl functional materials, polymeric rheology modifiers which impart shear thinning characteristics to the aqueous liquid phase matrix of the liquid detergent composition. The rheology and viscosity of the detergent may be modified and adjusted by methods known in the art, for example, as shown in EP 2169040.

Other suitable adjuvants include, but are not limited to, shrink proofing agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, suds suppressors, and solvents.

Protease inhibitors

The protease inhibitor may be any compound that stabilizes the protease or inhibits the protease such that the protease or other enzyme or enzymes in the laundry bar are not degraded. Examples of protease inhibitors are aprotinin, bestatin (bestatin), calpain inhibitors I and II, chymotrypsin inhibitors, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), boric acid, borates, borax, boric acid, phenyl boric acid (e.g.4-formylphenyl boronic acid (4-FPBA)), peptide aldehydes or sulfate or hemiacetal adducts thereof and peptide trifluoromethyl ketones. One or more protease inhibitors may be present, such as 5, 4, 3, 2 or 1 inhibitors, at least one of which is a peptide aldehyde, a bisulfite adduct or a hemiacetal adduct thereof.

Peptide aldehyde inhibitors

The peptide aldehyde can have the formula P- (A)_y-L-(B)_x-B⁰-H, or a bisulfite adduct or a hemiacetal adduct thereof, wherein:

i.H is hydrogen;

ii.B⁰is a single amino acid residue having the L-or D-configuration of formula-NH-ch (r) -C (═ O) -;

iii.(B)_xx is 1, 2 or 3 and B is independently linked to B via the C-terminus of the B amino acid⁰A single amino acid of

L is absent or L is independently a linker of formula-C (═ O) -, -C (═ O) -, -C (═ S) -or-C (═ S) -C (═ O) -;

v.(A)_yY of (a) is 0, 1 or 2, and a is independently a single amino acid residue linked to L via the N-terminus of the a amino acid, with the proviso that if L is absent, then a is absent;

p is selected from the group consisting of: hydrogen and an N-terminal protecting group, with the proviso that if L is absent, then P is an N-terminal protecting group;

r is independently selected from the group consisting of: c optionally substituted by one or more identical or different substituents R_1-6Alkyl radical, C_6-10Aryl or C_7-10Aralkyl group;

r' is independently selected from the group consisting of: halogen, -OH, -OR ", -SH, -SR", -NH₂、-NHR”、-NR”₂、-CO₂H、-CONH₂、-CONHR”、-CONR”₂、-NHC(＝N)NH₂(ii) a And

ix.R' is C_1-6An alkyl group.

x may be 1, 2 or 3 and thus B may be 1, 2 or 3 amino acid residues, respectively. Thus, B may represent B¹、B²-B¹Or B³-B²-B¹In which B is³、B²And B¹Each represents an amino acid residueAnd (4) a base. y may be 0, 1 or 2 and thus A may not be present, or is correspondingly of formula A¹Or A²-A¹1 or 2 amino acid residues of (A), wherein²And A¹Each represents an amino acid residue.

B⁰May be a single amino acid residue having L or D configuration, which is linked to H via the C-terminus of the amino acid, wherein R is C _1-6Alkyl radical, C_6-10Aryl or C_7-10Aralkyl side chains, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl or benzyl, and wherein R may optionally be substituted by one or more identical or different substituents R'. Particular examples are the D-or L-form of arginine (Arg), 3, 4-dihydroxyphenylalanine, isoleucine (Ile), leucine (Leu), methionine (Met), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), meta-tyrosine, para-tyrosine (Tyr) and valine (Val). A particular embodiment is when B⁰Leucine, methionine, phenylalanine, para-tyrosine and valine.

Via B¹The C-terminal end of the amino acid being linked to B⁰B of (A) to¹May be aliphatic, hydrophobic and/or neutral amino acids. B is¹Examples of (A) are alanine (Ala), cysteine (Cys), glycine (GIy), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (VaI). B is¹Specific examples of (a) are alanine, glycine, isoleucine, leucine and valine. A particular embodiment is when B¹Alanine, glycine or valine.

If present, via B²The C-terminal end of the amino acid being linked to B¹B of (A) to²May be aliphatic, hydrophobic, neutral and/or polar amino acids. B is²Examples of (A) are alanine (Ala), arginine (Arg), capreomycin (Cpd), cysteine (Cys), glycine (GIy), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr) and valine (V)Amino acid (VaI). B is²Specific examples of (a) are alanine, arginine, tendrilysin, glycine, isoleucine, leucine, phenylalanine and valine. A particular embodiment is when B²Arginine, glycine, leucine, phenylalanine, or valine.

Via B³The C-terminal end of the amino acid being linked to B²B of (A) to³And (if present) may be large, aliphatic, aromatic, hydrophobic, and/or neutral amino acids. B is³Examples of (A) are isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (VaI). B is³Specific examples of (a) are leucine, phenylalanine, tyrosine and tryptophan.

The linking group L may be absent or selected from the group consisting of: -C (═ O) -, -C (═ S) -or-C (═ S) -C (═ O) -. Particular examples include when L is absent or is a carbonyl group-C (═ O) -.

A linked to L via the N-terminus of the amino acid¹And (if present) may be aliphatic, aromatic, hydrophobic, neutral and/or polar amino acids. A. the¹Examples of (A) are alanine (Ala), arginine (Arg), tendriline (Cpd), glycine (GIy), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (VaI). A. the¹Specific examples of (a) are alanine, arginine, glycine, leucine, phenylalanine, tyrosine, tryptophan, and valine. A particular embodiment is when B²Leucine, phenylalanine, tyrosine or tryptophan.

Attachment to A via the N-terminus of the amino acid¹A of²The residue (if present) may be a large, aliphatic, aromatic, hydrophobic and/or neutral amino acid. A. the²Examples of (A) are arginine (Arg), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan Acids (Trp) and valine (VaI). A. the²Specific examples of (a) are phenylalanine and tyrosine.

The N-terminal protecting group P (if present) may be selected from formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups such as fluorenylmethoxycarbonyl (Fmoc), methoxycarbonyl, (fluoromethoxy) carbonyl, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), and adamantyloxycarbonyl; p-methoxybenzylcarbonyl (Moz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxyacetyl, methylaminocarbonyl, methylsulfonyl, ethylsulfonyl, benzylsulfonyl, methylphosphono (meop (oh) (═ O)), and benzylphosphoramido (PhCH)₂OP(OH)(＝O))。

The general formula of the peptide aldehyde can also be written: P-A²-A¹-L-B³-B² B¹-B⁰-H, wherein P, A²、A¹、L、B³、B²、B¹And B⁰As defined above.

In the case of a tripeptide aldehyde with a protecting group (i.e. x ═ 2, L is absent and a is absent), P is preferably acetyl, methoxycarbonyl, benzyloxycarbonyl, methylaminocarbonyl, methylsulfonyl, benzylsulfonyl and benzylphosphoramido. In the case of tetrapeptide aldehydes with protecting groups (i.e. x ═ 3, L is absent and a is absent), P is preferably acetyl, methoxycarbonyl, methylsulfonyl, ethylsulfonyl and methylphosphatamido.

Suitable peptide aldehydes are described in WO 94/04651, WO 95/25791, WO 98/13458, WO 98/13459, WO 98/13460, WO 98/13461, WO 98/13462, WO 07/141736, WO 07/145963, WO 09/118375, WO 10/055052 and WO 11/036153.

More specifically, the peptide aldehyde may be

Cbz-Arg-Ala-Tyr-H (L-alaninamide, N2- [ (phenylmethoxy) carbonyl ] -L-arginyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] -),

Ac-Gly-Ala-Tyr-H (L-alaninamide, N-acetylglycinyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] -),

Cbz-Gly-Ala-Tyr-H (L-alaninamide, N- [ (phenylmethoxy) carbonyl ] glycyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] -),

Cbz-Gly-Ala-Leu-H (L-alaninamide, N- [ (phenylmethoxy) carbonyl ] glycyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

Cbz-Val-Ala-Leu-H (L-alaninamide, N- [ (phenylmethoxy) carbonyl ] -L-valyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

Cbz-Gly-Ala-Phe-H (L-alaninamide, N- [ (phenylmethoxy) carbonyl ] glycyl-N- [ (1S) -1-formyl-2-phenylethyl ] -),

Cbz-Gly-Ala-Val-H (L-alaninamide, N- [ (phenylmethoxy) carbonyl ] glycyl-N- [ (1S) -1-formyl-2-methylpropyl ] -,

Cbz-Gly-Gly-Tyr-H (glycinamide, N- [ (phenylmethoxy) carbonyl ] glycyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] -,

Cbz-Gly-Gly-Phe-H (glycinamide, N- [ (phenylmethoxy) carbonyl ] glycyl-N- [ (1S) -1-formyl-2-phenylethyl ] -),

Cbz-Arg-Val-Tyr-H (L-valinamide, N2- [ (phenylmethoxy) carbonyl ] -L-arginyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] -),

Cbz-Leu-Val-Tyr-H (L-valinamide, N- [ (phenylmethoxy) carbonyl ] -L-leucyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] -)

Ac-Leu-Gly-Ala-Tyr-H (L-alaninamide, N-acetyl-L-leucylglycyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] -),

Ac-Phe-Gly-Ala-Tyr-H (L-alaninamide, N-acetyl-L-phenylalanyl glycyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] -),

Ac-Tyr-Gly-Ala-Tyr-H (L-alaninamide, N-acetyl-L-tyrosylglycyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] -),

Ac-Phe-Gly-Ala-Leu-H (L-alaninamide, N-acetyl-L-phenylalanyl glycyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

Ac-Phe-Gly-Ala-Phe-H (L-alaninamide, N-acetyl-L-phenylalanyl glycyl-N- [ (1S) -1-formyl-2-phenylethyl ] -),

Ac-Phe-Gly-Val-Tyr-H (L-valinamide, N-acetyl-L-phenylalanyl glycyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] -),

Ac-Phe-Gly-Ala-Met-H (L-alaninamide, N-acetyl-L-phenylalanyl glycyl-N- [ (1S) -1-formyl-3- (methylthio) propyl ] -),

Ac-Trp-Leu-Val-Tyr-H (L-valinamide, N-acetyl-L-tryptophanyl-L-leucyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] -),

MeO-CO-Val-Ala-Leu-H (L-alaninamide, N- (methoxycarbonyl) -L-valyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

MeNHCO-Val-Ala-Leu-H (L-alaninamide, N- (aminomethylcarbonyl) -L-valyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

MeO-CO-Phe-Gly-Ala-Leu-H (L-alaninamide, N- (methoxycarbonyl) -L-phenylalanyl glycyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

MeO-CO-Phe-Gly-Ala-Phe-H (L-alaninamide, N- (methoxycarbonyl) -L-phenylalanyl glycyl-N- [ (1S) -1-formyl-2-phenylethyl ] -),

MeSO2-Phe-Gly-Ala-Leu-H (L-alaninamide, N- (methylsulfonyl) -L-phenylalanyl glycyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

MeSO2-Val-Ala-Leu-H (L-alaninamide, N- (methylsulfonyl) -L-valyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

PhCH2O-P (OH) (O) -Val-Ala-Leu-H (L-alaninamide, N- [ hydroxy (phenylmethoxy) phosphinyl ] -L-valyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

EtSO2-Phe-Gly-Ala-Leu-H (L-alaninamide, N- (ethylsulfonyl) -L-phenylalanyl glycyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

PhCH2SO2-Val-Ala-Leu-H (L-alaninamide, N- [ (benzyl) sulfonyl ] -L-valyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

PhCH2O-P (OH) (O) -Leu-Ala-Leu-H (L-alaninamide, N- [ hydroxy (phenylmethoxy) phosphinyl ] -L-leucyl-N- [ (1S) -1-formyl-3-methylbutyl ] -),

PhCH2O-P (OH) (O) -Phe-Ala-Leu-H (L-alaninamide, N- [ hydroxy (phenylmethoxy) phosphinyl ] -L-benzamido-N- [ (1S) -1-formyl-3-methylbutyl ] -), or

MeO-P (OH) (O) -Leu-Gly-Ala-Leu-H; (L-alaninamide, N- (hydroxymethoxyphosphinyl) -L-leucylglycyl-N- [ (1S) -1-formyl-3-methylbutyl ] -).

A preferred example is Cbz-Gly-Ala-Tyr-H.

Additional examples of such peptide aldehydes include

α -MAPI (3,5,8, 11-tetraazatridecanoic acid, 6- [3- [ (aminoiminomethyl) amino ] propyl ] -12-formyl-9- (1-methylethyl) -4,7, 10-trioxo-13-phenyl-2- (phenylmethyl) -, (2S,6S,9S,12S) -

L-valinamide, N2- [ [ (1-carboxy-2-phenylethyl) amino ] carbonyl ] -L-arginyl-N- (1-formyl-2-phenylethyl) -, [1(S),2(S) ] -; l-valinamide, N2- [ [ [ (1S) -1-carboxy-2-phenylethyl ] amino ] carbonyl ] -L-arginyl-N- [ (1S) -1-formyl-2-phenylethyl ] - (9 CI); SP-chymotrypsin inhibitor B),

beta-MAPI (L-valinamide, N2- [ [ [ (1S) -1-carboxy-2-phenylethyl ] amino ] carbonyl ] -L-arginyl-N- [ (1R) -1-formyl-2-phenylethyl ] -L-valinamide, N2- [ [ (1-carboxy-2-phenylethyl) amino ] carbonyl ] -L-arginyl-N- (1-formyl-2-phenylethyl) -, [1(S),2(R) ] -,

Phe-C (═ O) -Arg-Val-Tyr-H (L-valinamide, N2- [ [ [ (1S) -1-carboxy-2-phenylethyl ] amino ] carbonyl ] -L-arginyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] - (9CI)),

Phe-C (═ O) -Gly-Tyr-H (3,5,8, 11-tetraazatridecanoic acid, 12-formyl-13- (4-hydroxyphenyl) -4,7, 10-trioxo-2- (phenylmethyl), (2S,12S) -),

Phe-C (═ O) -Gly-Ala-Phe-H (3,5,8, 11-tetraazatridecanoic acid, 12-formyl-9-methyl-4, 7, 10-trioxo-13-phenyl-2- (phenylmethyl) -, (2S,9S,12S) -),

Phe-C (═ O) -Gly-Ala-Tyr-H (3,5,8, 11-tetraazatridecanoic acid, 12-formyl-13- (4-hydroxyphenyl) -9-methyl-4, 7, 10-trioxo-2- (phenylmethyl) -, (2S,9S,12S) -),

Phe-C (═ O) -Gly-Ala-Leu-H (3,5,8, 11-tetraazapentadecanoic acid, 12-formyl-9, 14-dimethyl-4, 7, 10-trioxo-2- (benzyl) -, (2S,9S,12S) -),

Phe-C (═ O) -Gly-Ala-Nva-H (3,5,8, 11-tetraazapentadecanoic acid, 12-formyl-9-methyl-4, 7, 10-trioxo-2- (phenylmethyl) -, (2S,9S,12S) -),

Phe-C (═ O) -Gly-Ala-Nle-H (3,5,8, 11-tetraazahexadecanoic acid, 12-formyl-9-methyl-4, 7, 10-trioxo-2- (phenylmethyl) -, (2S,9S,12S) -),

Tyr-C (═ O) -Arg-Val-Tyr-H (L-valinamide, N2- [ [ [ (1S) -1-carboxy-2- (4-hydroxyphenyl) ethyl ] amino ] carbonyl ] -L-arginyl-N- [ (1S) -1-formyl-2- (4-hydroxyphenyl) ethyl ] - (9CI))

Tyr-C (═ O) -Gly-Ala-Tyr-H (3,5,8, 11-tetraazatridecanoic acid, 12-formyl-13- (4-hydroxyphenyl) -2- [ (4-hydroxyphenyl) methyl ] -9-methyl-4, 7, 10-trioxo-, (2S,9S,12S) -)

Phe-C (═ S) -Arg-Val-Phe-H (3,5,8, 11-tetraazatridecanoic acid, 6- [3- [ (aminoiminomethyl) amino ] propyl ] -12-formyl-9- (1-methylethyl) -7, 10-dioxo-13-phenyl-2- (phenylmethyl) -4-thioxo-, (2S,6S,9S,12S) -),

Phe-C (═ S) -Arg-Val-Tyr-H (3,5,8, 11-tetraazatridecanoic acid, 6- [3- [ (aminoiminomethyl) amino ] propyl ] -12-formyl-13- (4-hydroxyphenyl) -9- (1-methylethyl) -7, 10-dioxo-2- (benzyl) -4-thioxo-, (2S,6S,9S,12S) -),

Phe-C (═ S) -Gly-Ala-Tyr-H (3,5,8, 11-tetraazatridecanoic acid, 12-formyl-13- (4-hydroxyphenyl) -9-methyl-7, 10-dioxo-2- (benzyl) -4-thio-, (2S,9S,12S) -),

antipodicin (L-valinamide, N2- [ [ (1-carboxy-2-phenylethyl) amino ] carbonyl ] -L-arginyl-N- [4- [ (aminoiminomethyl) amino ] -1-formylbutyl ] -),

GE20372A (L-valinamide, N2- [ [ [ (1S) -1-carboxy-2- (4-hydroxyphenyl) ethyl ] amino ] carbonyl ] -L-arginyl-N- [ (1S) -1-formyl-2-phenylethyl ] -

L-valinamide, N2- [ [ [ 1-carboxy-2- (4-hydroxyphenyl) ethyl ] amino ] carbonyl ] -L-arginyl-N- (1-formyl-2-phenylethyl) -, [1(S),2(S) ] -),

GE20372B (L-valinamide, N2- [ [ [ (1S) -1-carboxy-2- (4-hydroxyphenyl) ethyl ] amino ] carbonyl ] -L-arginyl-N- [ (1R) -1-formyl-2-phenylethyl ] -

L-valinamide, N2- [ [ [ 1-carboxy-2- (4-hydroxyphenyl) ethyl ] amino ] carbonyl ] -L-arginyl-N- (1-formyl-2-phenylethyl) -, [1(S),2(R) ] -),

chymotrypsin inhibitor A (L-leucinamide, (2S) -2- [ (4S) -2-amino-3, 4,5, 6-tetrahydro-4-pyrimidinyl ] -N- [ [ [ (1S) -1-carboxy-2-phenylethyl ] amino ] carbonyl ] glycyl-N- (1-formyl-2-phenylethyl) -

L-leucinamide, (2S) -2- [ (4S) -2-amino-1, 4,5, 6-tetrahydro-4-pyrimidinyl ] -N- [ [ [ (1S) -1-carboxy-2-phenylethyl ] amino ] carbonyl ] glycyl-N- (1-formyl-2-phenylethyl) - (9 CI); l-leucinamide, L-2- (2-amino-1, 4,5, 6-tetrahydro-4-pyrimidinyl) -N- [ [ (1-carboxy-2-phenylethyl) amino ] carbonyl ] glycyl-N- (1-formyl-2-phenylethyl) -, a stereoisomer),

Chymotrypsin inhibitor B (L-valinamide, (2S) -2- [ (4S) -2-amino-3, 4,5, 6-tetrahydro-4-pyrimidinyl ] -N- [ [ [ (1S) -1-carboxy-2-phenylethyl ] amino ] carbonyl ] glycyl-N- (1-formyl-2-phenylethyl) -

L-valinamide, (2S) -2- [ (4S) -2-amino-1, 4,5, 6-tetrahydro-4-pyrimidinyl ] -N- [ [ [ (1S) -1-carboxy-2-phenylethyl ] amino ] carbonyl ] glycyl-N- (1-formyl-2-phenylethyl) - (9 CI); l-valinamide, L-2- (2-amino-1, 4,5, 6-tetrahydro-4-pyrimidinyl) -N- [ [ (1-carboxy-2-phenylethyl) amino ] carbonyl ] glycyl-N- (1-formyl-2-phenylethyl) -, a stereoisomer), and

chymotrypsin inhibitor C (L-isoleucinamide, (2S) -2- [ (4S) -2-amino-3, 4,5, 6-tetrahydro-4-pyrimidinyl ] -N- [ [ [ (1S) -1-carboxy-2-phenylethyl ] amino ] carbonyl ] glycyl-N- (1-formyl-2-phenylethyl) -

L-isoleucamide, (2S) -2- [ (4S) -2-amino-1, 4,5, 6-tetrahydro-4-pyrimidinyl ] -N- [ [ [ (1S) -1-carboxy-2-phenylethyl ] amino ] carbonyl ] glycyl-N- (1-formyl-2-phenylethyl) - (9 CI); l-isoleucinamide, L-2- (2-amino-1, 4,5, 6-tetrahydro-4-pyrimidinyl) -N- [ [ (1-carboxy-2-phenylethyl) amino ] carbonyl ] glycyl-N- (1-formyl-2-phenylethyl) -, a stereoisomer.

Peptide aldehyde adducts

Instead of a peptide aldehyde, the protease inhibitor may be an adduct of a peptide aldehyde. The adduct may be of the formula P- (A)_y-L-(B)_x-N(H)-CHR-CH(OH)-SO₃Bisulfite adducts of M, wherein P, A, y, L, B, x and R are as defined above, and M is H or an alkali metal, preferably Na or K. Alternatively, the adduct may be of the formula P- (A)_y-L-(B)_xHemiacetals of N (H) -CHR-CH (OH) -OR, wherein P, A, y, L, B, x, and R are as defined above. A preferred embodiment is the bisulfite adduct, where P ═ Cbz, B²＝Gly；B¹＝Ala；B⁰Tyr (hence R ═ PhCH)₂R' ═ OH), x ═ 2, y ═ 0, L ═ a ═ absent and M ═ Na (Cbz-Gly-Ala-n (h) -CH (CH) — (CH)₂-p-C₆H₄OH)-CH(OH)-SO₃Na, L-alaninamide, N- [ (phenylmethoxy) carbonyl]glycyl-N- [ 2-hydroxy-1- [ (4-hydroxyphenyl) methyl group]-2-sulfoethyl]-, sodium salt (1: 1)).

The bisulfite adduct of a peptide aldehyde can also be written as: P-A²-A¹-L-B³-B²-B¹-N(H)-CHR-CH(OH)-SO₃M, wherein P, A²、A¹、L、B³、B²、B¹R and M are as defined above.

Alternatively, the adduct of the peptide aldehyde may be Cbz-Gly-Ala-N (H) -CH (CH)₂-p-C₆H₄OH)-CH(OH)-SO₃Na ((2S) - [ (N- { N- [ ((benzyloxy)) carbonyl)]Glycyl } -L-alanyl) amino]-1-hydroxy-3- (4-hydroxyphenyl) propane-1-sulfonic acid sodium) or Cbz-Gly-Ala-N (H) -CH (CH2Ph) -CH (OH) -SO₃Na ((2S) - [ (N- { N- [ ((benzyloxy)) carbonyl) ]Glycyl } -L-alanyl) amino]-1-hydroxy-3- (phenyl) propane-1-sulfonic acid sodium salt) or "MeO-CO _ Val-Ala-N (H) -CH (CH2CH (CH)₃)₂)-CH(OH)-SO₃Na((2S)-[(N- { N- [ ((benzyloxy)) carbonyl]Glycyl } -L-alanyl) amino]-1-hydroxy-3- (2-propyl) propane-1-sulfonic acid sodium salt).

Other preferred peptide aldehyde bisulfite salts are

Cbz-Arg-Ala-NHCH(CH₂C₆H₄OH)C(OH)(SO₃M) -H, wherein M ═ Na,

Ac-Gly-Ala-NHCH(CH₂C₆H₄OH)C(OH)(SO₃m) -H, wherein M ═ Na,

Cbz-Gly-Ala-NHCH(CH₂C₆H₄OH)C(OH)(SO₃m) -H, wherein M ═ Na (L-alaninamide, N- [ (phenylmethoxy) carbonyl]glycyl-N- [ 2-hydroxy-1- [ (4-hydroxyphenyl) methyl group]-2-sulfoethyl]-, sodium salt (1:1)),

Cbz-Gly-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

Cbz-Val-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

Cbz-Gly-Ala-NHCH(CH₂Ph)C(OH)(SO₃m) -H, wherein M ═ Na,

Cbz-Gly-Ala-NHCH(CH(CH₃)₂)C(OH)(SO₃m) -H, wherein M ═ Na,

Cbz-Gly-Gly-NHCH(CH₂C₆H₄OH)C(OH)(SO₃m) -H, wherein M ═ Na,

Cbz-Gly-Gly-NHCH(CH₂Ph)C(OH)(SO₃m) -H, wherein M ═ Na,

Cbz-Arg-Val-NHCH(CH₂C₆H₄OH)C(OH)(SO₃m) -H, wherein M ═ Na,

Cbz-Leu-Val-NHCH(CH₂C₆H₄OH)C(OH)(SO₃m) -H, wherein M ═ Na,

Ac-Leu-Gly-Ala-NHCH(CH₂C₆H₄OH)C(OH)(SO₃m) -H, wherein M ═ Na,

Ac-Phe-Gly-Ala-NHCH(CH₂C₆H₄OH)C(OH)(SO₃m) -H, wherein M ═ Na,

Ac-Tyr-Gly-Ala-NHCH(CH₂C₆H₄OH)C(OH)(SO₃m) -H, wherein M ═ Na,

Ac-Phe-Gly-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

Ac-Phe-Gly-Ala-NHCH(CH₂Ph)C(OH)(SO₃m) -H, wherein M ═ Na,

Ac-Phe-Gly-Val-NHCH(CH₂C₆H₄OH)C(OH)(SO₃m) -H, wherein M ═ Na,

Ac-Phe-Gly-Ala-NHCH(CH₂CH₂SCH₃)(SO₃m) -H, wherein M ═ Na,

Ac-Trp-Leu-Val-NHCH(CH₂C₆H₄OH)C(OH)(SO₃m) -H, wherein M ═ Na,

MeO-CO-Val-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

MeNCO-Val-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

MeO-CO-Phe-Gly-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

MeO-CO-Phe-Gly-Ala-NHCH(CH₂Ph)C(OH)(SO₃m) -H, wherein M ═ Na,

MeSO₂-Phe-Gly-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

MeSO₂-Val-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

PhCH₂O(OH)(O)P-Val-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

EtSO₂-Phe-Gly-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

PhCH₂SO₂-Val-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

PhCH₂O(OH)(O)P-Leu-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃M) -H, wherein M ═ Na,

PhCH₂O(OH)(O)P-Phe-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na,

MeO(OH)(O)P-Leu-Gly-Ala-NHCH(CH₂CH(CH₃)₂))C(OH)(SO₃m) -H, wherein M ═ Na, and

Phe-urea-Arg-Val-NHCH (CH)₂C₆H₄OH)C(OH)(SO₃M) -H, wherein M ═ Na.

Salt (salt)

The salt used in the bars is a salt of a monovalent cation with an organic anion. The monovalent cation may be, for example, Na⁺、K⁺Or NH₄ ⁺. The organic anion may be, for example, formate, acetate, citrate, or lactate. Thus, the salt of the monovalent cation with the organic anion may be, for example, sodium formate, potassium formate, ammonium formate, sodium acetate, potassium acetate, ammonium acetate, sodium lactate, potassium lactate, ammonium lactate, sodium mono-citrate, sodium di-citrate, sodium tri-citrate, sodium potassium citrate, ammonium citrate, and the like. A particular example is sodium formate.

Formulation of detergent products

The detergent composition of the invention may be in any conventional form, such as a bar, a homogeneous tablet, a tablet with two or more layers, a pouch with one or more compartments, a regular or compressed powder, a granule, a paste, a gel, or a regular, compressed or concentrated liquid.

The bag may be configured as a single chamber or as multiple chambers. It may be of any form, shape and material suitable for holding the composition, e.g. not allowing the composition to be released from the bag before contact with water. The pouch is made of a water-soluble film that contains an interior volume. The interior volume may be divided into chambers of bags. Preferred films are polymeric materials, preferably polymers that form films or sheets. Preferred polymers, copolymers or derivatives thereof are selected from polyacrylates, and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers and Hydroxypropylmethylcellulose (HPMC). Preferably, the level of polymer in the film, e.g., PVA, is at least about 60%. Preferred average molecular weights will typically be from about 20,000 to about 150,000. The films may also be blend compositions comprising hydrolytically degradable and water soluble polymer blends such as polylactic acid and polyvinyl alcohol (known under trade reference number M8630, as sold by MonoSol LLC of indiana, usa) plus plasticizers like glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. The pouch may contain a solid laundry cleaning composition or a part component and/or a liquid cleaning composition or a part component separated by a water-soluble film. The compartment for the liquid component may be different in composition from the compartment containing the solid. Reference: (US 2009/0011970A 1).

The detergent ingredients may be physically separated from each other by a compartment in a water-soluble pouch or in a different layer of the tablet. Thus, poor storage interactions between the components can be avoided. The different dissolution profiles of each compartment may also cause delayed dissolution of the selected component in the wash liquor.

Non-unit dose liquid or gel detergents may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water. Other types of liquids including, but not limited to, alkanols, amines, glycols, ethers, and polyols may be included in the aqueous liquid or gel. Aqueous liquid or gel detergents may contain from 0 to 30% of organic solvents.

The liquid or gel detergent may be non-aqueous.

Method of producing a composition

The invention also relates to methods of producing the compositions.

Use of

The present invention is also directed to methods for using the compositions thereof.

Use in detergents.

The polypeptides of the invention may be added to and thus made a component of a detergent composition.

The detergent compositions of the present invention may be formulated, for example, as hand or machine laundry detergent compositions comprising a laundry additive composition suitable for pre-treating stained fabrics or for rejuvenating textiles (e.g., by removing fuzz or pills) to restore some of the visual and sensory characteristics of the fabrics after extended use to match new textiles, and a rinse added fabric softener composition, or as a detergent composition for general household hard surface cleaning operations, or for hand or machine dishwashing operations.

In a particular aspect, the invention provides a detergent additive comprising a polypeptide of the invention as described herein.

Paragraph (b)

Paragraph 1. a variant of a parent polypeptide having glycoside hydrolase (EC 3.2.1.-) activity, wherein the variant comprises a catalytic domain, a proline-rich linker region, and a Carbohydrate Binding Module (CBM), and wherein the variant has glycoside hydrolase activity.

Paragraph 2. a variant of a parent polypeptide having cellulase activity, wherein the variant comprises a catalytic domain, a proline-rich linker region and a Carbohydrate Binding Module (CBM), and wherein the variant has cellulase activity.

Paragraph 3. a variant of a parent polypeptide having endoglucanase activity, wherein the variant comprises a catalytic domain, a proline-rich linker region and a Carbohydrate Binding Module (CBM), and wherein the variant has endoglucanase activity.

Paragraph 4. the variant according to any of paragraphs 1-3, wherein the variant has improved stability in an aqueous composition comprising the protease compared to the parent.

Paragraph 5. a variant of a parent polypeptide having glycoside hydrolase (EC 3.2.1.-) activity, wherein the variant comprises a catalytic domain, an engineered linker region and a Carbohydrate Binding Module (CBM), and wherein the variant has glycoside hydrolase activity, wherein the variant has improved stability in an aqueous composition comprising a protease compared to the parent.

Paragraph 6. a variant of a parent polypeptide having cellulase activity, wherein the variant comprises a catalytic domain, an engineered linker region and a Carbohydrate Binding Module (CBM), and wherein the variant has cellulase activity, wherein the variant has improved stability in an aqueous composition comprising a protease compared to the parent.

Paragraph 7. a variant of a parent polypeptide having endoglucanase activity, wherein the variant comprises a catalytic domain, an engineered linker region and a Carbohydrate Binding Module (CBM), and wherein the variant has endoglucanase activity, wherein the variant has improved stability in an aqueous composition comprising a protease compared to the parent.

Paragraph 8 a variant which is a hybrid polypeptide having a glycoside hydrolase activity, e.g., endoglucanase activity, preferably GH45 endoglucanase activity, comprising (a) a catalytic domain from a polypeptide having a glycoside hydrolase activity, e.g., endoglucanase activity, preferably GH45 endoglucanase activity, (b) a linker selected from the group consisting of: PPPPPPPP (SEQ ID NO:31), PPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) and SPSPSPSPSPG (SEQ ID NO:25), and (c) a Carbohydrate Binding Module (CBM), preferably CBM 1.

Paragraph 9. the hybrid polypeptide according to paragraph 8, which has an improved stability in an aqueous composition comprising a protease compared to the parent.

Paragraph 10. the variant according to any of the preceding paragraphs, wherein improved stability is determined according to the assay described in example 2 and/or example 7.

A variant according to any preceding claim, which is a family GH45 endoglucanase.

Paragraph 12. the variant according to any of the preceding paragraphs, wherein the CBM is CBM 1.

Paragraph 13. the variant according to any of the preceding paragraphs, wherein the variant comprises an N-terminal catalytic domain and a C-terminal CBM.

Paragraph 14. the variant according to any of the preceding paragraphs, wherein the variant comprises a C-terminal catalytic domain and an N-terminal CBM.

Paragraph 15. the variant according to any of the preceding paragraphs, wherein the variant exhibits improved fabric or textile care and/or improved wash performance relative to the parent, e.g., upon storage in the presence of a protease.

Paragraph 16. the variant according to any of the preceding paragraphs, wherein the linker comprises at least 25% proline, e.g., at least 28% proline, at least 30% proline, at least 35% proline, at least 40% proline, at least 50% proline, e.g., at least 60%, at least 66%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% proline.

Paragraph 17. the variant according to any of the preceding paragraphs, wherein the linker has a length of at least four amino acids and comprises one or more of the following optional repeat motifs:

a.[P/S/T/R/K/D/E]P, preferably [ P/S/T]P; most preferably (SP)_aA is 2-10 or P_bB is 4-20, preferably 4-15

b.P [ S/T/R/K/D/E/N/Q ] P [ S/T/R/K/D/E ] (SEQ ID NO:102), preferably P [ S/E ] PT (SEQ ID NO: 109).

Paragraph 18. the variant according to any of the preceding paragraphs, wherein the linker has a length of at least four amino acids and comprises the following optional repeat motif: [ S/T/R/K/D/E ] P [ S/T/R/K/D/E/N/Q ] [ P/S/T/R/K/D/E ] [ P/S/T/R/K/D/E ] P and/or P [ P/S/T/R/K/D/E ] [ P/S/T/R/K/D/E ].

Paragraph 19. the variant according to any of the preceding paragraphs, wherein the linker comprises:

a.(SP)_a，a＝2-10；

b.(PS)_a，a＝2-10；

c.P_bb is 4-20, preferably 4-15;

d.(PEPT(SEQ ID NO:125))_c，c＝2-5；

e.(PSPT(SEQ ID NO:104))_d，d＝2-5；

f.(P[S/T/R/K/D/E/N/Q]P[S/T/R/K/D/E](SEQ ID NO:102))_e，e＝2-5；

g.([S/T/R/K/D/E]P)_ff is 2 to 10, preferably 2 to 5;

h.([S/T/R/K/D/E/N/Q]P[S/T/R/K/D/E])_g，g＝2-6；

i.([S/T/R/K/D/E/N/Q][S/T/R/K/D/E/N/Q]P)_h，h＝2-5；

j.(TP)_i，i＝2-10；

k.([S/T/P][S/T/P][S/T/P])_j，j＝2-11；

and/or combinations thereof, wherein combinations of individual monomer units are contemplated.

The variant according to any of the preceding claims, wherein the linker comprises:

a.(SP)_a，a＝2-10；

b.(PS)_a，a＝2-10；

c.P_bb is 4-20, preferably 4-15; or

d.(PEPT(SEQ ID NO:125))_c，c＝2-5。

Paragraph 21, the variant according to any of the preceding paragraphs, wherein the linker has a length of at least 4 amino acids and not more than 30 amino acids, such as 4-28 amino acids, preferably 4-20 amino acids, or even 4-10 amino acids, such as 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids or 10 amino acids.

Paragraph 22. the variant according to any of the preceding paragraphs, wherein the linker comprises SPSP (SEQ ID NO:130), SPSPSP (SEQ ID NO:131), SPSPSPSP (SEQ ID NO:132), SPSPSPSPSP (SEQ ID NO:58), SPSPSPSPSPSP (SEQ ID NO:133), SPSPSPSPSPSPSP (SEQ ID NO:134), SPSPSPSPSPSPSPSP (SEQ ID NO:135), PPPP (SEQ ID NO:27), PPPPP (SEQ ID NO:28), PPPPPP (SEQ ID NO:29), PPPPPPP (SEQ ID NO:31), PPPPPPPP (SEQ ID NO:136), PPPPPPPPP (SEQ ID NO:137), PPPPPPPPPP (SEQ ID NO:138), PPPPPPPPPPP (SEQ ID NO:139), PPPPPPPPPPPP (SEQ ID NO:140), PPPPPPPPPPPPP (SEQ ID NO:141), PPPPPPPPPPPPPP (SEQ ID NO:142), PPPPPPPPPPPPPPP (SEQ ID NO:143), TPEPT (SEQ ID NO:144), PEPTPEPTPEPT (SEQ ID NO:145), PEPTPEPTPEPTPEPT (SEQ ID NO:146), PEPTPEPTPEPTPEPTPEPT (SEQ ID NO:79), PSPTPSPT (SEQ ID NO:147), PSPTPSPTPSPT (SEQ ID NO:148), PSPTPSPTPSPTPSPT (SEQ ID NO:149), PSPTPSPTPSPTPSPTPSPT (SEQ ID NO:150), SPSSPS (SEQ ID NO:151), SPSSPSSPS (SEQ ID NO:152), SPSSPSSPSSPS (SEQ ID NO:153), SPSSPSSPSSPSSPS (SEQ ID NO:154), TPTTPT (SEQ ID NO:155), TPTTPTG (SEQ ID NO:96), TPTTPTTPT (SEQ ID NO:156), TPTTPTTPTTPT (SEQ ID NO:157), TPTTPTTPTTPTTPT (SEQ ID NO:158), PEPTPRPTPEPTPRPT (SEQ ID NO:159), PEPTPKPTPEPTPKPT (SEQ ID NO:160), PEPTPQPTPEPTPQPT (SEQ ID NO:161), PRPTPEPTPRPT (SEQ ID NO:162), PKPTPEPTPKPT (SEQ ID NO:163), TPQPT (SEQ ID NO:164), PEPTPQPTPEPT (SEQ ID NO:165), PEPTPRPTPEPTPRPTG (SEQ ID NO: 68685), SEQ ID NO: PEPTPKPTPEPTPKPTG (PEP NO:87), PEPTPQPTPEPTPQPTG (SEQ ID NO:88), PRPTPEPTPRPTG (SEQ ID NO:89), PKPTPEPTPKPTG (SEQ ID NO:90), PEPTPQPTG (SEQ ID NO:91), PEPTPQPTPEPTG (SEQ ID NO:92), PPPGGPGGPGTPTSTAPGSGPTSPGGGSG (SEQ ID NO: 82).

Paragraph 23. the variant according to any of the preceding paragraphs, wherein the linker further comprises a glycine at the C-terminal position.

Paragraph 24. the variant according to any of the preceding paragraphs, wherein the linker is PPPPP (SEQ ID NO:31), PPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO: 25).

Paragraph 25. the variant according to any of the preceding paragraphs, wherein the linker is PPPPP (SEQ ID NO:31), PPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is CBM 1.

Paragraph 26. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g. at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, to the amino acid sequence as depicted at positions 1-212 of SEQ ID No. 1, positions 1-211 of SEQ ID No. 2, positions 1-210 of SEQ ID No. 3, positions 1-211 of SEQ ID No. 4. Paragraph 27.a variant according to any of the preceding paragraphs, wherein the CBM comprises a variant of any of SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 180, SEQ ID NO 181, SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 184, SEQ ID NO 185, SEQ ID NO 186, SEQ ID NO 187, SEQ ID NO 188, SEQ ID NO 189, SEQ ID NO 190, SEQ ID NO 191, SEQ ID NO 192, SEQ ID NO 193, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, SEQ ID NO 197, 198, 199, 200, such as, for example, an amino acid sequence having at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity.

Paragraph 28. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is CBM 1.

Paragraph 29. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 6.

Paragraph 30. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 7.

Paragraph 31. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 8.

Paragraph 32. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 9.

Paragraph 33. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 173.

Paragraph 34. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 174.

Paragraph 35. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 175.

Paragraph 36. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 176.

Paragraph 37. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 177.

Paragraph 38. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 178.

Paragraph 39. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 179.

Paragraph 40. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 5, the linker is PPPPPPP (SEQ ID NO:31), PPPPPPP (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 180.

Paragraph 41. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 181.

Paragraph 42. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 182.

Paragraph 43. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 183.

Paragraph 44. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 184.

Paragraph 45. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 5, the linker is PPPPPPP (SEQ ID NO:31), PPPPPPP (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 185.

Paragraph 46. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 186.

Paragraph 47, the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 187.

Paragraph 48. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 188.

Paragraph 49. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 5, the linker is PPPPPPP (SEQ ID NO:31), PPPPPPP (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 189.

Paragraph 50. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 5, the linker is PPPPPPP (SEQ ID NO:31), PPPPPPP (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 190.

Paragraph 51. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 5, the linker is PPPPPPP (SEQ ID NO:31), PPPPPPP (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 191.

Paragraph 52. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 192.

Paragraph 53. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 193.

Paragraph 54. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 194.

Paragraph 55, the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 195.

Paragraph 56. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 5, the linker is PPPPPPP (SEQ ID NO:31), PPPPPPP (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 196.

Paragraph 57. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 5, the linker is PPPPPPP (SEQ ID NO:31), PPPPPPP (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 197.

Paragraph 58. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 198.

Paragraph 59. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 5, the linker is PPPPPPP (SEQ ID NO:31), PPPPPPP (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID NO: 199.

Paragraph 60. the variant according to any of the preceding paragraphs, wherein the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5, the linker is ppppppppppp (SEQ ID NO:31), PPPPPPPG (SEQ ID NO:30), SPSPSPSPSP (SEQ ID NO:58) or SPSPSPSPSPG (SEQ ID NO:25) and the CBM is SEQ ID No. 200.

Paragraph 61. the variant according to any of the preceding paragraphs, further comprising a substitution selected from the group consisting of:

Q147R+Q156E；

Q147R+Q169Y；

S56A+Q147R；

Q147R+A162E；

Q147R+Q156E+A162E；

A25G+S56A+Q147R；

N134D+Q156E+A162E；

S56A +N134D+Q156E+A162E；

A25G+S56A+Q156E+A162E；

A25G+N134D+Q156E+A162E；

A25G+S56A+N134D+Q169Y；

S56A+N134D+A162E；

S56A+Q147R+Q169Y；

N134D+Q147R；

Q156E+Q169Y；

S56A+N134D+Q147R；

S56A+N134D+Q156E+Q169Y；

S56A+A146D+Q147R+Q169Y；

S56A+N134D+Q147R+Q169Y；

S56A+Q147R+A162E+Q169Y；

S2*+S56A+Q147R+Q169Y；

S41T+S56A+Q147R+Q169Y；

S56A+S77N+Q147R+Q169Y；

S56A+T104K+Q147R+Q169Y；

S56A+Q147R+K165Q+Q169Y；

S56A+Q147R+Q169Y+I194L；

S56A+Q147R+Q169Y+K201R；

S56A+Q147R+Q169Y+G219W；

N44D+S56A+Q147R+Q169Y；

N50E+S56A+Q147R+Q169Y；

A32S+S56A+Q147R+Q169Y；

N44D+S56A+Q147R+Q169Y；

S56A+Q147R+Q169Y+Q186R；

S56A+Q147R+Q169Y+F183V；

S56A+A146S+Q147R+A162E+Q169Y；

S56A+N134D+Q147R；

S56A+N134D+Q147R+A162E；

A32S+S56A+N134D+Q147R+Q169Y+F183V；

S56A+N134D+Q147R+A162E+Q169Y+F183V；

A32S+S56A+S77N+N134D+Q147R+A162E+Q169Y；

A32S+S56A+N134D+A146D+Q147R+Q169Y+F183V；

A32S+S56A+N134D+Q147R+Q169Y；

S56A+N134D+Q147R+A162E+Q169Y；

A32S+S56A+N134D+A146S+Q147R+Q169Y；

A32S+S56A +N134D+A146D+Q147R+Q169Y；

A32S+S56A +N134D+Q147R+Q169Y+F183V；

A32S+S56A +N134D+Q147R+Q169Y+K201R；

S56A+N134D+A146D+Q147R+Q169Y+F183V；

S56A+N134D+A146D+Q147R+A162E+Q169Y；

S56A+N134D+A146D+Q147R+Q169Y+K201R；

S56A+N134D+Q147R+A162E+Q169Y+F183V；

S56A+N134D+Q147R+Q169Y+F183V+K201R；

A32S+S56A+S77N+N134D+Q147R+Q169Y+F183V；

A32S+S56A+S77N+N134D+Q147R+A162E+Q169Y；

A32S+S56A+N134D+A146S+Q147R+Q169Y+F183V；

a32S + S56A + N134D + a146D + Q147R + Q169Y + F183V; or

A32S+S56A+N134D+A146D+Q147R+A162E+Q169Y。

Paragraph 62. the variant according to any of the preceding paragraphs, wherein the linker is as shown in table a.

Paragraph 63. the variant according to any of the preceding paragraphs, wherein the variant is as shown in table B1, table B2, table C1, table C2, table D.

Paragraph 64 an isolated polynucleotide encoding a variant according to any one of paragraphs 1-63.

Paragraph 65. a nucleic acid construct comprising the polynucleotide according to paragraph 64.

Paragraph 66. an expression vector comprising the polynucleotide according to paragraph 64.

Paragraph 67. a host cell comprising the polynucleotide of paragraph 64.

Paragraph 68. A method of producing a variant having glycoside hydrolase (EC 3.2.1.-), cellulase or endoglucanase activity, the method comprising

a. Culturing the host cell according to paragraph 67 under conditions suitable for expression of the variant; and

b. recovering the variant.

Paragraph 69. a method for obtaining a variant having glycoside hydrolase (EC 3.2.1.-), cellulase or endoglucanase activity, the method comprising introducing a proline-rich linker region into a parent glycoside hydrolase; and recovering the variant.

Paragraph 70. a whole broth formulation or cell culture composition comprising the variant according to any of paragraphs 1-63.

Paragraph 71 a composition comprising a variant according to any of paragraphs 1-63.

Paragraph 72 the composition according to paragraph 71, further comprising a protease.

Paragraph 73. the composition according to any one of paragraphs 71-72, further comprising one or more additional enzymes selected from the group consisting of: (further) protease, lipase, cutinase, amylase, (further) carbohydrase, (further) cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, nuclease, lichenase, oxidase, e.g., laccase, and/or peroxidase, and combinations thereof.

Paragraph 74. the composition according to any of paragraphs 71-73, further comprising an amylase.

Paragraph 75. the composition according to any one of paragraphs 71-74, further comprising another carbohydrase.

Paragraph 76. the composition according to any one of paragraphs 71-75, further comprising lichenase.

Paragraph 77. the composition according to any of paragraphs 71-76, which is a detergent composition.

Paragraph 78. the composition according to any one of paragraphs 71-77, further comprising one or more compounds selected from surfactants, builders and co-builders, and polymers.

Paragraph 79. the composition according to any of paragraphs 71-78, which is a liquid detergent composition.

Paragraph 80. use of the variant according to any of paragraphs 1-63 for cleaning a fabric, textile or hard surface.

Paragraph 81. use of the variant according to any of paragraphs 1-63 or the composition according to any of paragraphs 71-78 for fabric or textile care, for example for pretreating stained fabrics or for rejuvenating textiles (e.g. by removing fuzz or pills) to restore the visual and sensory properties of the fabric after prolonged use to match new textiles.

Paragraph 82. the use according to paragraphs 80-81, comprising the use of a variant according to any of paragraphs 1-63 or a composition according to any of paragraphs 71-79 for laundering.

Paragraph 83. the use according to paragraph 82, comprising the use of a variant according to any of paragraphs 1-63 or a composition according to any of paragraphs 71-79 as a rinse added fabric softener composition.

Paragraph 84. a method for reducing or preventing soil redeposition comprising contacting a polypeptide or composition or detergent composition according to any of the preceding paragraphs.

Paragraph 85. a method for fabric or textile care using a polypeptide or composition or detergent composition according to any of the preceding paragraphs.

Paragraph 86. A method for laundering an object such as a fabric or textile, the method comprising

(a) Providing a wash liquor by dissolving/mixing the variant according to any of paragraphs 1-63 or the composition according to any of paragraphs 71-79 in water;

(b) washing the object in the washing liquid;

(d) the object is rinsed and optionally dried.

Paragraph 87. A method for laundering an object such as a fabric or textile, the method comprising

(a) Providing water and rinsing the object;

(b) optionally, draining the water and providing fresh water;

(c) dosing a variant according to any of paragraphs 1-63 or a composition according to any of paragraphs 71-79 to form a wash liquor;

(d) agitating the washing liquid, thereby washing the object, optionally heating the washing liquid; and

(e) the washing solution was drained off.

Examples of the invention

Materials and methods

General methods of PCR, cloning, linking nucleotides, etc. are well known to those of ordinary skill in the art and may be found, for example, in "Molecular cloning: A laboratory manual [ Molecular cloning: a laboratory Manual ] ", Sambrook et al (1989), Cold Spring Harbor lab, [ Cold Spring Harbor laboratory ], Cold Spring Harbor, N.Y. (Cold Spring Harbor, NY); ausubel, f.m. et al (editors); "Current protocols in Molecular Biology [ Molecular Biology Experimental guidelines ]", John Wiley and Sons [ John Willi-father, Inc ], (1995); harwood, c.r. and Cutting, S.M. (editors); "DNA Cloning: APractcal application [ DNA Cloning: methods of use ], volumes I and II ", d.n. glover editions (1985); "Oligonucleotide Synthesis", edited by m.j.gait (1984); "Nucleic Acid Hybridization," edited by B.D.Hames and S.J.Higgins (1985); "adaptive Guide To Molecular Cloning [ Molecular Cloning Guide for practical use ]", B.Perbal, (1984).

Determination of cellulolytic Activity

The cellulolytic activity was determined using the cellulase assay kit (CellG5 method) supplied by Megazyme (Megazyme) following the manufacturer's instructions (Wicklow, Ireland); product code: K-CellG 5-4V).

The reagent for the CellG5 assay for measuring endo-cellulase (endo-1, 4-. beta. -glucanase) contained two components;

1)4,6-O- (3-ketobutylidene) -4-nitrophenyl-beta-D-fibropentaglycoside (BPNPG5) and 2) thermostable beta-glucosidase. The ketone blocking group prevents any hydrolysis by the beta-glucosidase on BPNPG 5. Incubation with endo-cellulase produces a non-blocking colorimetric oligosaccharide that is rapidly hydrolyzed by the helper β -glucosidase. Thus, the rate of formation of 4-nitrophenol is directly related to the endocellulase hydrolysis of BPNPG 5.

Composition (liquid) of Standard detergent A

Composition of detergent a (liquid): the components: 12% LAS, 11% AEO Biosoft N25-7(NI), 7% AEOS (SLES), 6% MPG (propylene glycol), 3% ethanol, 3% TEA, 2.75% cocoa soap, 2.75% soy soap, 2% glycerol, 2% sodium hydroxide, 2% sodium citrate, 1% sodium formate, 0.2% DTMPA and 0.2% PCA (all percentages are w/w).

Protease enzyme

The protease used in the examples is the protease of SEQ ID NO 10. Other proteases include those of SEQ ID NO 11 or of SEQ ID NO 11 with the mutations S9E + N42R + N74D + V199I + Q200L + Y203W + S253D + N255W + L256E.

Washing assay

Launder-O-Meter (LOM) mode washing system

Launder-O-meter (lom) is a medium scale standard washing system that can be applied to simultaneously test up to 20 different washing conditions. The LOM is basically a large temperature controlled water bath with 20 closed metal beakers rotating therein. Each beaker constitutes a small washing machine and during an experiment each beaker will contain the solution of the particular detergent/enzyme system to be tested together with the soiled and unsoiled fabrics on which the test is carried out. The mechanical stress is achieved by a beaker rotating in a water bath and by a metal ball included in the beaker.

The LOM standard wash system is used primarily for medium scale testing of detergents and enzymes under european wash conditions. In the LOM experiment, factors such as the ratio of ballast to soil and the ratio of fabric to wash liquor can be varied. Thus, LOM provides a link between small scale experiments (such as AMSA and mini-wash) and more time consuming full scale experiments in front loading washing machines.

Mini laundry-O-Meter (Mini LOM) Standard washing System

Mini-LOM is a modified mini-wash system of Launder-O-Meter (LOM), which is a medium-scale standard wash system that can be applied to simultaneously test up to 20 different wash conditions. The LOM is basically a large temperature controlled water bath with 20 closed metal beakers rotating therein. Each beaker constitutes a small washing machine and during an experiment each beaker will contain the solution of the particular detergent/enzyme system to be tested together with the soiled and unsoiled fabrics on which the test is carried out. The mechanical stress is achieved by a beaker rotating in a water bath and by a metal ball included in the beaker.

In mini-LOM, washing is performed in 50ml tubes placed in a stewart (Stuart) rotator.

Terg-O-Tometer (TOM) Wash assay

Terg-O-meter (TOM) is a medium scale standard washing system that can be applied to test 12 different washing conditions simultaneously. TOM is basically a large temperature controlled water bath in which up to 12 open metal beakers are immersed. Each beaker constitutes a small top-loading washing machine and during the experiment, each beaker will contain a solution of the specific detergent/enzyme system and test its performance on soiled and unsoiled fabrics. Mechanical stress is obtained by rotating stirring arms that stir the liquid in each beaker. Because the TOM beaker has no lid, it is possible to retrieve the sample during the TOM experiment and analyze the information online during the wash.

The TOM standard wash system is primarily used for medium scale testing of detergents and enzymes under US or LA/AP wash conditions. In TOM experiments, factors such as the ratio of ballast to soil and the ratio of fabric to wash liquor can vary. Thus, TOM provides a link between small scale experiments and more time consuming full scale experiments in top loading washing machines.

Example 1: determination of stability of cellulase variants (core stability method)

The stability of the cellulase variants was measured in 90% liquid detergent a containing protease. After incubation of the protease-containing enzyme-detergent mixture, the activity of the variants was measured by CellG5 kit to assess stability in detergent.

Temperature/protease stress conditions in 90% detergent a:

in a 96-well microplate (polystyrene), 20. mu.L of 1000ppm purified endo-cellulase diluted in buffer (100mM HEPES; 0.01% Tween-20; pH 7.5) was mixed with 180. mu.L of detergent A containing 0.3mg/mL of active enzyme protease protein.

15 μ L of enzyme/detergent mixture was transferred to two new 384 well microplates and sealed. One of the two identical plates was stored at 5 ℃ (reference) and the other was incubated at high temperature (stress) for 16 or 17 hours. See the stress-temperature results table used. After incubation, 60 μ L of assay buffer (100mM HEPES; 0.01% Tween-20; pH 7.5) was added to the samples of both plates and mixed vigorously for subsequent activity measurements.

Measurement sample of cellulolytic Activity (CellG5 kit):

enzyme activity was measured by mixing 20 μ L of the diluted enzyme-detergent mixture with 10 μ L of assay buffer (100mM HEPES; 0.01% tween-20; pH 7.5) and 10 μ L of freshly prepared substrate solution in a UV-transparent 384-well microplate. The substrate solution of the CellG5 assay kit was prepared by mixing 10. mu.L of bottle #2 with 300. mu.L of bottle # 1.

UV-absorbance (405nm) was measured dynamically (every 2 minutes for 44 minutes) using a microplate reader (Tecan; Infinite M1000, professional). The portion of the curve that shows a constant increase in absorbance is used to calculate the enzyme activity (mOD/min) of the sample. The residual activity was then calculated as the enzyme activity of the samples incubated at high temperature for 16 or 17 hours relative to the enzyme activity of the corresponding samples stored at 5 ℃.

Residual activity (%) - (activity of sample incubated at high temperature/activity of sample incubated at 5 ℃) × 100

Example 2: determination of Joint stability

Principle of

The joint stability was measured by: (A) incubating cellulase in a detergent comprising a protease, and then (B) determining the ability of the incubated cellulase to bind to cellulose fibers. If the linker or cellulose binding domain is affected by a protease, the binding affinity of the cellulase to the cellulose fiber will be reduced.

Binding was determined by adding a dilution of incubated cellulase to a suspension of cellulose fibers. After incubation at 5 ℃, cellulose-bound cellulase was removed by centrifugation and the amount of cellulose-unbound cellulase was determined by: measuring the cellulase activity in the supernatant of (C). The activity of cellulase not bound to cellulose relative to the activity of a parallel sample incubated under similar conditions but in the absence of cellulase is a measure of linker stability.

This activity is based on the hydrolysis of soluble carboxymethylcellulose (CMC) and subsequent (D) detection of the number of reducing ends formed. The substrates for both intact cellulases and cellulases without a cellulose binding domain are CMC.

A.In detergents containing proteaseIn (1) performing incubation

Chemical product

A detergent: standard detergent A

Protease: SEQ ID NO 10

HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, Sigma H3375

Reagent

Dilution buffer: 50mM HEPES, pH 8

Protease stock, e.g., protease of SEQ ID NO 10

Detergent with 0.3mg/mL protease: 300ppm in Standard detergent A

Standard detergent A as above

Procedure

1) Detergents with 0.3mg/mL protease were prepared by: the protease stock was added to 100mL of detergent to a final protease concentration of 300ppm active protease protein in the detergent and mixed by magnetic stirring at room temperature for 1 hour.

2) Cellulase was diluted to 300ppm in dilution buffer.

3) Pipet 270 μ L of the detergent with protease from (1) to a 96-well polypropylene microplate (Thermo Scientific)^TM249944) in the hole positions a1 to D12.

4) 30 μ L of diluted cellulase from (2) was added to each well (positions a1 to D12). Each cellulase was tested in triplicate and positions D4 to D6 were used as blank controls to which 30 μ L Milli Q water was added instead of cellulase.

5) A few small magnets were added to each well (positions A1 to D12) and the plate was heat sealed (Thermo Scientific)^TMViscous PCR plate seal AB0558) and then mixed by magnetic stirring for 30 minutes.

6) After mixing, the plates were incubated for the times and temperatures shown in the examples.

B.Bound to cellulosic fibres

Chemical product

Cellulose fiber:PH-101 (Sigma 11365)

HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, Sigma H3375

Reagent:

binding buffer: 50mM HEPES, pH 8

Avicel suspension: 1.25g/100mL Avicel in binding buffer, mix for 1 hour before use

The procedure is as follows:

1) add 180. mu.L of Avicel suspension to a new 96-well microplate (Thermo Scientific)^TMDirectory number 269620) location a1->D12 and 180. mu.L of binding buffer was added to position E1->In H12

2) A 20 μ Ι _ aliquot of sample from each well in the incubated plate of step a was then added to the wells at positions a1- > D12 and E1- > H12, respectively.

3) The board was shaken at 5 ℃ for 1 hour at a speed sufficient to keep the cellulose fibers in suspension to allow the cellulase to bind to the cellulose

4) After binding, plates were centrifuged at 1500rpm for 10 seconds and supernatants were diluted 2.5 fold in binding buffer (40 μ L sample +60 μ L buffer). Both the supernatant from the Avicel well and the supernatant from the corresponding Avicel-free well were diluted.

C.CMC Activity assay

Chemical products:

CMC: sodium carboxymethylcellulose (Sigma C5678)

K-Na-tartaric acid: merck 8087

Beta-glucosidase, Megazyme (Thermotoga maritima; accession No. Q08638, catalogue No. E-BGOSTM), diluted to 0.1mg/mL (specific activity 70U/mg and activity in product about 460U/mL- >6,57mg/mL)

PAHBAH 4-hydroxybenzoyl hydrazine (Sigma H9882)

NaOH sodium hydroxide (J.T.Baker 0402.1000)

HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, Sigma H3375

Reagent:

determination of buffer: 50mM HEPES, pH 8

CMC substrate: 1.25g CMC/100mL assay buffer, mixed for 1 hour prior to use

PAHBAH buffer: 50g/L K-Na-tartaric acid +20g/L NaOH

PAHBAH reagent: 15mg/mL PAHBAH in PAHBAH buffer

Beta-glucosidase solution: 0.1mg/mL beta-glucosidase in assay buffer

The procedure is as follows:

1) pipet 160. mu.L CMC substrate to a new 96-well microplate (Thermo Scientific)^TMCatalog number 269620)

2) mu.L of the diluted supernatant from step B was added with 20. mu.L of beta-glucosidase solution

3) The plates were heat sealed (Thermo Scientific)^TMViscous PCR plate seal AB0558) and incubated at 40 ℃ for 45 minutes

4) After the reaction, 100. mu.L from each well was transferred to ThermoFast 96PCR plate (Thermo Scientific)^TMCatalog number AB-0600), followed by the addition of 75. mu.L of PAHBAH reagent

5) The plates from (4) were then sealed with a sealing foil (Greiner bio-one plate seal, catalog number 676001) and mounted on a BioRad T100^TMIncubation in a thermal cycler PCR machine at 95 ℃ for 10 minutes followed by cooling at 10 ℃ for 5 minutes

6) After cooling, 100 μ L aliquots were transferred to new 96-well microplates (Thermo Scientific)^TMCatalog number 269620) and read absorbance at 405nm (A)_405nm)。The absorbance is an indication of the cellulase activity in the supernatant.

D.Data processing

1) From the absorbance readings of step C, the average A of the 3 blanks from wells without Avicel was calculated_405nm(blank _ reference) (position H4- >H6) And the average a of 3 blanks from wells with Avicel was calculated_405nm(blank _ Avicel) (position D4->D6)。

2) The absorbance readings from the cellulase-containing wells were then corrected for their respective blanks (i.e., those from (1)).

3) Calculating the joint stability as

1- [ Activity_405nm(+ Avicel)/Activity_405nm(-Avicel)]，

Wherein the activity is_405nm(+ Avicel) and Activity_405nm(-Avicel) is the activity in wells with supernatant incubated with and without Avicel (i.e. absorbance corrected for blank), respectively.

4) Linker stability reported in the examples is the average of triplicates analyzed.

This assay clearly distinguishes binding in the presence and absence of the core, as further demonstrated in example 3 below.

Example 3: cellulose binding assay-protease free

Principle of

The cellulase (a) was allowed to bind cellulose by incubation with Avicel in a dilute detergent solution for 60 minutes at 5 ℃. After incubation, the activity of cellulase not bound to cellulose is determined in supernatant (B) and compared to a parallel cellulase sample incubated in the absence of cellulase. The temperature during incubation with Avicel was kept low to ensure that the catalytic activity of the cellulase during the binding step did not have a significant effect on the binding assay.

A.Bound to cellulose

Chemical product

A detergent: standard detergent A

HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, Sigma H3375

Cellulose fiber:PH-101 (Sigma 11365)

Reagent

Binding buffer: 50mM HEPES, pH 8

Avicel suspension: 1.25g/100mL Avicel in binding buffer, mix for 1 hour before use

Standard detergent A as above

Procedure

1) Cellulase was diluted to 300ppm in binding buffer.

2) Pipet 270 μ L of detergent to a 96-well polypropylene microplate (Thermo Scientific)^TM249944) in the hole positions a1 to D12.

3) 30 μ L of diluted cellulase from (1) was added to each well (positions a1 to D12). Each cellulase was tested in triplicate and positions D4 to D6 were used as blank controls to which 30 μ L Milli Q water was added instead of cellulase.

4) A few small magnets were added to each well (positions a1 to D12) and the plate was sealed with a heat seal (Thermo Scientific adhesive PCR plate seal AB0558), followed by mixing by magnetic stirring for 30 minutes.

5) 160. mu.L of binding buffer was pipetted into a new 96-well microplate (Thermo Scientific)^TM Nunc^TM96-well polypropylene deep well^TMStock plates (positions a1 to D12)) and 160 μ Ι of Avicel suspension were pipetted to positions E1 to H12.

6) mu.L Milli Q water was added to all wells (A1-H12)

7) Aliquots of 20 μ L cellulase-detergent samples from (4) were added to wells with Avicel (a1 to D12) and without (E1 to H12).

8) The plates were then incubated in a 5 ℃ cold room on a Haidaofu (Heidolph) Titramax 101 shaker for 60 minutes to allow binding of cellulase to cellulose. The oscillation speed was adjusted to ensure that the cellulose remained suspended during the incubation.

9) After binding, plates were centrifuged at 1500rpm for 10 seconds and supernatants were diluted 2.5 fold in binding buffer (40 μ L sample +60 μ L buffer). Both the supernatant from the Avicel well and the supernatant from the corresponding Avicel-free well were diluted.

B.CMC Activity assay

Chemical products:

CMC: sodium carboxymethylcellulose (Sigma C5678)

K-Na-tartaric acid: merck 8087

Beta-glucosidase, Megazyme (Thermotoga maritima; accession No. Q08638, catalogue No. E-BGOSTM), diluted to 0.1mg/mL (specific activity 70U/mg and activity in product about 460U/mL- >6,57mg/mL)

PAHBAH 4-hydroxybenzoyl hydrazine (Sigma H9882)

NaOH sodium hydroxide (J.T.Baker 0402.1000)

HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, Sigma H3375

Reagent:

determination of buffer: 50mM HEPES, pH 8

CMC substrate: 1.25g CMC/100mL assay buffer, mixed for 1 hour prior to use

PAHBAH buffer: 50g/L K-Na-tartaric acid +20g/L NaOH

PAHBAH reagent: 15mg/mL PAHBAH in PAHBAH buffer

Beta-glucosidase solution: 0.1mg/mL beta-glucosidase in assay buffer

The procedure is as follows:

1) pipetting 160. mu.L of CMC substrate into a new 96-well microplate (catalog number 269620, Seimer science Co., Ltd.)

2) Add 20. mu.L of diluted supernatant from step A along with 20. mu.L of beta-glucosidase solution

3) The plates were sealed using heat sealing (Seimer science technologies adhesive PCR plate seal AB0558) and incubated at 40 ℃ for 45 minutes

4) After the reaction, 100. mu.L from each well was transferred to ThermoFast 96PCR plate (catalog number AB-0600 of Saimer science) followed by addition of 75. mu.L of PAHBAH reagent

6) After cooling, 100 μ L aliquots were transferred to new 96-well microplates (catalog number 269620, Seimer science) and the absorbance read at 405nm (A) _405nm). The absorbance is an indication of the cellulase activity in the supernatant.

C.Data processing

1) From the absorbance readings of step B, the average A of 3 blanks from wells without Avicel was calculated_405nm(blank _ reference) (position H4->H6) And the average a of 3 blanks from wells with Avicel was calculated_405nm(blank _ Avicel) (position D4->D6)。

2) The absorbance readings from the cellulase-containing wells were then corrected for their respective blanks (i.e., those from (1)).

3) Calculating the combination as

1- [ Activity_405nm(+ Avicel)/Activity_405nm(-Avicel)]，

Wherein the activity is_405nm(+ Avicel) and Activity_405nm(-Avicel) is the activity in wells with supernatant incubated with and without Avicel (i.e. absorbance corrected for blank), respectively.

Linker stability reported in the examples is the average of triplicates analyzed.

To demonstrate this, cellulase samples with and without CBM were tested for binding to cellulose as described in this example. The ratio was calculated as the binding of the whole cellulase (i.e. the cellulase with the catalytic domain, linker and CBM) compared to the binding of the cellulase with the catalytic domain only.

Sequence length reflecting catalytic domains, as annotated by bioinformatic processing

This data clearly demonstrates that in the absence of CBM, binding to cellulose is significantly reduced.

Example 4: construction of variants

The cellulase variant was constructed from an Escholtzia californica cellulase (SEQ ID NO: 1). These variants were prepared by conventional Cloning of DNA fragments using PCR together with correctly designed oligonucleotides which introduce the desired mutations in the resulting sequences (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor, 1989). Alternatively, the native DNA sequence is replaced with a new desired DNA sequence using a synthetic gene fragment purchased from a supplier, such as integrated DNA technology corporation (IDTDNA).

Oligonucleotides are designed corresponding to the DNA sequence flanking the desired mutation site or mutations or DNA segment to be replaced, separated by DNA base pairs defining the insertion/deletion/substitution/synthesis DNA sequence, and purchased from suppliers of oligonucleotides, such as IDTDNA. To test the variants of the invention, mutant DNA comprising the variants of the invention was integrated by homologous recombination into competent aspergillus oryzae strains, fermented using standard protocols (yeast extract based medium, 4-5 days, 30 ℃) and purified as follows.

The culture broth was filtered through a Nalgene 0.2 μm filtration unit to remove the aspergillus host cells. Using 20% CH₃COOH, adjusting the pH of the filtrate to pH 4.0 and applying the pH adjusted filtrate to 20mM CH₃COOH/NaOH、1mM CaCl₂Equilibrated Capto MMC column (from GE Healthcare) in pH 4.0. After the column was washed well with equilibration buffer, the cellulase was washed with 50mM Tris base, 1mM CaCl₂(unbuffered) elution. Fractions from the column were analyzed for cellulase activity. Cellulase peaks were pooled and applied to a Q-sepharose FF column (from the general electro-medical group) equilibrated in 50mM Tris/HCl, pH 9.0. After the column was washed well with the equilibration buffer, cellulase was applied to the equilibration buffer with 50mM Tris/HCl, 5mM CaCl₂A linear NaCl gradient between 500mM NaCl, pH 9.0 eluted through three column volumes. Fractions from the column were analyzed for cellulase activity and cellulase peaks were combined into a purified product. The purified variants were analyzed by SDS-PAGE. Since the cellulase variants are glycosylated, they give discrete bands on coomassie stained SDS-PAGE gels. The purified product was used for further characterization.

Example 5: variant stability

Variants were prepared as described in example 4. Stability was determined using the assay described in example 2 (linker stability assay-in the presence of protease), where the stress conditions were incubation with protease (SEQ ID NO:10) at 20 ℃ for 21 hours, followed by analysis of residual activity. The results are shown in Table 1.

TABLE 1 linker stability after 21 hours incubation at 20 ℃

Example 6: variant stability

Variants were prepared as described in example 4. Stability was determined using the assay described in example 2, where the stress condition was incubation with protease (SEQ ID NO:10) at 37 ℃ for 21 hours, and then residual activity was analyzed. The results are shown in Table 2.

TABLE 2 linker stability after incubation at 37 ℃ for 21 hours

Example 7: in-wash linker stability assay using protease

Principle of

The joint stability was measured by: (A) incubating cellulase in a detergent wash solution comprising a protease, and then (B) determining the ability of the incubated cellulase to bind to cellulose fibers. If the linker or cellulose binding domain is affected by a protease, the binding affinity of the cellulase to the cellulose fiber will be reduced.

E.Incubation in detergents with protease

Chemical product

A detergent: standard detergent A

Protease: protease having SEQ ID NO 10

HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, Sigma H3375

Reagent

Dilution buffer: 50mM HEPES, pH 8

Detergents with protease: 0.3. mu.g/mL active protease protein in Standard detergent A

Detergent washing solution: 3.3g/L detergent with protease in water with 15 ℃ dH water hardness.

Procedure

7) Preparation of detergent washing solutions

8) Cellulase was diluted to 300ppm in dilution buffer.

9) Transfer 270. mu.L of the detergent wash solution from (1) to a 96-well polypropylene microplate (Thermo Scientific)^TM249944) in the hole positions a1 to D12.

10) 30 μ L of diluted cellulase from (2) was added to each well (positions a1 to D12). Each cellulase was tested in triplicate and positions D4 to D6 were used as blank controls to which 30 μ L Milli Q water was added instead of cellulase.

11) A few small magnets were added to each well (positions A1 to D12) and the plate was heat sealed (Thermo Scientific)^TMViscous PCR plate seal AB0558) and then mixed by magnetic stirring for 30 minutes.

12) After mixing, the plates were incubated for the times and temperatures shown in the examples (e.g., at 40 ℃, 2 hours).

F.Bound to cellulosic fibres

Chemical product

Cellulose fiber:PH-101 (Sigma 11365)

HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, Sigma H3375

Reagent:

binding buffer: 50mM HEPES, pH 8

Avicel suspension: 1.25g/100mL Avicel in binding buffer, mix for 1 hour before use

The procedure is as follows:

5) add 180. mu.L of Avicel suspension to a new 96-well microplate (Thermo Scientific)^TMDirectory number 269620) location a1->D12 and 180. mu.L of binding buffer was added to position E1->In H12

6) A 20 μ Ι _ aliquot of sample from each well in the incubated plate of step a was then added to the wells at positions a1- > D12 and E1- > H12, respectively.

7) The board was shaken at 5 ℃ for 1 hour at a speed sufficient to keep the cellulose fibers in suspension to allow the cellulase to bind to the cellulose

8) After binding, plates were centrifuged at 1500rpm for 10 seconds and supernatants were diluted 2.5 fold in binding buffer (40 μ L sample +60 μ L buffer). Both the supernatant from the Avicel well and the supernatant from the corresponding Avicel-free well were diluted.

G.CMC Activity assay

Chemical products:

CMC: sodium carboxymethylcellulose (Sigma C5678)

K-Na-tartaric acid: merck 8087

Beta-glucosidase, Megazyme (Thermotoga maritima; accession No. Q08638, catalogue No. E-BGOSTM), diluted to 0.1mg/mL (specific activity 70U/mg and activity in product about 460U/mL- >6,57mg/mL)

PAHBAH 4-hydroxybenzoyl hydrazine (Sigma H9882)

NaOH sodium hydroxide (J.T.Baker 0402.1000)

HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, Sigma H3375

Reagent:

determination of buffer: 50mM HEPES, pH 8

CMC substrate: 1.25g CMC/100mL assay buffer, mixed for 1 hour prior to use

PAHBAH buffer: 50g/L K-Na-tartaric acid +20g/L NaOH

PAHBAH reagent: 15mg/mL PAHBAH in PAHBAH buffer

Beta-glucosidase solution: 0.1mg/mL beta-glucosidase in assay buffer

The procedure is as follows:

7) pipet 160. mu.L CMC substrate to a new 96-well microplate (Thermo Scientific)^TMCatalog number 269620)

8) mu.L of the diluted supernatant from step B was added with 20. mu.L of beta-glucosidase solution

9) The plates were heat sealed (Thermo Scientific)^TMViscous PCR plate seal AB0558) and incubated at 40 ℃ for 45 minutes

10) After the reaction, 100. mu.L from each well was transferred to ThermoFast 96PCR plate (Thermo Scie)ntific^TMCatalog number AB-0600), followed by the addition of 75. mu.L of PAHBAH reagent

11) The plates from (4) were then sealed with a sealing foil (Greiner bio-one plate seal, catalog number 676001) and mounted on a BioRad T100^TMIncubation in a thermal cycler PCR machine at 95 ℃ for 10 minutes followed by cooling at 10 ℃ for 5 minutes

12) After cooling, 100 μ L aliquots were transferred to new 96-well microplates (Thermo Scientific)^TMCatalog number 269620) and read absorbance at 405nm (A)_405nm). The absorbance is an indication of the cellulase activity in the supernatant.

H.Data processing

5) From the absorbance readings of step C, the average A of the 3 blanks from wells without Avicel was calculated_405nm(blank _ reference) (position H4->H6) And the average a of 3 blanks from wells with Avicel was calculated_405nm(blank _ Avicel) (position D4->D6)。

6) The absorbance readings from the cellulase-containing wells were then corrected for their respective blanks (i.e., those from (1)).

7) Calculating the joint stability as

1- [ Activity_405nm(+ Avicel)/Activity_405nm(-Avicel)]，

Wherein the activity is_405nm(+ Avicel) and Activity_405nm(-Avicel) is the activity in wells with supernatant incubated with and without Avicel (i.e. absorbance corrected for blank), respectively.

8) Linker stability reported in the examples is the average of triplicates analyzed.

Example 8: variant stability

Variants were prepared as described in example 4. Stability was determined using the assay described in example 2, where the stress condition was incubation with protease (SEQ ID NO:10) at 20 ℃ for 20 hours, and then residual activity was analyzed.

Linker stability (linker stability relative to control) is shown in table 3.

In the context of Table 3, the following examples are,

sequence listing

<110> Novozymes corporation (Novozymes A/S)

<120> stabilized variants

<130> 14915-WO-PCT

<160> 201

<170> PatentIn 3.5 edition

<210> 1

<211> 278

<212> PRT

<213> Thielavia terrestris Spreng Shell

<400> 1

Ala Ser Gly Ser Gly Gln Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro

1 5 10 15

Ser Cys Ala Trp Pro Gly Lys Ala Ala Val Ser Gln Pro Val Tyr Ala

20 25 30

Cys Asp Ala Asn Phe Gln Arg Leu Ser Asp Phe Asn Val Gln Ser Gly

35 40 45

Cys Asn Gly Gly Ser Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala

50 55 60

Val Asn Asp Asn Leu Ala Tyr Gly Phe Ala Ala Thr Ser Ile Ala Gly

65 70 75 80

Gly Ser Glu Ser Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr Phe Thr

85 90 95

Ser Gly Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr Ser Thr

100 105 110

Gly Gly Asp Leu Gly Ser Asn His Phe Asp Ile Ala Met Pro Gly Gly

115 120 125

Gly Val Gly Ile Phe Asn Gly Cys Ser Ser Gln Phe Gly Gly Leu Pro

130 135 140

Gly Ala Gln Tyr Gly Gly Ile Ser Ser Arg Asp Gln Cys Asp Ser Phe

145 150 155 160

Pro Ala Pro Leu Lys Pro Gly Cys Gln Trp Arg Phe Asp Trp Phe Gln

165 170 175

Asn Ala Asp Asn Pro Thr Phe Thr Phe Gln Gln Val Gln Cys Pro Ala

180 185 190

Glu Ile Val Ala Arg Ser Gly Cys Lys Arg Asn Asp Asp Ser Ser Phe

195 200 205

Pro Val Phe Thr Pro Pro Ser Gly Gly Asn Gly Gly Thr Gly Thr Pro

210 215 220

Thr Ser Thr Ala Pro Gly Ser Gly Gln Thr Ser Pro Gly Gly Gly Ser

225 230 235 240

Gly Cys Thr Ser Gln Lys Trp Ala Gln Cys Gly Gly Ile Gly Phe Ser

245 250 255

Gly Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Gln Lys Leu Asn Asp

260 265 270

Tyr Tyr Ser Gln Cys Leu

275

<210> 2

<211> 283

<212> PRT

<213> Humicola insolens

<400> 2

Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys

1 5 10 15

Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro Val Phe Ser Cys Asn

20 25 30

Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala Lys Ser Gly Cys Glu

35 40 45

Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala Val

50 55 60

Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr Ser Ile Ala Gly Ser

65 70 75 80

Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu Leu Thr Phe Thr Ser

85 90 95

Gly Pro Val Ala Gly Lys Lys Met Val Val Gln Ser Thr Ser Thr Gly

100 105 110

Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn Ile Pro Gly Gly Gly

115 120 125

Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe Gly Gly Leu Pro Gly

130 135 140

Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu Cys Asp Arg Phe Pro

145 150 155 160

Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe Asp Trp Phe Lys Asn

165 170 175

Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val Gln Cys Pro Ala Glu

180 185 190

Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp Asp Gly Asn Phe Pro

195 200 205

Ala Val Gln Ile Ser Ser Ser Thr Ser Ser Pro Val Asn Gln Pro Thr

210 215 220

Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr Ser Ser Pro Pro Val Gln

225 230 235 240

Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu Arg Trp Ala Gln Cys Gly

245 250 255

Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys Val Ala Gly Ser Thr Cys

260 265 270

Thr Lys Ile Asn Asp Trp Tyr His Gln Cys Leu

275 280

<210> 3

<211> 295

<212> PRT

<213> Staphylotrichum coccosporum (Staphylotrichum coccosporum)

<400> 3

Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys

1 5 10 15

Ser Trp Pro Gly Lys Ala Ser Val Asn Gln Pro Val Phe Ala Cys Ser

20 25 30

Ala Asn Phe Gln Arg Ile Ser Asp Pro Asn Val Lys Ser Gly Cys Asp

35 40 45

Gly Gly Ser Ala Tyr Ala Cys Ala Asp Gln Thr Pro Trp Ala Val Asn

50 55 60

Asp Asn Phe Ser Tyr Gly Phe Ala Ala Thr Ser Ile Ser Gly Gly Asn

65 70 75 80

Glu Ala Ser Trp Cys Cys Gly Cys Tyr Glu Leu Thr Phe Thr Ser Gly

85 90 95

Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr Ser Thr Gly Gly

100 105 110

Asp Leu Gly Thr Asn His Phe Asp Leu Ala Met Pro Gly Gly Gly Val

115 120 125

Gly Ile Phe Asp Gly Cys Ser Pro Gln Phe Gly Gly Leu Ala Gly Asp

130 135 140

Arg Tyr Gly Gly Val Ser Ser Arg Ser Gln Cys Asp Ser Phe Pro Ala

145 150 155 160

Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe Asp Trp Phe Lys Asn Ala

165 170 175

Asp Asn Pro Thr Phe Thr Phe Arg Gln Val Gln Cys Pro Ser Glu Leu

180 185 190

Val Ala Arg Thr Gly Cys Arg Arg Asn Asp Asp Gly Asn Phe Pro Val

195 200 205

Phe Thr Pro Pro Ser Gly Gly Gln Ser Ser Ser Ser Ser Ser Ser Ser

210 215 220

Ser Ala Lys Pro Thr Ser Thr Ser Thr Ser Thr Thr Ser Thr Lys Ala

225 230 235 240

Thr Ser Thr Thr Ser Thr Ala Ser Ser Gln Thr Ser Ser Ser Thr Gly

245 250 255

Gly Gly Cys Ala Ala Gln Arg Trp Ala Gln Cys Gly Gly Ile Gly Phe

260 265 270

Ser Gly Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Asn Lys Gln Asn

275 280 285

Asp Trp Tyr Ser Gln Cys Leu

290 295

<210> 4

<211> 277

<212> PRT

<213> Acremonium thermophilum

<400> 4

Ala Leu Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser

1 5 10 15

Cys Gly Trp Pro Gly Lys Ala Ser Val Asn Gln Pro Val Phe Ser Cys

20 25 30

Ser Ala Asp Trp Gln Arg Ile Ser Asp Phe Asn Ala Lys Ser Gly Cys

35 40 45

Asp Gly Gly Ser Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala Val

50 55 60

Asn Asp Asn Phe Ser Tyr Gly Phe Ala Ala Thr Ala Ile Ala Gly Gly

65 70 75 80

Ser Glu Ser Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr Phe Asn Ser

85 90 95

Gly Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr Ser Thr Gly

100 105 110

Gly Asp Leu Gly Ser Asn Gln Phe Asp Leu Ala Ile Pro Gly Gly Gly

115 120 125

Val Gly Ile Phe Asn Gly Cys Ala Ser Gln Phe Gly Gly Leu Pro Gly

130 135 140

Ala Gln Tyr Gly Gly Ile Ser Asp Arg Ser Gln Cys Ser Ser Phe Pro

145 150 155 160

Ala Pro Leu Gln Pro Gly Cys Gln Trp Arg Phe Asp Trp Phe Gln Asn

165 170 175

Ala Asp Asn Pro Thr Phe Thr Phe Gln Arg Val Gln Cys Pro Ser Glu

180 185 190

Leu Thr Ser Arg Thr Gly Cys Lys Arg Asp Asp Asp Ala Ser Tyr Pro

195 200 205

Val Phe Asn Pro Pro Ser Gly Gly Ser Pro Ser Thr Thr Ser Thr Thr

210 215 220

Thr Ser Ser Pro Ser Gly Pro Thr Gly Asn Pro Pro Gly Gly Gly Gly

225 230 235 240

Cys Thr Ala Gln Lys Trp Ala Gln Cys Gly Gly Thr Gly Phe Thr Gly

245 250 255

Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Gln Val Gln Asn Gln Trp

260 265 270

Tyr Ser Gln Cys Leu

275

<210> 5

<211> 212

<212> PRT

<213> Artificial sequence

<220>

<223> catalytic domain of SEQ ID NO:1

<400> 5

Ala Ser Gly Ser Gly Gln Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro

1 5 10 15

Ser Cys Ala Trp Pro Gly Lys Ala Ala Val Ser Gln Pro Val Tyr Ala

20 25 30

Cys Asp Ala Asn Phe Gln Arg Leu Ser Asp Phe Asn Val Gln Ser Gly

35 40 45

Cys Asn Gly Gly Ser Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala

50 55 60

Val Asn Asp Asn Leu Ala Tyr Gly Phe Ala Ala Thr Ser Ile Ala Gly

65 70 75 80

Gly Ser Glu Ser Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr Phe Thr

85 90 95

Ser Gly Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr Ser Thr

100 105 110

Gly Gly Asp Leu Gly Ser Asn His Phe Asp Ile Ala Met Pro Gly Gly

115 120 125

Gly Val Gly Ile Phe Asn Gly Cys Ser Ser Gln Phe Gly Gly Leu Pro

130 135 140

Gly Ala Gln Tyr Gly Gly Ile Ser Ser Arg Asp Gln Cys Asp Ser Phe

145 150 155 160

Pro Ala Pro Leu Lys Pro Gly Cys Gln Trp Arg Phe Asp Trp Phe Gln

165 170 175

Asn Ala Asp Asn Pro Thr Phe Thr Phe Gln Gln Val Gln Cys Pro Ala

180 185 190

Glu Ile Val Ala Arg Ser Gly Cys Lys Arg Asn Asp Asp Ser Ser Phe

195 200 205

Pro Val Phe Thr

210

<210> 6

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> carbohydrate binding module of SEQ ID NO:1

<400> 6

Cys Thr Ser Gln Lys Trp Ala Gln Cys Gly Gly Ile Gly Phe Ser Gly

1 5 10 15

Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Gln Lys Leu Asn Asp Tyr

20 25 30

Tyr Ser Gln Cys Leu

<210> 7

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> carbohydrate binding module of SEQ ID NO: 2

<400> 7

Cys Thr Ala Glu Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly

1 5 10 15

Cys Thr Thr Cys Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp

20 25 30

Tyr His Gln Cys Leu

<210> 8

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> carbohydrate binding module of SEQ ID NO. 3

<400> 8

Cys Ala Ala Gln Arg Trp Ala Gln Cys Gly Gly Ile Gly Phe Ser Gly

1 5 10 15

Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Asn Lys Gln Asn Asp Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 9

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> carbohydrate binding module of SEQ ID NO: 4

<400> 9

Cys Thr Ala Gln Lys Trp Ala Gln Cys Gly Gly Thr Gly Phe Thr Gly

1 5 10 15

Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Gln Val Gln Asn Gln Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 10

<211> 311

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 10

Ala Val Pro Ser Thr Gln Thr Pro Trp Gly Ile Lys Ser Ile Tyr Asn

1 5 10 15

Asp Gln Ser Ile Thr Lys Thr Thr Gly Gly Lys Gly Ile Lys Val Ala

20 25 30

Val Leu Asp Thr Gly Val Tyr Thr Ser His Leu Asp Leu Ala Gly Ser

35 40 45

Ala Glu Gln Cys Lys Asp Phe Thr Gln Ser Asn Pro Leu Val Asp Gly

50 55 60

Ser Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val

65 70 75 80

Leu Ala His Gly Gly Ser Asn Gly Gln Gly Val Tyr Gly Val Ala Pro

85 90 95

Gln Ala Lys Leu Trp Ala Tyr Lys Val Leu Gly Asp Lys Gly Glu Gly

100 105 110

Tyr Ser Asp Asp Ile Ala Ala Ala Ile Arg His Val Ala Asp Glu Ala

115 120 125

Ser Arg Thr Gly Ser Lys Val Val Ile Asn Met Ser Leu Gly Ser Ser

130 135 140

Ala Lys Asp Ser Leu Ile Ala Ser Ala Val Asp Tyr Ala Tyr Gly Lys

145 150 155 160

Gly Val Leu Ile Val Ala Ala Ala Gly Asn Glu Gly Pro Lys Pro Asn

165 170 175

Thr Ile Gly Tyr Pro Ala Gly Phe Val Asn Ala Val Ala Val Ala Ala

180 185 190

Leu Glu Asn Val Gln Glu Lys Gly Thr Tyr Arg Val Ala Asp Phe Ser

195 200 205

Ser Arg Gly Asn Pro Ala Thr Ala Gly Asp Tyr Ile Ile Gln Glu Arg

210 215 220

Asp Ile Glu Val Ser Ala Pro Gly Ala Ser Val Glu Ser Thr Trp Tyr

225 230 235 240

Thr Gly Gly Tyr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His

245 250 255

Val Ala Gly Leu Ala Ala Lys Ile Trp Ser Ala Asn Thr Ser Leu Ser

260 265 270

His Ser Gln Leu Arg Thr Glu Leu Gln Asn Arg Ala Lys Val Tyr Asp

275 280 285

Ile Lys Gly Gly Ile Gly Ala Gly Pro Gly Asp Asp Tyr Ala Ser Gly

290 295 300

Phe Gly Tyr Pro Arg Val Lys

305 310

<210> 11

<211> 269

<212> PRT

<213> Bacillus lentus

<400> 11

Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala

1 5 10 15

His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

50 55 60

His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu

65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

85 90 95

Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

115 120 125

Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser

145 150 155 160

Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln

165 170 175

Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile

180 185 190

Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr

195 200 205

Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

210 215 220

Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile

225 230 235 240

Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu

245 250 255

Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265

<210> 12

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 12

Thr Thr Pro Pro Thr Pro Thr Pro Thr Pro Thr Pro Gly

1 5 10

<210> 13

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 13

Thr Thr Pro Thr Pro Pro Thr Pro Thr Pro Thr Pro Thr Pro Gly

1 5 10 15

<210> 14

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 14

Thr Thr Pro Thr Pro Thr Pro Pro Thr Pro Thr Pro Thr Pro Thr Pro

1 5 10 15

Gly

<210> 15

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 15

Thr Thr Pro Thr Pro Thr Pro Thr Pro Pro Thr Pro Thr Pro Thr Pro

1 5 10 15

Thr Pro Gly

<210> 16

<211> 34

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 16

Thr Pro Pro Thr Pro Pro Thr Pro Pro Thr Pro Pro Thr Pro Pro Thr

1 5 10 15

Pro Pro Thr Pro Pro Thr Pro Pro Thr Pro Pro Thr Pro Pro Thr Pro

20 25 30

Pro Gly

<210> 17

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 17

Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Gly

1 5 10

<210> 18

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 18

Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr

1 5 10 15

Pro Thr Gly

<210> 19

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 19

Ser Pro Ser Ser Pro Ser Ser Pro Ser Ser Pro Ser Gly

1 5 10

<210> 20

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 20

Ser Pro Ser Ser Pro Ser Ser Pro Ser Ser Pro Ser Ser Pro Ser Gly

1 5 10 15

<210> 21

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 21

Ser Pro Pro Ser Pro Pro Ser Pro Pro Ser Pro Pro Ser Pro Pro Gly

1 5 10 15

<210> 22

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 22

Ser Pro Pro Ser Pro Pro Ser Pro Pro Ser Pro Pro Ser Pro Pro Ser

1 5 10 15

Pro Pro Ser Pro Pro Ser Pro Pro Ser Pro Pro Ser Pro Pro Gly

20 25 30

<210> 23

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 23

Pro Pro Ser Ser Pro Ser Ser Pro Ser Ser Pro Ser Ser Pro Ser Ser

1 5 10 15

Pro Ser Ser Pro Ser Gly

<210> 24

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 24

Ser Pro Ser Pro Gly

1 5

<210> 25

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 25

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Gly

1 5 10

<210> 26

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 26

Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Gly

1 5 10

<210> 27

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 27

Pro Pro Pro Pro

<210> 28

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 28

Pro Pro Pro Pro Pro

1 5

<210> 29

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 29

Pro Pro Pro Pro Pro Pro

1 5

<210> 30

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 30

Pro Pro Pro Pro Pro Pro Pro Gly

1 5

<210> 31

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 31

Pro Pro Pro Pro Pro Pro Pro

1 5

<210> 32

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 32

Pro Pro Pro Pro Pro Pro Pro Pro Gly

1 5

<210> 33

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 33

Pro Pro Pro Pro Pro Pro Pro Pro Pro Gly

1 5 10

<210> 34

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 34

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gly

1 5 10

<210> 35

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 35

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gly

1 5 10

<210> 36

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 36

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gly

1 5 10

<210> 37

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 37

Pro Glu Pro Thr Pro Glu Pro Thr Gly

1 5

<210> 38

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 38

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Gly

1 5 10

<210> 39

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 39

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Gly

<210> 40

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 40

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Glu Pro Thr Gly

<210> 41

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 41

Pro Ser Pro Thr Pro Ser Pro Thr Pro Ser Pro Thr Pro Ser Pro Thr

1 5 10 15

Gly

<210> 42

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 42

Pro Ser Pro Thr Pro Ser Pro Thr Pro Ser Pro Thr Pro Ser Pro Thr

1 5 10 15

Pro Ser Pro Thr Gly

<210> 43

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 43

Pro Gln Pro Thr Pro Gln Pro Thr Gly

1 5

<210> 44

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 44

Pro Asp Pro Thr Pro Asp Pro Thr Gly

1 5

<210> 45

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 45

Pro Arg Pro Thr Pro Glu Pro Thr Gly

1 5

<210> 46

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 46

Pro Gln Pro Thr Pro Glu Pro Thr Gly

1 5

<210> 47

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 47

Pro Ser Pro Asn Ser Pro Asn Ser Pro Asn Gly

1 5 10

<210> 48

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 48

Pro Glu Pro Thr Pro Arg Pro Thr Gly

1 5

<210> 49

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 49

Pro Gln Pro Thr Pro Glu Pro Thr Pro Gln Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Gln Pro Thr Pro Glu Pro Thr Pro Gln Pro Thr Gly

20 25

<210> 50

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 50

Pro Asp Pro Thr Pro Asp Pro Thr Pro Asp Pro Thr Gly

1 5 10

<210> 51

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 51

Pro Gln Pro Thr Pro Gln Pro Thr Pro Gln Pro Thr Pro Gln Pro Thr

1 5 10 15

Gly

<210> 52

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 52

Pro Gln Pro Thr Pro Glu Pro Thr Pro Gln Pro Thr Pro Glu Pro Thr

1 5 10 15

Gly

<210> 53

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 53

Ser Pro Ser Pro Ser Pro Ser Pro Pro Pro Gly

1 5 10

<210> 54

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 54

Ser Pro Ser Pro Ser Pro Ser Pro Asp Pro Gly

1 5 10

<210> 55

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 55

Ser Pro Ser Pro Ser Pro Ser Pro Lys Pro Gly

1 5 10

<210> 56

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 56

Ser Pro Ser Pro Ser Pro Ser Pro Ala Pro Gly

1 5 10

<210> 57

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 57

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Ser Gly

1 5 10

<210> 58

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 58

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro

1 5 10

<210> 59

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 59

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Ser

1 5 10

<210> 60

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 60

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Pro

1 5 10

<210> 61

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 61

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Glu

1 5 10

<210> 62

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 62

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Asn

1 5 10

<210> 63

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 63

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Gly Gly

1 5 10

<210> 64

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 64

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Lys

1 5 10

<210> 65

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 65

Pro Glu Pro Thr Pro Glu Pro Thr Pro

1 5

<210> 66

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 66

Pro Glu Pro Thr Pro Glu Pro Thr Arg

1 5

<210> 67

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 67

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro

1 5 10

<210> 68

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 68

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Ser Pro Thr Gly

<210> 69

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 69

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Thr Pro Thr Gly

<210> 70

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 70

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Gly Pro Thr Gly

<210> 71

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 71

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Asp Pro Thr Gly

<210> 72

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 72

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Glu Thr Gly

<210> 73

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 73

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Glu Pro Thr Asp

<210> 74

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 74

Pro Glu Pro Thr Pro Glu Pro Thr Glu

1 5

<210> 75

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 75

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Glu Pro

<210> 76

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 76

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Ser Pro Thr

<210> 77

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 77

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Arg Pro Thr Thr

<210> 78

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 78

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Glu Pro Thr Thr

<210> 79

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 79

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Glu Pro Thr

<210> 80

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 80

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Glu Pro Thr Ser

<210> 81

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 81

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

Pro Glu Pro Thr Arg

<210> 82

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 82

Pro Pro Pro Gly Gly Pro Gly Gly Pro Gly Thr Pro Thr Ser Thr Ala

1 5 10 15

Pro Gly Ser Gly Pro Thr Ser Pro Gly Gly Gly Ser Gly

20 25

<210> 83

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 83

Pro Pro Pro Gly Gly Pro Gly Gly Thr Gly Thr Pro Thr Ser Thr Ala

1 5 10 15

Pro Gly Ser Gly Pro Thr Ser Pro Gly Gly Gly Ser Gly

20 25

<210> 84

<211> 29

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 84

Pro Pro Ser Gly Gly Pro Gly Gly Pro Gly Thr Pro Thr Ser Thr Ala

1 5 10 15

Pro Gly Ser Gly Pro Thr Ser Pro Gly Gly Gly Ser Gly

20 25

<210> 85

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 85

Pro Glu Pro Thr Pro Arg Pro Thr Pro Glu Pro Thr Pro Arg Pro Thr

1 5 10 15

Gly

<210> 86

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 86

Pro Lys Pro Thr Pro Glu Pro Thr Pro Lys Pro Thr Pro Glu Pro Thr

1 5 10 15

Gly

<210> 87

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 87

Pro Glu Pro Thr Pro Lys Pro Thr Pro Glu Pro Thr Pro Lys Pro Thr

1 5 10 15

Gly

<210> 88

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 88

Pro Glu Pro Thr Pro Gln Pro Thr Pro Glu Pro Thr Pro Gln Pro Thr

1 5 10 15

Gly

<210> 89

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 89

Pro Arg Pro Thr Pro Glu Pro Thr Pro Arg Pro Thr Gly

1 5 10

<210> 90

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 90

Pro Lys Pro Thr Pro Glu Pro Thr Pro Lys Pro Thr Gly

1 5 10

<210> 91

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 91

Pro Glu Pro Thr Pro Gln Pro Thr Gly

1 5

<210> 92

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 92

Pro Glu Pro Thr Pro Gln Pro Thr Gly

1 5

<210> 93

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 93

Thr Pro Pro Thr Pro Pro Gly

1 5

<210> 94

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 94

Ser Pro Ser Ser Pro Ser Gly

1 5

<210> 95

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 95

Ser Pro Ser Ser Pro Ser Ser Pro Ser Gly

1 5 10

<210> 96

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 96

Thr Pro Thr Thr Pro Thr Gly

1 5

<210> 97

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 97

Thr Pro Thr Thr Pro Thr Thr Pro Thr Gly

1 5 10

<210> 98

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<220>

<221> features not yet classified

<222> (2)..(2)

<223> Xaa = any amino acid

<220>

<221> features not yet classified

<222> (4)..(4)

<223> Xaa = any amino acid

<400> 98

Pro Xaa Pro Xaa

<210> 99

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<220>

<221> features not yet classified

<222> (1)..(1)

<223> Xaa = any amino acid

<220>

<221> features not yet classified

<222> (3)..(3)

<223> Xaa = any amino acid

<400> 99

Xaa Pro Xaa Pro

<210> 100

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<220>

<221> features not yet classified

<222> (1)..(1)

<223> Xaa = any amino acid

<220>

<221> features not yet classified

<222> (3)..(3)

<223> Xaa = any amino acid

<220>

<221> features not yet classified

<222> (4)..(4)

<223> Xaa = any amino acid

<220>

<221> features not yet classified

<222> (6)..(6)

<223> Xaa = any amino acid

<400> 100

Xaa Pro Xaa Xaa Pro Xaa

1 5

<210> 101

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<220>

<221> features not yet classified

<222> (1)..(1)

<223> Xaa = any amino acid

<220>

<221> features not yet classified

<222> (2)..(2)

<223> Xaa = any amino acid

<220>

<221> features not yet classified

<222> (4)..(4)

<223> Xaa = any amino acid

<220>

<221> features not yet classified

<222> (5)..(5)

<223> Xaa = any amino acid

<400> 101

Xaa Xaa Pro Xaa Xaa Pro

1 5

<210> 102

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<220>

<221> features not yet classified

<222> (2)..(2)

<223> Xaa = Ser、Thr、Arg、Lys、Asp、Glu、Asn、Gln

<220>

<221> features not yet classified

<222> (4)..(4)

<223> Xaa = Ser、Thr、Arg、Lys、Asp、Glu

<400> 102

Pro Xaa Pro Xaa

<210> 103

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 103

Pro Ser Pro Ser

<210> 104

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 104

Pro Ser Pro Thr

<210> 105

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 105

Pro Ser Pro Arg

<210> 106

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 106

Pro Ser Pro Lys

<210> 107

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 107

Pro Ser Pro Asp

<210> 108

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 108

Pro Ser Pro Glu

<210> 109

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<220>

<221> features not yet classified

<222> (2)..(2)

<223> Xaa = Ser、Glu

<400> 109

Pro Xaa Pro Thr

<210> 110

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 110

Pro Pro Ser Pro Thr Pro

1 5

<210> 111

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 111

Pro Pro Thr Pro Thr Pro

1 5

<210> 112

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 112

Pro Pro Ser Pro Ser Pro

1 5

<210> 113

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 113

Ser Pro Pro Pro Thr Pro

1 5

<210> 114

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 114

Ser Pro Thr Pro Pro Pro

1 5

<210> 115

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 115

Ser Pro Pro Pro Pro Pro

1 5

<210> 116

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 116

Ser Pro Thr Pro Thr Pro

1 5

<210> 117

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 117

Thr Pro Pro Pro Ser Pro

1 5

<210> 118

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 118

Thr Pro Ser Pro Pro Pro

1 5

<210> 119

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 119

Thr Pro Pro Pro Pro Pro

1 5

<210> 120

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 120

Thr Pro Ser Pro Ser Pro

1 5

<210> 121

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 121

Pro Ser Pro Thr Pro Glu Pro Thr

1 5

<210> 122

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 122

Pro Ser Pro Thr Pro Glu Pro Thr Pro Ser Pro Thr Pro Glu Pro Thr

1 5 10 15

<210> 123

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 123

Pro Glu Pro Thr Pro Ser Pro Thr

1 5

<210> 124

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 124

Pro Glu Pro Thr Pro Ser Pro Thr Pro Ser Pro Thr

1 5 10

<210> 125

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 125

Pro Glu Pro Thr

<210> 126

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 126

Ser Pro Pro Glu Pro Thr

1 5

<210> 127

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 127

Ser Pro Pro Ser Pro Thr

1 5

<210> 128

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 128

Pro Ser Pro Glu Pro Thr

1 5

<210> 129

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 129

Pro Ser Pro Ser Pro Thr

1 5

<210> 130

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 130

Ser Pro Ser Pro

<210> 131

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 131

Ser Pro Ser Pro Ser Pro

1 5

<210> 132

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 132

Ser Pro Ser Pro Ser Pro Ser Pro

1 5

<210> 133

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 133

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro

1 5 10

<210> 134

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 134

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro

1 5 10

<210> 135

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 135

Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro Ser Pro

1 5 10 15

<210> 136

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 136

Pro Pro Pro Pro Pro Pro Pro Pro

1 5

<210> 137

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 137

Pro Pro Pro Pro Pro Pro Pro Pro Pro

1 5

<210> 138

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 138

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro

1 5 10

<210> 139

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 139

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro

1 5 10

<210> 140

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 140

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro

1 5 10

<210> 141

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 141

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro

1 5 10

<210> 142

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 142

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro

1 5 10

<210> 143

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 143

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro

1 5 10 15

<210> 144

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 144

Pro Glu Pro Thr Pro Glu Pro Thr

1 5

<210> 145

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 145

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10

<210> 146

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 146

Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr Pro Glu Pro Thr

1 5 10 15

<210> 147

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 147

Pro Ser Pro Thr Pro Ser Pro Thr

1 5

<210> 148

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 148

Pro Ser Pro Thr Pro Ser Pro Thr Pro Ser Pro Thr

1 5 10

<210> 149

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 149

Pro Ser Pro Thr Pro Ser Pro Thr Pro Ser Pro Thr Pro Ser Pro Thr

1 5 10 15

<210> 150

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 150

Pro Ser Pro Thr Pro Ser Pro Thr Pro Ser Pro Thr Pro Ser Pro Thr

1 5 10 15

Pro Ser Pro Thr

<210> 151

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 151

Ser Pro Ser Ser Pro Ser

1 5

<210> 152

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 152

Ser Pro Ser Ser Pro Ser Ser Pro Ser

1 5

<210> 153

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 153

Ser Pro Ser Ser Pro Ser Ser Pro Ser Ser Pro Ser

1 5 10

<210> 154

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 154

Ser Pro Ser Ser Pro Ser Ser Pro Ser Ser Pro Ser Ser Pro Ser

1 5 10 15

<210> 155

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 155

Thr Pro Thr Thr Pro Thr

1 5

<210> 156

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 156

Thr Pro Thr Thr Pro Thr Thr Pro Thr

1 5

<210> 157

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 157

Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr

1 5 10

<210> 158

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 158

Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr

1 5 10 15

<210> 159

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 159

Pro Glu Pro Thr Pro Arg Pro Thr Pro Glu Pro Thr Pro Arg Pro Thr

1 5 10 15

<210> 160

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 160

Pro Glu Pro Thr Pro Lys Pro Thr Pro Glu Pro Thr Pro Lys Pro Thr

1 5 10 15

<210> 161

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 161

Pro Glu Pro Thr Pro Gln Pro Thr Pro Glu Pro Thr Pro Gln Pro Thr

1 5 10 15

<210> 162

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 162

Pro Arg Pro Thr Pro Glu Pro Thr Pro Arg Pro Thr

1 5 10

<210> 163

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 163

Pro Lys Pro Thr Pro Glu Pro Thr Pro Lys Pro Thr

1 5 10

<210> 164

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 164

Pro Glu Pro Thr Pro Gln Pro Thr

1 5

<210> 165

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 165

Pro Glu Pro Thr Pro Gln Pro Thr Pro Glu Pro Thr

1 5 10

<210> 166

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 166

Thr Thr Pro Pro Thr Pro Thr Pro Thr Pro Thr Pro

1 5 10

<210> 167

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 167

Thr Thr Pro Thr Pro Pro Thr Pro Thr Pro Thr Pro Thr Pro

1 5 10

<210> 168

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 168

Thr Thr Pro Thr Pro Thr Pro Pro Thr Pro Thr Pro Thr Pro Thr Pro

1 5 10 15

<210> 169

<211> 33

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 169

Thr Pro Pro Thr Pro Pro Thr Pro Pro Thr Pro Pro Thr Pro Pro Thr

1 5 10 15

Pro Pro Thr Pro Pro Thr Pro Pro Thr Pro Pro Thr Pro Pro Thr Pro

20 25 30

Pro

<210> 170

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 170

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gly

1 5 10

<210> 171

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 171

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gly

1 5 10 15

<210> 172

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 172

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gly

1 5 10 15

<210> 173

<211> 37

<212> PRT

<213> Chaetomium thermophilum)

<400> 173

Cys Thr Thr Gln Lys Trp Gly Gln Cys Gly Gly Ile Gly Tyr Thr Gly

1 5 10 15

Cys Thr Asn Cys Val Ala Gly Thr Thr Cys Thr Gln Leu Asn Pro Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 174

<211> 37

<212> PRT

<213> Chaetomium thermophilum)

<400> 174

Cys Thr Thr Gln Lys Trp Gly Gln Cys Gly Gly Ile Gly Tyr Thr Gly

1 5 10 15

Cys Thr Asn Cys Val Ala Gly Thr Thr Cys Thr Glu Leu Asn Pro Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 175

<211> 36

<212> PRT

<213> Mycothermus thermophilus

<400> 175

Cys Ala Ser Lys Trp Gly Gln Cys Gly Gly Gln Gly Trp Ala Gly Pro

1 5 10 15

Thr Cys Cys Glu Ala Gly Ser Thr Cys Thr Arg Gln Asn Glu Trp Tyr

20 25 30

Ser Gln Cys Leu

<210> 176

<211> 37

<212> PRT

<213> Ovatospora medusarum

<400> 176

Cys Thr Ala Ala Arg Trp Gln Gln Cys Gly Gly Ile Gly Tyr Ser Gly

1 5 10 15

Cys Lys Ala Cys Ala Ser Pro Trp Thr Cys Gln Lys Leu Asn Asp Trp

20 25 30

Tyr His Gln Cys Leu

<210> 177

<211> 37

<212> PRT

<213> Thielavia heteroclita (Thermoelomyces heterothilotallica)

<400> 177

Cys Thr Val Ala Lys Trp Gly Gln Cys Gly Gly Gln Gly Tyr Ser Gly

1 5 10 15

Cys Thr Val Cys Ala Ala Gly Ser Thr Cys Gln Lys Thr Asn Asp Tyr

20 25 30

Tyr Ser Gln Cys Leu

<210> 178

<211> 38

<212> PRT

<213> Ovatospora medusarum

<400> 178

Cys Ser Val Gln Ala Phe Gly Gln Cys Gly Gly Thr Gly Tyr Ser Gly

1 5 10 15

Cys Thr Gln Cys Ala Asp Gly Tyr Thr Cys Lys Asp Val Ser Pro Pro

20 25 30

Tyr Tyr Ser Gln Cys Val

<210> 179

<211> 37

<212> PRT

<213> Thielavia arenaria shell (Thielavia arenaria)

<400> 179

Cys Thr Val Ala Lys Trp Gly Gln Cys Gly Gly Leu Gly Trp Thr Gly

1 5 10 15

Cys Thr Thr Cys Ala Ala Gly Ser Thr Cys Asn Lys Ala Asn Asp Phe

20 25 30

Tyr Ser Gln Cys Val

<210> 180

<211> 37

<212> PRT

<213> Ovatospora medusarum

<400> 180

Cys Thr Val Ala Lys Tyr Gly Gln Cys Gly Gly Asn Asn Tyr Ser Gly

1 5 10 15

Cys Thr Thr Cys Ala Ala Gly Ser Thr Cys Ser Arg Thr Asn Glu Tyr

20 25 30

Tyr Ser Gln Cys Val

<210> 181

<211> 37

<212> PRT

<213> Chaetomium thermophilum

<400> 181

Cys Val Thr Gln Lys Trp Ala Gln Cys Gly Gly Asn Gly Phe Ser Gly

1 5 10 15

Cys Arg Thr Cys Ala Ser Gly Ser Thr Cys Gln Val Leu Asn Glu Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 182

<211> 37

<212> PRT

<213> Chaetomium thermophilum

<400> 182

Cys Thr Val Ala Lys Trp Ala Gln Cys Gly Gly Ile Gly Tyr Ser Gly

1 5 10 15

Cys Thr Thr Cys Glu Ala Gly Ser Thr Cys Arg Arg Thr Asn Asp Tyr

20 25 30

Tyr Ser Gln Cys Val

<210> 183

<211> 36

<212> PRT

<213> Ovatospora medusarum

<400> 183

Cys Thr Ala Ala Gln Trp Gln Gln Cys Gly Gly Thr Asn Phe Asn Gly

1 5 10 15

Cys Thr Thr Cys Ala Ala Gly Tyr Asn Cys Lys Leu Ile Asn Glu Tyr

20 25 30

Tyr Ser Gln Cys

<210> 184

<211> 38

<212> PRT

<213> Chaetomium thermophilum

<400> 184

Cys Thr Ala Gln Arg Tyr Gln Gln Cys Gly Gly Asn Gly Tyr Thr Gly

1 5 10 15

Cys Thr Asn Cys Ala Ala Gly Ser Thr Cys Ser Ala Val Ser Pro Pro

20 25 30

Tyr Tyr Ser Gln Cys Leu

<210> 185

<211> 37

<212> PRT

<213> Thielavia heteroclita (Thermoelomyces heterothilotallica)

<400> 185

Cys Thr Ala Ala Gln Trp Ala Gln Cys Gly Gly Ile Asn Phe Thr Gly

1 5 10 15

Cys Thr Thr Cys Ala Ser Pro Tyr Lys Cys Asn Phe Ile Asn Asp Tyr

20 25 30

Tyr Ser Gln Cys Tyr

<210> 186

<211> 37

<212> PRT

<213> Thielavia species (Thielavia species)

<400> 186

Cys Val Ala Gln Lys Trp Ala Gln Cys Gly Gly Ser Gly Phe Thr Gly

1 5 10 15

Cys Thr Thr Cys Ala Ser Gly Ser Thr Cys Gln Lys Gln Asn Asp Phe

20 25 30

Tyr Ser Gln Cys Leu

<210> 187

<211> 38

<212> PRT

<213> Thielavia arenaria shell (Thielavia arenaria)

<400> 187

Cys Thr Val Gln Arg Tyr Gly Gln Cys Gly Gly Gln Gly Tyr Thr Gly

1 5 10 15

Cys Thr Thr Cys Ala Ser Gly Ser Thr Cys Thr Gly Val Ser Ala Pro

20 25 30

Tyr Tyr Tyr Gln Cys Ile

<210> 188

<211> 37

<212> PRT

<213> Thielavia arenaria shell (Thielavia arenaria)

<400> 188

Cys Ala Ala Ala Lys Tyr Gly Gln Cys Asp Gly Lys Asn Trp Asn Gly

1 5 10 15

Cys Lys Ser Cys Val Ala Gly Thr Thr Cys Arg Tyr Gln Asn Asp Tyr

20 25 30

Tyr Ser Gln Cys Leu

<210> 189

<211> 37

<212> PRT

<213> Ovatospora medusarum

<400> 189

Cys Val Ala Gln Lys Trp Ala Gln Cys Gly Gly Lys Gly Phe Thr Gly

1 5 10 15

Cys Lys Asn Cys Val Ser Gly Thr Thr Cys Gln Glu Gln Asn Gln Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 190

<211> 37

<212> PRT

<213> Thielavia arenaria shell (Thielavia arenaria)

<400> 190

Cys Asn Val Ala Gln Trp Gln Gln Cys Gly Gly Ser Thr Tyr Thr Gly

1 5 10 15

Cys Thr Gln Cys Ala Ser Pro Tyr Thr Cys Lys Asn Ile Asn Thr Tyr

20 25 30

Tyr Ser Gln Cys Gln

<210> 191

<211> 37

<212> PRT

<213> Chaetomium thermophilum

<400> 191

Cys Thr Ala Ala Arg Trp Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly

1 5 10 15

Cys Thr Ala Cys Ala Ser Pro Trp Thr Cys Gln Arg Ile Ser Asp Trp

20 25 30

Tyr His Gln Cys Leu

<210> 192

<211> 36

<212> PRT

<213> Bdeltoid rotifer (Adineta vaga)

<400> 192

Cys Asn Asn Ile Tyr Asn Gln Cys Gly Gly Asn Gly Trp Asn Gly Thr

1 5 10 15

Thr Asn Cys Cys Ser Gly Leu Ser Cys Val Tyr Lys Asn Ser Ser Tyr

20 25 30

Ser Gln Cys Leu

<210> 193

<211> 37

<212> PRT

<213> Chaetomium species

<400> 193

Cys Val Ala Gln Lys Trp Ala Gln Cys Gly Gly Lys Gly Phe Thr Gly

1 5 10 15

Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Lys Glu His His Glu Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 194

<211> 37

<212> PRT

<213> Achaetomium strumarium

<400> 194

Cys Thr Ala Gln Arg Trp Ser Gln Cys Gly Gly Asn Gly Phe Thr Gly

1 5 10 15

Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Gln Lys Gln Asn Asp Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 195

<211> 36

<212> PRT

<213> Chaetomium thermophilum

<400> 195

Cys Thr Val Pro Gln Trp Ala Gln Cys Gly Gly Val Asn Tyr Thr Gly

1 5 10 15

Cys Thr Thr Cys Ala Pro Gly Tyr Thr Cys Lys Tyr Thr Asn Asp Tyr

20 25 30

Tyr Ser Gln Cys

<210> 196

<211> 37

<212> PRT

<213> Ovatospora medusarum

<400> 196

Cys Val Ser Gln Lys Trp Ala Gln Cys Gly Gly Asn Gly Tyr Thr Gly

1 5 10 15

Cys Thr Gln Cys Val Ser Gly Thr Thr Cys Asn Lys Leu Asn Asp Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 197

<211> 37

<212> PRT

<213> Chaetomium olivicolor (Chaetomium olivicolor)

<400> 197

Cys Thr Ala Gln Lys Trp Ala Gln Cys Gly Gly Ser Gly Phe Ser Gly

1 5 10 15

Cys Thr Ser Cys Val Ser Gly Thr Thr Cys Gln Lys Gln Asn Asp Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 198

<211> 37

<212> PRT

<213> Dabaozi (Taifangliania major)

<400> 198

Cys Val Ala Gln Lys Trp Ala Gln Cys Gly Gly Asn Gly Phe Thr Gly

1 5 10 15

Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Thr Lys Ser Asn Asp Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 199

<211> 37

<212> PRT

<213> Helminthosporium crassimum (Trichocaulum asperum)

<400> 199

Cys Phe Ala Gln Lys Trp Ala Gln Cys Gly Gly Asn Gly Phe Thr Gly

1 5 10 15

Cys Thr Ser Cys Val Ser Gly Thr Thr Cys Gln Lys Gln Asn Asp Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 200

<211> 37

<212> PRT

<213> Ovatospora brasiliensis

<400> 200

Cys Val Ala Gln Lys Trp Ala Gln Cys Gly Gly Asn Gly Phe Ser Gly

1 5 10 15

Cys Thr Thr Cys Val Ser Gly Ser Thr Cys Gln Lys Gln Asn Asp Trp

20 25 30

Tyr Ser Gln Cys Leu

<210> 201

<211> 262

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 201

Ala Ser Gly Ser Gly Gln Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro

1 5 10 15

Ser Cys Ala Trp Pro Gly Lys Ala Ala Val Ser Gln Pro Val Tyr Ala

20 25 30

Cys Asp Ala Asn Phe Gln Arg Leu Ser Asp Phe Asn Val Gln Ser Gly

35 40 45

Cys Asn Gly Gly Ser Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala

50 55 60

Val Asn Asp Asn Leu Ala Tyr Gly Phe Ala Ala Thr Ser Ile Ala Gly

65 70 75 80

Gly Ser Glu Ser Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr Phe Thr

85 90 95

Ser Gly Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr Ser Thr

100 105 110

Gly Gly Asp Leu Gly Ser Asn His Phe Asp Ile Ala Met Pro Gly Gly

115 120 125

Gly Val Gly Ile Phe Asn Gly Cys Ser Ser Gln Phe Gly Gly Leu Pro

130 135 140

Gly Ala Gln Tyr Gly Gly Ile Ser Ser Arg Asp Gln Cys Asp Ser Phe

145 150 155 160

Pro Ala Pro Leu Lys Pro Gly Cys Gln Trp Arg Phe Asp Trp Phe Gln

165 170 175

Asn Ala Asp Asn Pro Thr Phe Thr Phe Gln Gln Val Gln Cys Pro Ala

180 185 190

Glu Ile Val Ala Arg Ser Gly Cys Lys Arg Asn Asp Asp Ser Ser Phe

195 200 205

Pro Val Phe Thr Thr Thr Pro Pro Thr Pro Thr Pro Thr Pro Thr Pro

210 215 220

Gly Cys Thr Ser Gln Lys Trp Ala Gln Cys Gly Gly Ile Gly Phe Ser

225 230 235 240

Gly Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Gln Lys Leu Asn Asp

245 250 255

Tyr Tyr Ser Gln Cys Leu

260

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