Alpha-amylase variants and polynucleotides encoding same

文档序号：1191667 发布日期：2020-08-28 浏览：30次中文

阅读说明：本技术 α-淀粉酶变体以及对其进行编码的多核苷酸 (Alpha-amylase variants and polynucleotides encoding same ) 是由 K.斯塔灵斯 C.安德森 V.P.拉奥 R.塞基亚 V.斯里瓦斯塔瓦于 2018-12-07 设计创作，主要内容包括：本发明涉及α-淀粉酶变体,所述α-淀粉酶变体包含在对应于位置188的位置处的取代和在对应于SEQ ID NO:1的位置242或279或275的位置处的至少一个另外的取代,特别是选自由以下组成的组的取代中的一个或多个组合：E188P+S242Y、E188P+S242F、E188P+S242H、E188P+S242W、E188P+S242P、E188P+S242I、E188P+S242T、E188P+S242L、E188P+K279W、E188P+K279Y、E188P+K279F、E188P+K279H、E188P+K279I、E188P+K279L、E188P+K279D、E188P+K279M、E188P+K279S、E188P+K279T、E188P+K279N、E188P+K279Q、E188P+K279V、E188P+K279A、E188P+N275F、E188P+N275Y、E188P+N275W、和E188P+N275H,其中所述变体与选自由以下组成的组的亲本α-淀粉酶具有至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、或至少99％、但小于100％的序列同一性：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、和SEQ ID NO:27。本发明还涉及编码所述变体的多核苷酸；包含所述多核苷酸的核酸构建体、载体和宿主细胞；以及使用所述变体的方法。(The present invention relates to alpha-amylase variants comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations of substitutions selected from the group consisting of: e188 + S242, E188 + K279, E188 + N275, and E188 + N275, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to a parent α -amylase selected from the group consisting of: SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, and SEQ ID NO 27. The invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.)

1. An alpha-amylase variant comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID NO:1, in particular one or more combinations of substitutions selected from the group consisting of: e188 + S242, E188 + K279, E188 + N275, and E188 + N275, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to a parent α -amylase selected from the group consisting of: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 27.

2. The variant alpha-amylase of claim 1, wherein the variant has increased thermostability at pH4.5 over the parent alpha-amylase.

3. The variant of claim 1, wherein said variant has increased chelator stability in model detergent a over said parent alpha-amylase.

4. The variant of claim 1, wherein the variant is capable of producing a liquefact having a Dextrose Equivalent (DE) value greater than the DE value produced by the parent alpha-amylase.

5. The variant of claim 1, wherein said variant is capable of producing a liquefact having a reduced viscosity as compared to a liquefact produced by a parent alpha-amylase.

6. The variant alpha-amylase of claim 1, wherein said variant has increased thermostability, in particular increased stability, at pH4.5 over the parent alpha-amylase as determined by an Improvement Factor (IF), wherein the IF is determined as: the residual activity of the variant alpha-amylase (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃) versus the residual activity of the parent alpha-amylase (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃), in particular the variant has an IF of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0.

7. The variant according to claim 1, wherein said variant has increased chelator stability, in particular increased stability, over said parent alpha-amylase in model detergent a, as determined by an Improvement Factor (IF), wherein said IF is determined as: the residual activity of the variant (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃) versus the residual activity of the parent alpha-amylase (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃), in particular the variant has an IF of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0.

8. The variant according to any of the preceding claims, wherein said variant further comprises a deletion of two amino acids in the region corresponding to position 179-182, numbered with SEQ ID NO 1.

9. The variant of claim 8, wherein said deletion is selected from the group consisting of: 179 × 180, 179 × 181, 179 × 182, 180 × 181 × 182, and 181 × 182, in particular 181 × 182.

10. The variant according to any of claims 1-9, wherein said parent alpha-amylase is SEQ ID NO 3, and wherein said variant comprises specific substitutions corresponding to:

G48A + T49I + H68W + G107A + H156Y + A181T + A209V + Q264S + K176L + F201Y + H205Y + K213T + E255P + Q360S + D416V + R437W, numbered with SEQ ID NO: 2; or

G48A + T49I + H68W + G107A + T116Q + H156Y + A181T + A209V + Q264S + K176L + F201Y + H205Y + K213T + E255P + Q360S + D416V + R437W, numbered with SEQ ID NO: 2; and wherein said variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO. 3.

11. The variant of claim 10, said variant further comprising N190F, numbered using SEQ ID No. 2.

12. The variant according to any of claims 1-9, wherein said parent alpha-amylase is SEQ ID NO:1, and wherein said variant further comprises specific substitutions corresponding to:

V59A + E129V + E177L + R179E + Q254S + M284V + V212T + Y268G + N293Y + T297N, and optionally the deletion of two amino acids in the region corresponding to positions 179-182, in particular 181 +182, numbering with SEQ ID NO:1, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO:1 or SEQ ID NO: 27.

13. The variant of claim 12, said variant further comprising N193F, numbered with SEQ ID No. 1.

14. The variant according to any of the preceding claims, which comprises a substitution at a position corresponding to position 188 and further substitutions at positions corresponding to positions 242 and 279, particularly in a specific combination selected from:

E188P+S242Y+K279I；

E188P+S242L+K279W；

E188P+S242P+K279W；

E188P+S242L+K279I；

E188P+S242Y+K279W；

E188P+S242Y+K279F；

E188P+S242Y+K279H；

E188P+S242Y+K279L；

E188P+S242Y+K279Y；

E188P+S242P+K279I；

E188P+S242F+K279W；

E188P+S242H+K279W；

E188P+S242W+K279W。

15. the variant according to claim 1, said variant further comprising a substitution corresponding to I204Y, numbered using SEQ ID NO:1, in particular selected from the following specific combinations:

E188P+I204Y+S242Y；

E188P+I204Y+S242F；

E188P+I204Y+K279W；

E188P+I204Y+K279Y；

E188P+I204Y+K279F；

E188P+I204Y+K279H；

E188P+I204Y+K279I；

E188P+I204Y+K279L。

16. the variant of any of claims 1-15, wherein said variant alpha-amylase is isolated.

17. The variant according to any of claims 1-16, wherein the number of alterations is 1-20, such as 1-10 and 1-5, such as 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 alterations.

18. A composition comprising the variant alpha-amylase of any of claims 1-17.

19. The composition of claim 18, further comprising a surfactant.

20. The composition of any one of claims 18 or 19, wherein the composition comprises a surfactant or surfactant system, wherein the surfactant may be selected from the group consisting of nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof.

21. Composition according to claim 20, wherein the composition comprises anionic surfactants, in particular linear alkyl benzene sulphonate (LAS) and/or alcohol ethoxy sulphate (AEOS).

22. The composition of claim 20, wherein the composition comprises a non-ionic surfactant, such as an Alcohol Ethoxylate (AEO).

23. The composition of any one of claims 19-22, wherein the composition comprises one or more anions and/or one or more nonionic surfactants.

24. The composition of any one of claims 19-23, wherein the composition comprises one or more of a surfactant, in particular linear alkyl benzene sulphonic acid (LAS), sodium laureth sulfate (SLES) and/or Alcohol Ethoxylate (AEO).

25. The composition of claim 18, further comprising a protease, particularly an S8 protease, more particularly an S8 protease from the genus pyrococcus or thermophilus.

26. The composition of claim 25, wherein the protease has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 19.

27. A polynucleotide encoding the variant according to any one of claims 1-17.

28. A nucleic acid construct comprising the polynucleotide of claim 27.

29. An expression vector comprising the polynucleotide of claim 27.

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

31. A method of producing an alpha-amylase variant according to claims 1-17, the method comprising:

a) culturing the host cell of claim 30 under conditions suitable for expression of the variant; and

b) optionally recovering the variant.

32. Use of the variant according to claims 1-17 or the composition according to claims 18 or 25-26 for liquefying starch-containing material.

33. Use of the variant according to claims 1-17 in a detergent.

34. A method for producing a syrup from starch-containing material, the method comprising the steps of:

a) liquefying the starch-containing material at a temperature above an initial gelatinization temperature in the presence of the variant alpha-amylase of claims 1-17 or the composition of claims 18 or 25-26; and

b) saccharifying the product of step a) in the presence of glucoamylase.

35. The method of claim 34, wherein step b) is performed in conjunction with a glucoamylase and:

i) a fungal alpha-amylase;

ii) an isoamylase; or

iii) fungal alpha-amylase and isoamylase.

36. The method according to claims 34-35, wherein a pullulanase is present in step a) and/or b).

37. The method according to any one of claims 34-36, the method further comprising:

c) fermenting the product of step b) with a fermenting organism to produce a fermentation product.

38. The method of claim 37, wherein the fermenting organism is a yeast and the fermentation product is an alcohol.

39. The method of claim 38, wherein the yeast is saccharomyces cerevisiae and the alcohol is ethanol.

40. The method of claim 37, wherein steps b) and c) are performed simultaneously.

41. A method for increasing the stability of a parent alpha-amylase, said method comprising introducing a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations of substitutions selected from the group consisting of: e188 + S242, E188 + K279, E188 + K275, E188 + N275, and E188 + N275.

42. The method of claim 41, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to a parent alpha-amylase selected from the group consisting of: SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, and SEQ ID NO 27.

43. The method of any one of claims 41-42, wherein said variant has increased thermostability at pH4.5 over the parent alpha-amylase.

44. The method of any one of claims 41-42, wherein said variant has increased chelator stability in model detergent A over the parent alpha-amylase.

Technical Field

The present invention relates to alpha-amylase variants, polynucleotides encoding the variants, methods of producing the variants, and methods of using the variants.

Background

Disclosure of Invention

In accordance with the present invention, it has been unexpectedly found that substitutions at positions corresponding to positions 242, 279, or 275 (numbered using SEQ id no: 1) when used alone will result in reduced performance, but in combination with the E188P substitution will result in a synergistic improvement.

In a first aspect, the present invention relates to an alpha-amylase variant comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations of substitutions selected from the group consisting of: e188 + S242, E188 + K279, E188 + N275, and E188 + N275, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to a parent α -amylase selected from the group consisting of: SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, and SEQ ID NO 27.

The invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing the variants. Furthermore, the present invention relates to a composition comprising the alpha-amylase variant of the invention.

The present invention also relates to methods of producing the alpha-amylase variants of the invention, comprising:

a) culturing a host cell of the invention under conditions suitable for expression of the variant; and

b) optionally recovering the variant.

The present invention also relates to a process for producing a syrup from starch-containing material, said process comprising the steps of:

a) liquefying the starch-containing material at a temperature above the initial gelatinization temperature in the presence of the variant alpha-amylase according to the invention or the composition according to the invention; and

b) saccharifying the product of step a) in the presence of glucoamylase.

In another aspect, the present invention relates to a method for increasing the stability of a parent alpha-amylase, said method comprising introducing a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations of substitutions selected from the group consisting of: e188 + S242, E188 + K279, E188 + K275, E188 + N275, and E188 + N275.

Definition of

Alpha-amylase variants: alpha-amylases (e.c.3.2.1.1) are a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1, 4-glycoside oligosaccharides and polysaccharides. The skilled person will know how to determine the alpha-amylase activity. It can be determined according to the procedure described in the examples, for example by the PNP-G7 assay, the enzyme detection (EnzCheck) assay, or the Phadebas activity assay. In one aspect, a variant of the invention has at least 20%, e.g., 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 alpha-amylase activity of the polypeptide of SEQ ID Nos. 1-18. In one aspect, a variant of the present application has at least 20%, e.g., 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 alpha-amylase activity of its parent.

In further embodiments, the variant alpha-amylases of the invention have increased stability compared to the parent alpha-amylase, in particular the parent as disclosed in SEQ ID NO 1-18 and 27. In particular, any suitable alpha-amylase or wash assay may be used to determine increased stability. The skilled person will know how to select a suitable assay. Examples of suitable assays and conditions have been provided in the examples herein. Such increased stability may include increased thermostability at pH4.5 over the parent alpha-amylase, or chelator stability in model detergent (model detergent) a over the parent alpha-amylase. In a specific embodiment, the variant alpha-amylase according to the invention has an increased thermostability, in particular an increased stability, at pH4.5 over the parent alpha-amylase, as determined by an Improvement Factor (IF), wherein IF is determined as: the residual activity of the variant alpha-amylase (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃) versus the residual activity of the parent alpha-amylase (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃), in particular the variant has an IF of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0.

In another embodiment, the variant alpha-amylase according to the invention has increased chelator stability, in particular increased stability, over the parent alpha-amylase in a model detergent a, as determined by an Improvement Factor (IF), wherein IF is determined as: the residual activity of the variant (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃) versus the residual activity of the parent alpha-amylase (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃), in particular the variant has an IF of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0. Residual activity may be determined using any suitable alpha-amylase assay known to the skilled person, for example any of the assays disclosed in the examples herein. In one embodiment, the residual activity can be determined using the Phadebas activity assay.

In another embodiment, the variant alpha-amylases of the invention are capable of producing a liquefact having a Dextrose Equivalent (DE) value higher than the DE value produced by the parent alpha-amylase (particularly the parent disclosed in SEQ ID NO:1-18 and 27). In particular, the DE value is at least 1.5X, 2X higher than the DE value produced by the parent alpha amylase.

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, before it is presented as mature spliced mRNA.

A 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.

And (3) 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. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding the variant.

Expressing: 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.

Fragment (b): the term "fragment" means a polypeptide having one or more (e.g., several) amino acids not present at the amino and/or carboxy terminus of the mature polypeptide; wherein the fragment has alpha-amylase activity.

High stringency conditions: the term "high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65 ℃.

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.

Improved properties: the term "improved property" means a characteristic associated with a variant that is improved relative to the parent. Such improved properties may include increased thermostability at pH4.5 over the parent alpha-amylase, or chelant stability in model detergent a over the parent alpha-amylase. In a specific embodiment, the variant alpha-amylase according to the invention has an increased thermostability, in particular an increased stability, at pH4.5 over the parent alpha-amylase, as determined by an Improvement Factor (IF), wherein IF is determined as: the residual activity of the variant alpha-amylase (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃) versus the residual activity of the parent alpha-amylase (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃), in particular the variant has an IF of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0.

In another embodiment, the variant alpha-amylase according to the invention has increased chelator stability, in particular increased stability, over the parent alpha-amylase in a model detergent a, as determined by an Improvement Factor (IF), wherein IF is determined as: the residual activity of the variant (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃) versus the residual activity of the parent alpha-amylase (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃), in particular the variant has an IF of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0. Residual activity may be determined using any suitable alpha-amylase assay known to the skilled person, for example any of the assays disclosed in the examples herein. In particular, Phadebas activity assay can be used to determine residual activity.

In other embodiments, the improved property comprises increased stability measured as 5ppm Ca2 at 90 ℃, pH4.5, and pH 90 ℃⁺Residual α -amylase activity determined by enzyme detection after 20min of incubation, and/or the variant is capable of producing a liquefact having a Dextrose Equivalent (DE) value higher than the DE value produced by the parent α -amylase, or the variant is capable of producing a liquefact having a reduced viscosity compared to a liquefact produced by the parent α -amylase.

Separating: 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 that is at least partially removed from one or more or all of the naturally occurring components with which it is associated in nature, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor; (3) any substance that is modified manually by man relative to that found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than a promoter naturally associated with the gene encoding the substance). The isolated material may be present in a sample of fermentation broth.

Low stringency conditions: the term "low stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard southern blotting procedures for 12 to 24 hours. Finally, the carrier material is washed three times each for 15 minutes using 2 XSSC, 0.2% SDS at 50 ℃.

Mature polypeptide: the term "mature polypeptide" means a polypeptide that is in its final form following translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and the like. In one aspect, the mature sequence is a polypeptide as disclosed in SEQ ID NO 1-18.

It is known in the art that host cells can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and/or N-terminal amino acids) expressed from the same polynucleotide.

Mature polypeptide coding sequence: the term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having glucoamylase activity.

Medium stringency conditions: the term "moderately stringent conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard southern blotting procedures for 12 to 24 hours. Finally, the carrier material is washed three times each for 15 minutes using 2 XSSC, 0.2% SDS at 55 ℃.

Medium-high stringency conditions: the term "medium-high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard southern blotting procedures for 12 to 24 hours. Finally, the carrier material is washed three times each for 15 minutes using 2 XSSC, 0.2% SDS at 60 ℃.

Mutant: the term "mutant" means a polynucleotide encoding a variant.

Nucleic acid construct: 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 expression of the coding sequence.

Parent or parent alpha-amylase: the term "parent" or "parent alpha-amylase" means any polypeptide having alpha-amylase activity that is altered to produce these enzyme variants of the invention.

S8A protease: the term "S8A protease" means a S8 protease belonging to subfamily a. Subtilisin (EC3.4.21.62) is a subset of subfamily S8A. S8A protease hydrolysis of the substrate Suc-Ala-Ala-Pro-Phe-pNA. The release of p-nitroaniline (pNA) resulted in an increase in absorbance at 405nm and was proportional to the enzyme activity.

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, sequence identity between two amino acid sequences is determined using The Needman-Wunsch algorithm (Needleman-Wunsch) (Needleman and Wunsch,1970, J.Mol.biol. [ J.Mol.Biol ]48:443-453), as implemented in The Needler program of The EMBOSS Software 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 more). 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 Needle labeled "longest identity" (obtained using the non-reduced option) is used as the percent identity and is calculated as follows:

(consensus residue x 100)/(alignment Length-Total number of vacancies in alignment)

For the purposes of the present invention, the sequence identity between two deoxynucleotide sequences is determined using the Needman-Wusch algorithm (Needleman and Wunsch,1970, supra) as implemented by the Nidel program of the EMBOSS software package (EMBOSS: European molecular biology open software suite, Rice et al, 2000, supra), preferably version 5.0.0 or more. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and EDNAFULL (EMBOSS version of NCBI NUC 4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the non-reduction option) is used as the percent identity and is calculated as follows:

(consensus deoxyribonucleotide x 100)/(alignment length-total number of gaps in alignment)

Variants: the term "variant" means a polypeptide having alpha-amylase activity comprising an alteration (i.e., a substitution, insertion, and/or deletion) at one or more (e.g., several) positions. Substitution means the substitution of an amino acid occupying a position with a different amino acid; deletion means the removal of an amino acid occupying a position; and an insertion means that an amino acid is added next to and immediately following the amino acid occupying a certain position. In one embodiment, the parent alpha-amylase is selected from the group consisting of: 1,2,3,4,5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17 and 18.

Very high stringency conditions: the term "very high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70 ℃.

Very low stringency conditions: the term "very low stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard southern blotting procedures for 12 to 24 hours. Finally, the carrier material is washed three times each for 15 minutes using 2 XSSC, 0.2% SDS at 45 ℃.

Wild-type α -amylase: the term "wild-type" alpha-amylase means an alpha-amylase expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.

Variant naming conventions

For the purposes of the present invention, unless otherwise indicated, the mature polypeptide disclosed in SEQ ID NO:1 is used to determine the corresponding amino acid residues in another alpha-amylase. The amino acid sequence of another alpha-amylase is aligned to The polypeptide disclosed in SEQ ID NO:1 and based on said alignment, The amino acid position numbering corresponding to any amino acid residue in The polypeptide disclosed in SEQ ID NO:1 is determined using The Needleman-Weng algorithm (Needleman and Wunsch,1970, J.mol.biol. [ J.Mol ]48:443-453), as implemented in The Nidel program of The EMBOSS Software 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 more. 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 the corresponding amino acid residue in another alpha-amylase can be determined by aligning the multiple polypeptide sequences using their respective default parameters using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log expectation; version 3.5 or later; Edgar,2004, Nucleic Acids Research [ Nucleic acid Research ]32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma,2002, Nucleic Acids Research [ Nucleic acid Research ]30: 3059-3066; Katoh et al, 2005, Nucleic Acids Research [ Nucleic acid Research ]33: 511-518; Katoh and Toh,2007, information, Bioinformatics [ Bioinformatics ]23: 372-374; Katoh et al, 2009, method Molecular Biology [ Molecular Biology ] 39-537; Biocoding [ Biocoding ] 2010, Biocoding [ Biocoding ]26: 2010, and Biocoding [ Biocoding ]26: 2010, Biocoding [ Biocoding ]26:18, Biocoding [ Biocoding ]26, and Biocoding [ Biocoding ]26, Biocoding [ Biocoding ]18, Biocoding ] using the same, 1994, Nucleic Acids Research [ Nucleic Acids Research ]22: 4673-4-4680) using their respective default parameters.

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: 797-815; McGuffin and Jones,2003, Bioinformatics [ Bioinformatics ]19:874-881) use information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to neural networks that predict the structural folding of query sequences. 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 the 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 can 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-.

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.

SubstitutionFor amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, 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" represents the substitution of glycine (G) and serine (S) at positions 205 and 411 with arginine (R) and phenylalanine (F), respectively.

Absence ofFor amino acid deletions, the following nomenclature is used: original amino acid, position,^*. Thus, 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 intoFor amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, insertion of a lysine after a 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 variationsVariants comprising multiple alterations are separated by a plus sign ("+"), e.g., "Arg 170Tyr + Gly195 Glu" or "R170Y + G195E" represents the substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

Different changesIn the case 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".

Detailed Description

The present invention relates to alpha-amylase variants comprising at least one substitution at a position corresponding to position 188 of SEQ ID No. 1 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1.

The substitution E188P in bacillus stearothermophilus alpha-amylase has previously been shown to improve stability (US 8,084,240). The same has been shown for the substitution at position 242 when S is substituted to A, Q, E, D, or M, while the other substitution C, F, G, H, I, K, L, N, P, R, T, V, W, Y results in less activity compared to the wild type (WO 2009061381).

Variants

The inventors have surprisingly found that substitution at position 242 is selected from S242Y, F, H, W, P, I, T, L, which when used alone will result in reduced performance, but in combination with the E188P substitution will result in a synergistic improvement.

Furthermore, the inventors have surprisingly found that the substitution at position 279 is selected from K279Y, F, H, W, I, T, L, D, M, S, N, Q, V, A, which when used alone will result in reduced performance, but in combination with the E188P substitution will result in a synergistic improvement.

Furthermore, the inventors have surprisingly found that substitution at position 275 is selected from N275Y, F, H, W, which when used alone will result in reduced performance, but in combination with the E188P substitution will result in a synergistic improvement.

In one embodiment, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, 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%, or at least 99%, but less than 100%, sequence identity to the amino acid sequence of the parent alpha-amylase.

In one embodiment, the present invention relates to an alpha-amylase variant comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations selected from the group consisting of: e188 + S242, E188 + K279, E188 + N275, and E188 + N275, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to a parent α -amylase selected from the group consisting of: SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, and SEQ ID NO 27.

In another embodiment, the alpha-amylase variant comprises a substitution at a position corresponding to position 185 and at least one further substitution at a position corresponding to position 239 or 276 or 272 of SEQ ID No. 2, in particular one or more combinations selected from the group consisting of: e185 + S239, E185 + K276, E185 + N272, and wherein the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, such as at least 96%, such as at least 97%, at least 98%, at least 99%, or 99%, but wherein the variant has at least a further substitution of the sequence corresponding to the sequence I + K + 201, G, preferably to the sequence I + K205, preferably to the sequence I + K + 201, preferably to the sequence I + K185 + K, preferably to the sequence of SEQ ID NO. sup.sub.sup.5, numbering is performed using SEQ ID NO 2.

In another embodiment, the alpha-amylase variant comprises a substitution at a position corresponding to position 185 and at least one further substitution at a position corresponding to position 239 or 276 or 272 of SEQ ID No. 2, in particular one or more combinations selected from the group consisting of: e185 + S239, E185 + K276, E185 + N272, and wherein the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 99%, but wherein the variant has at least a substitution of the sequence corresponding to the sequence of the I + T + K70, preferably the I + K205, G + K, and the variant, preferably the, numbering is performed using SEQ ID NO 2.

In another embodiment, the alpha-amylase variant comprises a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations of substitutions selected from the group consisting of: e188 + S242, E188 + K279, E188 + K275, E188 + N275, and wherein the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, such as at least 96%, such as at least 97%, at least 98%, or at least 99%, such as at least one additional substitution of the variant with the polypeptide of SEQ ID NO:1 or SEQ ID NO:27, such as a G188 + N85%, such as a variant comprising at least one of the substitution of the sequence of the T188 + V + K268, preferably of the T188 + N179 + K, preferably of the sequence of the type I + S242, numbering is performed using SEQ ID NO 1.

In one embodiment, the present invention relates to an alpha-amylase variant comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations selected from the group consisting of: e188 + S242, E188 + K279, E188 + K275, E188 + N275, and E188 + N275, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with a parent alpha-amylase selected from SEQ ID No. 12.

In one embodiment, the present invention relates to an alpha-amylase variant comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations selected from the group consisting of: e188 + S242, E188 + K279, E188 + K275, E188 + N275, and E188 + N275, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with a parent alpha-amylase selected from SEQ ID No. 13.

In one embodiment, the present invention relates to an alpha-amylase variant comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations selected from the group consisting of: e188 + S242, E188 + K279, E188 + K275, E188 + N275, and E188 + N275, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with a parent alpha-amylase selected from SEQ ID No. 14.

In one embodiment, the present invention relates to an alpha-amylase variant comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations selected from the group consisting of: e188 + S242, E188 + K279, E188 + K275, E188 + N275, and E188 + N275, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with a parent alpha-amylase selected from SEQ ID No. 15.

In one embodiment, the present invention relates to an alpha-amylase variant comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations selected from the group consisting of: e188 + S242, E188 + K279, E188 + K275, E188 + N275, and E188 + N275, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with a parent alpha-amylase selected from SEQ ID No. 16.

In one embodiment, the present invention relates to an alpha-amylase variant comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations selected from the group consisting of: e188 + S242, E188 + K279, E188 + K275, E188 + N275, and E188 + N275, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with a parent alpha-amylase selected from SEQ ID No. 17.

In one embodiment, the present invention relates to an alpha-amylase variant comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations selected from the group consisting of: e188 + S242, E188 + K279, E188 + K275, E188 + N275, and E188 + N275, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with a parent alpha-amylase selected from SEQ ID No. 18.

In another embodiment, the-amylase variant comprises one or more combinations of a substitution at a position corresponding to position 185 and at least one further substitution at a position corresponding to position 239 or 276 or 272 of SEQ ID No. 2, in particular a substitution selected from the group consisting of E185 + S239, E185 + S239, E185 + K276, E185 + N272, and wherein the variant has at least 60% of the same sequence as SEQ ID No. 3, at least 70% or at least 70 + K276, at least 80% of the sequence ID No. 2, at least 70, at least 90, at least 70, at least 10, at least 70, at least 10, preferably at least one or more preferably at least one more than the sequence of the sequence as the amino acid sequence as SEQ ID No. the variant as shown in SEQ ID No. 70, at least 70, preferably as the following SEQ ID No. 185 + S²⁺After a next incubation time of 20min, the variants had increased residual α -amylase activity as determined by enzyme assay.

In another embodiment, the α -amylase variant comprises a substitution at a position corresponding to position 185 and at a position corresponding to SEAt least one further substitution at position 239 or 276 or 272 of Q ID NO:2, in particular one or more combinations of substitutions selected from the group consisting of: e185 + S239, E185 + K276, E185 + N272, and wherein the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:3, and wherein the variant comprises a substitution of the I + T + K156 + K + T + 17, E185 + T +, numbering using SEQ ID NO:2 and wherein 5ppm Ca at 90 ℃, pH4.5, compared to SEQ ID NO:10²⁺After a next incubation time of 20min, the variants had increased residual α -amylase activity as determined by enzyme assay.

In another embodiment, the α -amylase variant comprises one or more combinations of a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular a substitution selected from the group consisting of E188P + S242P, E188P + K279P, E188 + K P, E188 + P + K279, E188 + K P, E188 + 36188 + P, E188 + x P + K279, E188N 188 + P, E188 + x P, at least one or at least one of the P, E188 + P, E, at least one of the P, E188, P, E P, the 36188 + P, the 36188 + P, the 36188 + P, the 36188At least 93%, at least 94%, at least 95%, e.g. at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity, and wherein said variant comprises specific substitutions corresponding to V59A + E129V + E177L + R179E + I181 + G182 + Q254S + M284V + V212T + Y268G + N293Y + T297N, and optionally N193F, numbering using SEQ ID NO:1, and wherein at 90 ℃, pH4.5, 5ppm Ca is compared to SEQ ID NO:11²⁺After a next incubation time of 20min, the variants had increased residual α -amylase activity as determined by enzyme assay.

In another embodiment, the alpha-amylase variant comprises a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations of substitutions selected from the group consisting of: e188 + S242, E188 + K279, E188 + N275, and wherein the variants have at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO. 4, and wherein the specific amino acid substitutions of the variants of S188 + S180 and the terminal G + S475 + S242, E188 + K279, E188 + K275, numbering using SEQ ID NO:4 and wherein the variant has increased thermostability and/or increased chelator stability compared to the alpha-amylase of SEQ ID NO:4 with a C-terminal deletion of the specific substitutions R180 + S181 + S243Q + G475K and amino acids 484-583.

In another embodiment, the alpha-amylase variant comprises a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations of substitutions selected from the group consisting of: e188 + S242, E188 + K279, E188 + N275, and wherein the variants have at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:5, and wherein the variant has further substitution of the G + R188 + G + R203 with the ID NO: G + R179, and wherein the variant has increased thermostability and/or increased chelator stability compared to an alpha-amylase of SEQ ID No. 5 having the specific substitution R178 x + G179 x + E187P + I203Y + R458N + T459S + D460T + G476K.

In another embodiment, the alpha-amylase variant comprises a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations of substitutions selected from the group consisting of: e188 + S242, E188 + K279, E188 + N275, and wherein the variants have at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the SEQ ID NO:5, and wherein the specific substitution of the variants with the G + G188 + G203 + G188 + G179, G188 + G180, and wherein the variant has increased thermostability and/or increased chelator stability compared to the alpha-amylase of seq id No. 5 with the specific substitutions N126Y + E132H + R178 + G179 + T180D + E187P + I203Y + Y303D + G476T + G477E.

In another embodiment, the alpha-amylase variant comprises a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations of substitutions selected from the group consisting of: e188 + S242, E188 + K279, E188 + N275, and wherein the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO. 5, and wherein the variant further comprises the substitution of the specific T188 + T203 + S178 with the F + K179, G + K179, or G188 + N179, and wherein the variant has increased thermostability and/or increased chelator stability compared to the alpha-amylase of SEQ ID NO:5 with the specific substitution N126Y + F153W + R178 + G179 + T180H + E187P + I203Y.

In another embodiment, the alpha-amylase variant comprises a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID No. 1, in particular one or more combinations of substitutions selected from the group consisting of: e188 + S242, E188 + K279, E188 + N275, and wherein the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity, and wherein the variant further comprises the substitution of the specific T + S239R + G239 with the sequence ID NO of SEQ ID NO:5, or SEQ ID NO: 80, and wherein the variant has increased thermostability and/or increased chelator stability compared to the alpha-amylase of SEQ ID NO:5 with the specific substitution N126Y + F153W + R178 + G179 + T180H + I203Y + S239Q.

In another aspect, the present invention relates to an alpha-amylase variant comprising a substitution at a position corresponding to position 188 and further substitutions at positions corresponding to positions 242 and 279 of SEQ ID No. 1, in particular a specific combination selected from the group consisting of:

E188P+S242Y+K279I；

E188P+S242L+K279W；

E188P+S242P+K279W；

E188P+S242L+K279I；

E188P+S242Y+K279W；

E188P+S242Y+K279F；

E188P+S242Y+K279H；

E188P+S242Y+K279L；

E188P+S242Y+K279Y；

E188P+S242P+K279I；

E188P+S242F+K279W；

E188P+S242H+K279W；

E188P+S242W+K279W；

wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to a parent alpha-amylase selected from the group consisting of: 1,2,3,4,5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18 and 27.

In a still further aspect, the present invention relates to an alpha-amylase variant comprising a substitution corresponding to E188P and I204Y and at least one further substitution at a position corresponding to position 242 or 279 of SEQ ID NO:1, in particular a specific combination selected from:

E188P+I204Y+S242Y；

E188P+I204Y+S242F；

E188P+I204Y+K279W；

E188P+I204Y+K279Y；

E188P+I204Y+K279F；

E188P+I204Y+K279H；

E188P+I204Y+K279I；

E188P + I204Y + K279L; and is

Wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to a parent alpha-amylase selected from the group consisting of: 1,2,3,4,5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18 and 27.

In one aspect, the number of alterations in a variant of the invention is 1-20, such as 1-10 and 1-5, such as 1,2,3,4,5,6,7,8, 9 or 10 alterations.

These amino acid changes may 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; a small deletion of typically 1 to 30 amino acids; a short amino-or carboxy-terminal extension, 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 (e.g., a polyhistidine segment, an epitope, or a binding domain).

Examples of conservative substitutions are within the following groups: 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 generally 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 a property that: altering the physicochemical properties of the polypeptide.

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, single alanine mutations are introduced at each residue in the molecule, and the resulting mutant molecules are tested for alpha-amylase activity 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, along with mutating putative contact site amino acids. See, for example, 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.

The variant may consist of a C-terminally truncated version, e.g.the variant is truncated, preferably having a length of about 490 amino acids, e.g.from 482-493 amino acids.

In another embodiment, the variant α -amylase is preferably truncated after position 484, particularly after position 485, particularly after position 486, particularly after position 487, particularly after position 488, particularly after position 489, particularly after position 490, particularly after position 491, particularly after position 492, more particularly after position 493 of SEQ ID No. 1.

The variant alpha-amylases of the invention have increased stability compared to the parent alpha-amylase, in particular the parent as disclosed in SEQ ID NO 1-18. In particular, any suitable alpha-amylase or wash assay may be used to determine increased stability. The skilled person will know how to select a suitable assay. Examples of suitable assays and conditions have been provided in the examples herein. Such increased stability may include increased thermostability at pH4.5 over the parent alpha-amylase, or chelant stability in model detergent a over the parent alpha-amylase. In a specific embodiment, the variant alpha-amylase according to the invention has an increased thermostability, in particular an increased stability, at pH4.5 over the parent alpha-amylase, as determined by an Improvement Factor (IF), wherein IF is determined as: the residual activity of the variant alpha-amylase (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃) versus the residual activity of the parent alpha-amylase (ratio of activity in a heat stressed sample compared to activity in a sample incubated at 4 ℃), in particular the variant has an IF of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0.

In another embodiment, 5ppm Ca at 90 ℃, pH4.5, as compared to a parent α -amylase, in particular a parent α -amylase selected from the group consisting of²⁺After incubation for 20min below, the variants have increased thermostability, in particular increased stability, as determined by enzyme detection, measured as residual α -amylase activity, of the polypeptides of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11.

In one embodiment, the variant is capable of producing a liquefact having a Dextrose Equivalent (DE) value higher than the DE value produced by a parent alpha-amylase, in particular a parent amylase selected from the group consisting of: 1,2,3,4,5,6,7,8, 9,10, and 11.

In one embodiment, the variant is capable of producing a liquefact having a reduced viscosity compared to a liquefact produced by a parent alpha-amylase not having the claimed double substitution, in particular a parent amylase selected from the group consisting of: 1,2,3,4,5,6,7,8, 9,10 and 11.

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

The variant may be a fusion polypeptide or a cleavable fusion polypeptide in which 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 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 also 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 [ World Drug Discovery ]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 the source or by a strain into which a polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

The parent may be a bacterial alpha-amylase. For example, the parent may be a gram-positive bacterial polypeptide, such as bacillus, geobacillus, cellulophaga.

In one aspect, the parent is a 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, or bacillus thuringiensis alpha-amylase.

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

The above probes can be used to identify parents and obtain them from other sources including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or to obtain DNA samples directly from natural materials (e.g., soil, compost, water, etc.). 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 of another microorganism or mixed DNA sample. Once the 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

Variants can be made using any mutagenesis procedure 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 the 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 the oligonucleotide are the same, allowing the sticky ends of the plasmid and the 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 conjunction 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. In one embodiment, the polynucleotide is isolated.

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, a polynucleotide 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 in Sambrook et al, 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.

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).

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 expression of the gene.

Examples of suitable mRNA stability regions are obtained from: bacillus thuringiensis cryIIIA gene (WO94/25612) and Bacillus subtilis SP82 gene (Hue et al, 1995, Journal of Bacteriology 177: 3465-.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a 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 the case 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. Additional signal peptides are described by Simonen and Palva,1993, Microbiological Reviews [ Microbiological review ]57:109- & 137.

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 referred to as a precursor enzyme or propolypeptide (or zymogen (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 aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

In the case where both the signal peptide sequence and the 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.

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 a precise location in the chromosome. 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 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 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 additionally 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.

More than one copy of a polynucleotide of the invention may be inserted into a host cell to increase production of the variant. The 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 containing amplified copies of the selectable marker gene, and thus 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. In one embodiment, the one or more control sequences are heterologous to the polynucleotide of the 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 (Geobacillus), Lactobacillus, lactococcus, Paenibacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia, Flavobacterium, Clostridium, helicobacter, Citrobacter, Neisseria, Pseudomonas, Salmonella, and Urethania.

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 genetics and genomics ]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-. Introduction of DNA into a Streptococcus cell can be achieved by: such as 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.

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 cells are cultured in a nutrient medium suitable for producing the variants 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 culture medium. If the variant is not secreted, it can be recovered from the cell lysate.

The variants can be detected using methods known in the art that are specific for the variants. 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 the variants.

The 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, 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 press (VCH Publishers), new york, 1989) to obtain substantially pure variants.

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.

Fermentation broth formulations or cell compositions

The invention also relates to fermentation broth formulations or cell compositions comprising the polypeptides of the invention. The fermentation broth product further comprises additional components used in the fermentation process, such as, for example, cells (including host cells containing a gene encoding a polypeptide of the invention, which host cells are used to produce the polypeptide of interest), cell debris, biomass, fermentation medium, and/or fermentation product. In some embodiments, the composition is a cell-killed whole broth comprising one or more organic acids, killed cells and/or cell debris, and culture medium.

The term "fermentation broth" as used herein refers to a preparation produced by fermentation of a cell that has not undergone or has undergone minimal recovery and/or purification. For example, a fermentation broth is produced when a microbial culture is grown to saturation by incubation under carbon-limited conditions that allow protein synthesis (e.g., expression of an enzyme by a host cell) and secretion of the protein into the cell culture medium. The fermentation broth may contain an unfractionated or fractionated content of the fermented material obtained at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises spent culture medium and cell debris present after removal of microbial cells (e.g., filamentous fungal cells), e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or non-viable microbial cells.

In one embodiment, the fermentation broth formulation and cell composition comprises a first organic acid component (comprising at least one organic acid of 1-5 carbons and/or salt thereof) and a second organic acid component (comprising at least one organic acid of 6 or more carbons and/or salt thereof). In a particular embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, salts thereof, or mixtures of two or more of the foregoing.

In one aspect, the composition contains one or more organic acids and optionally further contains killed cells and/or cell debris. In one embodiment, the killed cells and/or cell debris are removed from the cell-killed whole broth to provide a composition free of these components.

The fermentation broth formulations or cell compositions may further comprise preservatives and/or antimicrobial (e.g., bacteriostatic) agents, including but not limited to sorbitol, sodium chloride, potassium sorbate, and other agents known in the art.

The cell-killed whole broth or composition may contain unfractionated contents of the fermented material obtained at the end of fermentation. Typically, the cell-killing whole broth or composition contains spent culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon limiting conditions to allow protein synthesis. In some embodiments, the cell-killing whole broth or composition contains spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art may be used to permeabilize and/or lyse microbial cells present in a cell-killed whole broth or composition.

The whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, media components, and/or one or more insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.

The whole broth formulations and cell compositions of the invention may be produced by the methods described in WO 90/15861 or WO 2010/096673.

Composition comprising a metal oxide and a metal oxide

The present invention also relates to compositions comprising the variant alpha-amylases of the invention.

These compositions may comprise the variant alpha-amylase of the invention as a major enzyme component, e.g., a one-component composition. Alternatively, the composition may comprise a plurality of enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of: protease, glucoamylase, beta-amylase, pullulanase. In a particular embodiment, the composition comprises a variant alpha-amylase of the invention and a protease, in particular a protease from a pyrococcus species or a thermophilic coccus species, or a protease from thermoascus aurantiacus.

In one embodiment, the protease is selected from the group consisting of the S8 protease from Pyrococcus furiosus shown in SEQ ID NO. 19 or a protease having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO. 19.

In another embodiment, the protease is selected from the group consisting of a variant thermolysin orange, wherein the variant protease comprises one of the following combinations of mutations:

D79L+S87P+A112P+D142L；

D79L + S87P + D142L; or

A27K + D79L + Y82F + S87G + D104P + a112P + a126V + D142L; and said protease variant has at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the polypeptide of SEQ ID NO. 20.

For use of the alpha-amylase variants of the invention in detergent compositions, the non-limiting list of composition components set forth below is suitable for such use, for example, to aid or enhance cleaning performance, to treat a substrate to be cleaned, or to modify the aesthetics of the composition as is the case with perfumes, colorants, dyes or the like. The level of any such component incorporated into any composition is in addition to any material previously recited for incorporation. The precise nature of these additional components and the levels of incorporation thereof will depend on the physical form of the composition and the nature of the cleaning operation in which the composition is to be used. 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 contain additional functionality as will be appreciated by the skilled person. Amounts in percent are by weight (wt%) of the composition, unless otherwise indicated.

Suitable component materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, dispersants, enzymes, and enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay removal/anti-redeposition agents, brighteners, suds suppressors, dyes, hueing dyes, perfumes, perfume delivery systems, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, solvents and/or pigments. In addition to the following disclosure, suitable examples and levels of use of such other components are found in US 5576282, US6306812 and US6326348, which are hereby incorporated by reference.

Thus, in certain embodiments, the present invention does not contain one or more of the following adjunct materials: surfactants, soaps, builders, chelating agents, dye transfer inhibiting agents, dispersants, additional enzymes, enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, perfume delivery systems, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, solvents and/or pigments. However, when one or more components are present, such one or more components may be present as detailed below:

surface active agentThe composition according to the invention may comprise a surfactant or surfactant system, wherein the surfactant may be selected from the group consisting of non-ionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi-polar non-ionic surfactants and mixtures thereof. When present, the surfactant is typically present at a level of from 0.1 wt% to 60 wt%, from 0.2 wt% to 40 wt%, from 0.5 wt% to 30 wt%, from 1 wt% to 50 wt%, from 1 wt% to 40 wt%, from 1 wt% to 30 wt%, from 1 wt% to 20 wt%, from 3 wt% to 10 wt%, from 3 wt% to 5 wt%, from 5 wt% to 40 wt%, from 5 wt% to 30 wt%, from 5 wt% to 15 wt%, from 3 wt% to 20 wt%, from 3 wt% to 10 wt%, from 8 wt% to 12 wt%, from 10 wt% to 12 wt%, from 20 wt% to 25 wt%, or from 25 wt% to 60 wt%.

Suitable anionic detersive surfactants include sulphate and sulphonate detersive surfactants.

Suitable sulphonate detersive surfactants include alkyl benzene sulphonates, in one aspect C_10-13An alkylbenzene sulfonate. Suitable alkyl benzene sulfonates (LAS) can be obtained by sulfonating commercially available Linear Alkyl Benzenes (LAB); suitable LAB include low 2-phenyl LAB, e.g.

OrOther suitable LABs include high 2-phenyl LABs, e.g.Suitable anionic detersive surfactants are alkyl benzene sulphonates obtained by DETAL catalysed processes, but other synthetic routes (e.g. HF) may also be suitable. In one aspect, a magnesium salt of LAS is used.

Suitable sulphate detersive surfactants include alkyl sulphates, in one aspect C_8-18Alkyl sulfates, or predominantly C₁₂An alkyl sulfate.

Further suitable sulphate detersive surfactants are alkyl alkoxylated sulphates, in one aspect alkyl ethoxylated sulphates, in one aspect C_8-18Alkyl alkoxylated sulfates, in another aspect C_8-18Alkyl ethoxylated sulfates, typically alkyl alkoxylated sulfates having an average degree of alkoxylation of from 0.5 to 20 or from 0.5 to 10, typically the alkyl alkoxylated sulfate is C_8-18An alkyl ethoxylated sulfate having an average degree of ethoxylation of from 0.5 to 10, from 0.5 to 7, from 0.5 to 5, or from 0.5 to 3.

The alkyl sulfates, alkyl alkoxylated sulfates and alkylbenzene sulfonates may be linear or branched, substituted or unsubstituted.

The detersive surfactant may be a mid-chain branched detersive surfactant, in one aspect a mid-chain branched anionic detersive surfactant, in one aspect a mid-chain branched alkyl sulphate and/or a mid-chain branched alkyl benzene sulphonate, for example a mid-chain branched alkyl sulphate. In one aspect, the mid-chain branch is C_1-4Alkyl, typically methyl and/or ethyl.

Non-limiting examples of anionic surfactants include sulfates and sulfonates, particularly Linear Alkylbenzene Sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2, 3-diylbis (sulfates), hydroxyalkane sulfonates and disulfonates, Alkyl Sulfates (AS) such AS Sodium Dodecyl Sulfate (SDS), Fatty Alcohol Sulfates (FAS), Primary Alcohol Sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known AS alcohol ethoxy sulfates or fatty alcohol ether sulfates), Secondary Alkane Sulfonates (SAS), Paraffin Sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, alpha-sulfonated fatty acid methyl esters (alpha-SFMe or SES) (including methyl 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 soap, and combinations thereof.

Suitable nonionic detersive surfactants are selected from the group consisting of: c₈-C₁₈Alkyl ethoxylates, e.g.

C₆-C₁₂An alkylphenol alkoxylate, wherein the alkoxylate unit may be an ethyleneoxy unit, a propyleneoxy unit, or a mixture thereof; c₁₂-C₁₈Alcohol and C₆-C₁₂Condensates of alkylphenols with ethylene oxide/propylene oxide block polymers, e.g.

C₁₄-C₂₂Mid-chain branched alcohols; c₁₄-C₂₂Mid-chain branched alkyl alkoxylates, typically having an average degree of alkoxylation of from 1 to 30; an alkyl polysaccharide, in one aspect an alkyl polyglycoside; polyhydroxy fatty acid amides; ether-terminated poly (alkoxylated) alcohol surfactants; and mixtures thereof.

Suitable nonionic detersive surfactants include alkyl polyglycosides and/or alkyl alkoxylated alcohols.

In one aspect, the nonionic detersive surfactant comprises an alkyl alkoxylated alcohol, in one aspect C_8-18An alkyl alkoxylated alcohol, a fatty alcohol,e.g. C_8-18An alkyl alkoxylated alcohol, which may have an average degree of alkoxylation of from 1 to 50, from 1 to 30, from 1 to 20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may be C_8-18An alkyl ethoxylated alcohol having an average degree of ethoxylation of from 1 to 10, from 1 to 7, more typically from 1 to 5 or from 3 to 7. The alkyl alkoxylated alcohol may be linear or branched, and substituted or unsubstituted. Suitable nonionic surfactants include

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 Monoethanolamide (FAM), Fatty Acid Diethanolamide (FADA), Ethoxylated Fatty Acid Monoethanolamide (EFAM), Propoxylated Fatty Acid Monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (glucamide (GA), or Fatty Acid Glucamide (FAGA)), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

Suitable cationic detersive surfactants include alkyl pyridine compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl trisulfonium compounds, and mixtures thereof.

Suitable cationic detersive surfactants are quaternary ammonium compounds having the general formula: (R)₁)(R₂)(R₃)N⁺X^-Wherein R is a linear or branched, substituted or unsubstituted C_6-18Alkyl or alkenyl moieties, R₁And R₂Independently selected from methyl or ethyl moieties, R₃Is a hydroxy, hydroxymethyl or hydroxyethyl moiety, X is an anion providing charge neutrality, suitable anions include halides, such as chloride; a sulfate salt; and a sulfonate salt. Suitable cationsThe detersive surfactant is mono-C_6-18Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride. A highly suitable cationic detersive surfactant is mono-C_8-10Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride, mono C_10-12Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride and mono-C₁₀Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride.

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.

Suitable amphoteric/zwitterionic surfactants include amine oxides and betaines (e.g., alkyl dimethyl betaines, sulfobetaines), or combinations thereof. The amine neutralized anionic surfactant-anionic surfactant and adjunct anionic co-surfactant of the present invention may be present in the acid form and the acid form may be neutralized to form a surfactant salt which is desired for use in the detergent compositions of the present invention. Typical reagents for neutralization include metal counter-ion bases such as hydroxides, e.g., NaOH or KOH. Further preferred agents for neutralising the anionic surfactant of the invention and the co-anionic surfactant or co-surfactant in its acid form include ammonia, amines or alkanolamines. Alkanolamines are preferred. Suitable non-limiting examples include monoethanolamine, diethanolamine, triethanolamine, and other linear or branched alkanolamines known in the art; for example, highly preferred alkanolamines include 2-amino-1-propanol, 1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. The amine neutralization may be carried out to a full or partial extent, for example, part of the anionic surfactant mixture may be neutralized by sodium or potassium, and part of the anionic surfactant mixture may be neutralized by an amine or alkanolamine.

Non-limiting examples of semi-polar surfactants include Amine Oxides (AO), such as alkyl dimethylamine oxides.

Surfactant systems comprising a mixture of one or more anionic surfactants, and additionally one or more nonionic surfactants, and optionally additional surfactants such as cationic surfactants, may be preferred. Preferred weight ratios of anionic to nonionic surfactant are at least 2:1, or at least 1:1 to 1: 10.

In one aspect, the surfactant system can include a mixture of isoprenoid surfactants represented by formula a and formula B:

wherein Y is CH₂Or none, and Z may be selected such that the resulting surfactant is selected from the following surfactants: alkyl carboxylate surfactants, alkyl polyalkoxy surfactants, alkyl anionic polyalkoxy sulfate surfactants, alkyl glyceride sulfonate surfactants, alkyl dimethyl amine oxide surfactants, alkyl polyhydroxy-based surfactants, alkyl phosphate ester surfactants, alkyl glycerol sulfonate surfactants, alkyl polygluconate surfactants, alkyl polyphosphate surfactants, alkyl phosphonate surfactants, alkyl polyglycoside surfactants, alkyl monoglycoside surfactants, alkyl diglycoside surfactants, alkyl sulfosuccinate surfactants, alkyl disulfate surfactants, alkyl disulfonate surfactants, alkyl sulfosuccinamate surfactants, alkyl glucamide surfactants, alkyl taurate surfactants, alkyl sarcosinate surfactants, alkyl ester surfactants, alkyl amine sulfonates, alkyl amine, Alkyl glycinate surfactants, alkyl isethionate surfactants, alkyl dialkanolamide surfactants, alkyl monoalkanolamide sulfate surfactants, alkyl dihydroxyacetamide sulfate surfactants, alkyl glyceride sulfate surfactants, alkyl glycerol ether surfactantsSurfactants, alkyl glyceryl ether sulfate surfactants, alkyl methyl ester sulfonate surfactants, alkyl polyglycerol ether surfactants, alkyl amidopropyl betaine surfactants, alkyl allylated quaternary ammonium salt based surfactants, alkyl monohydroxyalkyl-di-alkylated quaternary ammonium salt based surfactants, alkyl di-hydroxyalkyl monoalkyl quaternary ammonium salt based surfactants, alkylated quaternary ammonium salt surfactants, alkyl trimethylammonium quaternary ammonium salt surfactants, alkyl polyhydroxyalkyl oxypropyl quaternary ammonium salt based surfactants, alkyl glyceride quaternary ammonium salt surfactants, alkyl ethyleneglycolamine quaternary ammonium salt surfactants, alkyl monomethylhydroxyethyl quaternary ammonium salt surfactants, alkyl dimethylmonohydroxyethyl quaternary ammonium salt surfactants, alkyl trimethylammonium ammonium surfactants, alkyl imidazoline based surfactants, alkene-2-yl-succinate surfactants, alkyl a-sulfonated carboxylic acid alkyl ester surfactants, alkyl triisoalkyl triisopropanolate surfactants, alkyl trimethylammonium sulfate based surfactants, alkyl imidazoline based surfactants, alkyl-2-yl-succinate surfactants, alkyl-sulfonated carboxylic acid alkyl-alcohol ether sulfonate surfactants, alkyl-sulfonic acid alkyl ester surfactants, alkyl-alcohol sulfonate surfactants, alkyl-alcohol-substituted-ammonium surfactants, such as alkyl-substituted-alkyl-substituted-ammonium surfactants, alkyl-substituted-ammonium surfactants, alkyl-substituted-ammonium surfactants, alkyl-substituted-ammonium surfactants, alkyl-substituted-ammonium surfactants, alkyl-substitutedL-amino-3-propanol, or mixtures thereof. In one embodiment, the composition contains from 5% to 97% of one or more non-isoprenoid surfactants, and one or more adjunct detergent additives, wherein the weight ratio of surfactant having formula a to surfactant having formula B is from 50:50 to 95: 5.

SoapThe compositions herein may contain soap. Without being limited by theory, it may be desirable to include soap because it acts partially as a surfactant and partially as a builder, and may be used to inhibit suds, and in addition, may advantageously interact with various cationic compounds of the composition to enhance softness of textile fabrics treated with the compositions of the present invention. Any soap known in the art for use in laundry detergents may be utilized. In one embodiment, the compositions contain from 0 wt% to 20 wt%, from 0.5 wt% to 20 wt%, from 4 wt% to 10 wt%, or from 4 wt% to 7 wt% soap.

Examples of soaps useful herein include oleic acid soaps, palmitic acid soaps, palm kernel fatty acid soaps, and mixtures thereof. Typical soaps are in the form of mixtures of fatty acid soaps having different chain lengths and degrees of substitution. One such mixture is topped palm kernel fatty acid.

In one embodiment, the soap is selected from free fatty acids. Suitable fatty acids are saturated and/or unsaturated and can be obtained from natural sources such as vegetable or animal esters (e.g., palm kernel oil, palm oil, coconut oil, babassu oil, safflower oil, tall oil, castor oil, tallow and fish oils, fats and oils, and mixtures thereof), or synthetically produced (e.g., carbon monoxide hydrogenated via oxidation of petroleum or via the fischer Tropsch process).

Examples of suitable saturated fatty acids for use in the compositions of the present invention include capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid and behenic acid. Suitable classes of unsaturated fatty acids include: palmitoleic, oleic, linoleic, linolenic, and ricinoleic acids. Examples of preferred fatty acids are saturated Cn fatty acids, saturated Ci₂-Ci₄Fatty acid, fatty acid,And saturated or unsaturated Cn to Ci₈Fatty acids and mixtures thereof.

When present, the weight ratio of fabric softening cationic co-surfactant to fatty acid is preferably from about 1:3 to about 3:1, more preferably from about 1:1.5 to about 1.5:1, most preferably about 1:1.

The levels of soap and non-soap anionic surfactant herein are percentages by weight of the detergent composition specified on an acid basis. However, as is generally understood in the art, in practice anionic surfactants and soaps are neutralized using sodium, potassium or alkanolammonium bases such as sodium hydroxide or monoethanolamine.

Hydrotropic agentThe composition of the invention may comprise one or more hydrotropes. 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 (so-called amphiphilic character, as known from surfactants); however, the molecular structure of hydrotropes generally does not favor spontaneous self-aggregation, see, e.g., Hodgdon and Kaler (2007), Current Opinion in Colloid&Interface Science (New Science of colloid and Interface)]12: 121-. Hydrotropes do not exhibit a critical concentration above which self-aggregation and lipid formation into micelles, lamellae or other well-defined mesophases as found with surfactants occur. In contrast, many hydrotropes exhibit 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 (as in the process of compressing liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

The detergent may contain from 0 to 10 wt%, for example from 0 to 5 wt%, 0.5 wt% to 5 wt%, or from 3 wt% to 5 wt% 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.

BuilderThe composition of the invention may comprise one or more builders, co-builders, builder systems or mixtures thereof. When a builder is used, the cleaning composition will typically comprise from 0 to 65 wt%, at least 1 wt%, from 2 wt% to 60 wt% or from 5 wt% to 10 wt% builder. In dishwashing cleaning compositions, the level of builder is typically from 40 wt% to 65 wt% or from 50 wt% to 65 wt%. The composition may be substantially free of builder; by substantially free is meant "no intentionally added" zeolite and/or phosphate. Typical zeolite builders include zeolite a, zeolite P and zeolite MAP. A typical phosphate builder is sodium tripolyphosphate.

The builder and/or co-builder may in particular be a chelating agent which forms a water-soluble complex with Ca and Mg. Any builder and/or co-builder known in the art for use in detergents may be used. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines (e.g., 2-aminoethan-1-ol (MEA), iminodiethanol (DEA), and 2, 2', 2 "-nitrilotriethanol (TEA)), and carboxymethyl inulin (CMI), and combinations thereof.

The cleaning composition may include a co-builder alone, or in combination with a builder (e.g., a zeolite builder). Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly (acrylic acid) (PAA) or co (acrylic acid/maleic acid) (PAA/PMA). Additional non-limiting examples include citrates, chelating agents (e.g., aminocarboxylates, aminopolycarboxylates, and phosphates), and alkyl 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-diylbis (phosphonic acid) (HEDP), ethylenediaminetetra (methylene) tetra (phosphonic acid) (EDTMPA), diethylenetriaminepenta (methylene) penta (phosphonic acid) (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- (hydroxyethyl) -ethylenediaminetriacetic acid (HEDTA), Diethanolglycine (DEG), diethylenetriaminepenta (methylenephosphonic acid) (DTPMP), aminotri (methylenephosphonic Acid) (ATMP), and combinations and salts thereof. Further exemplary builders and/or co-builders are described in e.g. WO09/102854, US 5977053.

Chelating agents and crystal growth inhibitorsThe compositions herein may contain a chelating agent and/or a crystal growth inhibitor. Suitable molecules include copper, ionic and/or manganese chelating agents and mixtures thereof. Suitable molecules include DTPA (diethylenetriaminepentaacetic acid), HEDP (hydroxyethane diphosphonic acid), DTPMP (diethylenetriaminepenta (methylenephosphonic acid)), 1, 2-dihydroxybenzene-3, 5-disulfonic acid disodium salt hydrate, ethylenediamine, diethylenetriamine, ethylenediamine disuccinic acid (EDDS), N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycinAcids (DHEG), ethylenediamine tetrapropionic acid (EDTP), carboxymethyl inulin, and 2-phosphonobutane 1,2, 4-tricarboxylic acid (C:)

AM) and derivatives thereof. Typically, the composition may comprise from 0.005 wt% to 15 wt%, or from 3.0 wt% to 10 wt% of the chelating agent or crystal growth inhibitor.

Bleaching componentBleach components suitable for incorporation in the methods and compositions of the present invention include one or a mixture of more than one bleach component. Suitable bleaching components include bleach catalysts, photobleaches, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, and mixtures thereof. Typically, when a bleach component is used, the compositions of the present invention may comprise from 0 to 30 wt%, from 0.00001 wt% to 90 wt%, from 0.0001 wt% to 50 wt%, from 0.001 wt% to 25 wt% or from 1 wt% to 20 wt%. Examples of suitable bleaching components include:

(1) preformed peracid: suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of: a preformed peroxyacid or salt thereof, typically a peroxycarboxylic acid or salt thereof, or a peroxysulfuric acid or salt thereof.

The preformed peroxyacid or salt thereof is preferably a peroxycarboxylic acid or salt thereof, typically having a chemical structure corresponding to the formula:

wherein: r¹⁴Selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic groups; r¹⁴The groups may be linear or branched, substituted or unsubstituted; and Y is any suitable counterion to achieve charge neutrality, preferably Y is selected from hydrogen, sodium or potassium. Preferably, R¹⁴Is a straight or branched, substituted or unsubstituted C_6-9An alkyl group. Preferably, the peroxyacid or salt thereof is selected from peroxycaproic acid, peroxyheptanoic acid, peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, and salts thereof, or any combination thereof. Especially preferred are peroxyThe acid is a phthalimido-peroxy-alkanoic acid, especially-phthalimido-peroxy caproic acid (PAP). Preferably, the peroxy acid or salt thereof has a melting point in the range of from 30 ℃ to 60 ℃.

The pre-formed peroxyacid or salt thereof may also be peroxysulfuric acid or salt thereof, typically having a chemical structure corresponding to the formula:

wherein: r¹⁵Selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic groups; r¹⁵The groups may be linear or branched, substituted or unsubstituted; and Z is any suitable counterion to achieve charge neutrality, preferably Z is selected from hydrogen, sodium or potassium. Preferably, R¹⁵Is a straight or branched, substituted or unsubstituted C_6-9An alkyl group. Preferably, such bleach components may be present in the compositions of the present invention in an amount of from 0.01 wt% to 50 wt% or from 0.1 wt% to 20 wt%.

(2) Sources of hydrogen peroxide include, for example, inorganic perhydrate salts including alkali metal salts such as perborate (usually monohydrate or tetrahydrate), percarbonate, persulfate, perphosphate, sodium salts of persilicate salts and mixtures thereof. In one aspect of the invention, the inorganic perhydrate salts are for example those selected from the group consisting of: perborate salts, sodium salts of percarbonate salts and mixtures thereof. When used, inorganic perhydrate salts are typically present in amounts of from 0.05% to 40% or from 1% to 30% by weight of the overall composition and are typically incorporated in such compositions as crystalline solids which may be coated. Suitable coatings include: inorganic salts, such as alkali metal silicates, carbonates or borates or mixtures thereof, or organic materials, such as water-soluble or water-dispersible polymers, waxes, oils or fatty soaps. Preferably, such bleach components may be present in the compositions of the present invention in an amount of from 0.01 wt% to 50 wt% or from 0.1 wt% to 20 wt%.

(3) The term bleach activator is herein intended to mean a compound that reacts with hydrogen peroxide to form a peracid via a perhydrolysis reaction. The peracid formed in this way constitutes an activated bleaching agent. Suitable bleach activators to be used herein include those belonging to the class of esters, amides, imides or anhydrides. Suitable bleach activators are those having R- (C ═ O) -L, where R is an alkyl group (preferably branched), from 6 to 14 carbon atoms or from 8 to 12 carbon atoms when the bleach activator is hydrophobic, and less than 6 carbon atoms or less than 4 carbon atoms when the bleach activator is hydrophilic; and L is a leaving group. Examples of suitable leaving groups are benzoic acid and derivatives thereof-especially benzenesulfonates. Suitable bleach activators include dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or salts thereof, 3,5, 5-trimethylhexanoyloxybenzenesulfonate, Tetraacetylethylenediamine (TAED), sodium 4- [ (3,5, 5-trimethylhexanoyl) oxy ] benzene-1-sulfonate (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 family of bleach activators is disclosed in EP 624154 and particularly preferred in that 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). Suitable bleach activators are also disclosed in WO 98/17767. Although any suitable bleach activator may be employed, in one aspect of the present invention, the subject cleaning compositions may comprise NOBS, TAED, or mixtures thereof. When present, the peracid and/or bleach activator is typically present in the composition in an amount of from 0.1 wt% to 60 wt%, from 0.5 wt% to 40 wt%, or from 0.6 wt% to 10 wt%, based on the fabric and home care composition. One or more hydrophobic peracids or precursors thereof may be used in combination with one or more hydrophilic peracids or precursors thereof. Preferably, such bleach components may be present in the compositions of the present invention in an amount of from 0.01 wt% to 50 wt% or from 0.1 wt% to 20 wt%.

The amounts of hydrogen peroxide source and peracid or bleach activator can be selected such that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or even from 2:1 to 10: 1.

(4) Diacyl peroxides-preferred diacyl peroxide bleaching species include those selected from diacyl peroxides having the general formula: r¹-C(O)-OO-(O)C-R²Wherein R is¹Is represented by C₆-C₁₈Alkyl, preferably straight chain containing at least 5 carbon atoms and optionally containing one or more substituents (e.g. -N)⁺(CH₃)₃-COOH or-CN) and/or C with one or more interrupting moieties (e.g. -CONH-or-CH-) interposed between adjacent carbon atoms of the alkyl group₆-C₁₂An alkyl group, and R²Denotes an aliphatic group which is partially compatible with peroxide, so that R¹And R²Together containing a total of from 8 to 30 carbon atoms. In a preferred aspect, R¹And R²Is a straight chain unsubstituted C₆-C₁₂An alkyl chain. Most preferably, R¹And R²Are the same. Diacyl peroxides (wherein R¹And R²Are all C₆-C₁₂Alkyl groups) are particularly preferred. Preferably, the R group (R)₁Or R₂) At least one, most preferably only one, does not contain a branching or pendant ring at position α, or preferably does not contain a branching or pendant ring at either position α or β, or most preferably does not contain a branching or pendant ring at either position α or β or gamma in a further preferred embodiment, the DAP may be asymmetric such that the R1 acyl group is preferably rapidly hydrolyzed to produce a peracid, but the hydrolysis of the R2 acyl group is slow.

The tetraacyl peroxide bleaching species is preferablyA tetraacylperoxide selected from the group consisting of the following formulas: r³-C(O)-OO-C(O)-(CH₂)n-C(O)-OO-C(O)-R³Wherein R is³Is represented by C₁-C₉Alkyl or C₃-C₇And n represents an integer from 2 to 12 or 4 to 10 inclusive.

Preferably, the diacyl and/or tetraacyl peroxide bleaching species is present in an amount sufficient to provide at least 0.5ppm, at least 10ppm, or at least 50ppm by weight of the wash liquor. In a preferred embodiment, these bleaching species are present in an amount sufficient to provide from 0.5ppm to 300ppm, from 30ppm to 150ppm by weight of the wash liquor.

Preferably, the bleach component comprises a bleach catalyst (5 and 6).

(5) Preferred are organic (non-metallic) bleach catalysts, including bleach catalysts capable of accepting an oxygen atom from a peroxyacid and/or salt thereof and transferring said oxygen atom to an oxidizable substrate. Suitable bleach catalysts include, but are not limited to: iminium cations and polyions; an imine zwitterion; a modified amine; a modified amine oxide; n-sulfonylimines; n-phosphoryl imine; an N-acylimine; thiadiazole dioxides; a perfluoroimine; cyclic sugar ketones and mixtures thereof.

Suitable iminium cations and polyions include, but are not limited to, N-methyl-3, 4-dihydroisoquinolinium tetrafluoroborates prepared as described in Tetrahedron (1992), 49(2), 423-38 (e.g., compound 4, page 433); n-methyl-3, 4-dihydroisoquinolinium p-toluenesulfonate salt, prepared as described in US 5360569 (e.g. column 11, example 1); and n-octyl-3, 4-dihydroisoquinolinium p-toluenesulfonate salt, prepared as described in US 5360568 (e.g. column 10, example 3).

Suitable iminium zwitterions include, but are not limited to, N- (3-sulfopropyl) -3, 4-dihydroisoquinolinium, inner salts, prepared as described in US 5576282 (e.g., column 31, example II); n- [2- (sulfoxy) dodecyl ] -3, 4-dihydroisoquinolinium, inner salt, prepared as described in US 5817614 (e.g., column 32, example V); 2- [3- [ (2-ethylhexyl) oxy ] -2- (sulfooxy) propyl ] -3, 4-dihydroisoquinolinium, inner salt, prepared as described in WO 05/047264 (e.g. page 18, example 8), and 2- [3- [ (2-butyloctyl) oxy ] -2- (sulfooxy) propyl ] -3, 4-dihydroisoquinolinium, inner salt.

Suitable modified amine oxygen transfer catalysts include, but are not limited to, 1,2,3, 4-tetrahydro-2-methyl-1-isoquinolinol, which can be prepared according to the procedure described in Tetrahedron Letters (1987),28(48), 6061-. Suitable modified amine oxide oxygen transfer catalysts include, but are not limited to, 1-hydroxy-N-oxo-N- [2- (sulfooxy) decyl ] -1,2,3, 4-tetrahydroisoquinoline sodium.

Suitable N-sulfonylimido oxygen transfer catalysts include, but are not limited to, 3-methyl-1, 2-benzisothiazole 1, 1-dioxide, which can be prepared according to the procedure described in Journal of Organic Chemistry (1990), 55(4), 1254-61.

Suitable N-phosphonoimine oxygen transfer catalysts include, but are not limited to, [ R- (E) ] -N- [ (2-chloro-5-nitrophenyl) methylene ] -p-phenyl-p- (2,4, 6-trimethylphenyl) phosphinic acid amide, which can be prepared according to the procedure described in Journal of the Chemical Society [ Journal of Chemical Society ], Chemical Communications [ Chemical communication ] (1994), (22), 2569-70.

Suitable N-acylimine oxygen transfer catalysts include, but are not limited to, N- (phenylmethylene) acetamides which may be prepared according to the procedures described in Polish Journal of Chemistry [ Journal of Polish Chemistry ] (2003),77(5), 577-.

Suitable thiadiazole dioxide oxygen transfer catalysts include, but are not limited to, 3-methyl-4-phenyl-1, 2, 5-thiadiazole 1, 1-dioxide, which may be prepared according to the procedures described in US 5753599 (column 9, example 2).

Suitable perfluoroimine oxygen transfer catalysts include, but are not limited to, (Z) -2,2,3,3,4,4, 4-heptafluoro-N- (nonafluorobutyl) butyrylimine fluoride, which can be prepared according to the procedure described in Tetrahedron Letters (1994),35(34), 6329-30.

Suitable cyclic sugar ketone oxygen transfer catalysts include, but are not limited to, 1,2:4, 5-di-O-isopropylidene-D-erythro-2, 3-hexanedione (hexodiuro) -2, 6-pyranose as prepared in US 6649085 (column 12, example 1).

Preferably, the bleach catalyst comprises an iminium and/or carbonyl functionality and is typically capable of forming an oxaziridinium and/or dioxirane functionality upon acceptance of an oxygen atom, particularly from a peroxyacid and/or salt thereof. Preferably, the bleach catalyst comprises a peroxyiminium functional group and/or is capable of forming a peroxyiminium functional group upon receipt of an oxygen atom, especially upon receipt of an oxygen atom from a peroxyacid and/or salt thereof. Preferably, the bleach catalyst comprises a cyclic iminium functional group, preferably wherein the cyclic moiety has a ring size of from five to eight atoms (including nitrogen atoms), preferably six atoms. Preferably, the bleach catalyst comprises an aryliminium functional group, preferably a bicyclic aryliminium functional group, preferably a3, 4-dihydroisoquinolinium functional group. Typically, the imine functional group is a quaternary imine functional group and is typically capable of forming a quaternary peroxoimine cationic functional group upon receiving an oxygen atom, in particular upon receiving an oxygen atom from a peroxyacid and/or salt thereof. In another aspect, the detergent composition comprises a detergent having a logP of no greater than 0, no greater than-0.5, no greater than-1.0, no greater than-1.5, no greater than-2.0, no greater than-2.5, no greater than-3.0, or no greater than-3.5_o/wThe bleaching component of (1). The method for determining logP is described in more detail below_o/wThe method of (1).

Typically, the bleaching ingredient is capable of producing a product having an X of from 0.01 to 0.30, from 0.05 to 0.25, or from 0.10 to 0.20_SOThe bleaching species of (2). The method for determining X is described in more detail below_SOThe method of (1). For example, bleaching components having an isoquinolinium structure are capable of producing bleaching species having a peroxyimine cation structure. In this example, X_SOX being a peroxyimine positive ion bleaching species_SO。

Preferably, the bleach catalyst has a chemical structure corresponding to the formula:

wherein: n and m are independently 0 to 4, preferably both n and m are 0; each R¹Independently selected from substituted or unsubstituted groups selected from the group consisting of: hydrogen, alkyl, cycloalkyl, aryl, fused aryl, heterocycle, fused heterocycle, nitro, halo, cyano, sulfonate, alkoxy, keto, carboxy, and alkoxycarbonyl; and any two vicinal R¹The substituents may be combined to form a fused aryl, fused carbocyclic ring, or fused heterocyclic ring; each R²Independently selected from substituted or unsubstituted groups independently selected from the group consisting of: hydrogen, hydroxyl, alkyl, cycloalkyl, alkaryl, aryl, aralkyl, alkylene, heterocyclic, alkoxy, arylcarbonyl, carboxyalkyl, and amide groups; any R²May be combined with any other R²Joined together to form a portion of a common ring; any geminal R²May be combined to form a carbonyl group; and any two R²May be combined to form substituted or unsubstituted fused unsaturated moieties; r³Is C₁To C₂₀Substituted or unsubstituted alkyl; r⁴Is hydrogen or Q_t-part a, wherein: q is a branched or unbranched alkene, t ═ 0 or 1, and a is an anionic group selected from the group consisting of: OSO₃ ^-、SO₃ ^-、CO₂ ^-、OCO₂ ^-、OPO₃ ^2-、OPO₃H^-And OPO₂ ^-；R⁵Is hydrogen or moiety-CR¹¹R¹²-Y-G_b-Y_c-[(CR⁹R¹⁰)_y-O]_k-R⁸Wherein: each Y is independently selected from the group consisting of: o, S, N-H or N-R⁸(ii) a And each R⁸Independently selected from the group consisting of: alkyl, aryl and heteroaryl, said moieties being substituted or unsubstituted, and said moieties, whether substituted or unsubstituted, having less than 21 carbons; each G is independently selected from the group consisting of: CO, SO₂SO, PO and PO₂；R⁹And R¹⁰Independently selected from the group consisting ofThe group consisting of: h and C₁-C₄An alkyl group; r¹¹And R¹²Independently selected from the group consisting of: h and alkyl, or when taken together may combine to form a carbonyl; b is 0 or 1; c may be 0 or 1, but if b is 0, c must be 0; y is an integer from 1 to 6; k is an integer from 0 to 20; r⁶Is H, or is an alkyl, aryl or heteroaryl moiety; the moiety is substituted or unsubstituted; and if X is present, it is a suitable charge balancing counterion, when R is present⁴X is preferably present when hydrogen, suitable X include, but are not limited to: chloride, bromide, sulfate, methosulfate, sulfonate, p-toluenesulfonate, boron tetrafluoride phosphate.

In one embodiment of the invention, the bleach catalyst has a structure corresponding to the general formula:

wherein R is¹³Is a branched alkyl group containing from three to 24 carbon atoms (including branched carbon atoms) or a straight alkyl group containing from one to 24 carbon atoms; preferably, R¹³Is a branched alkyl group containing from eight to 18 carbon atoms or a straight alkyl group containing from eight to eighteen carbon atoms; preferably, R¹³Selected from the group consisting of: 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, isononyl, isodecyl, isotridecyl and isotentadecyl; preferably, R¹³Selected from the group consisting of: 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

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

When present, the peracid and/or bleach activator is typically present in the composition in an amount of from 0.1 wt% to 60 wt%, from 0.5 wt% to 40 wt%, or from 0.6 wt% to 10 wt%, based on the composition. One or more hydrophobic peracids or precursors thereof may be used in combination with one or more hydrophilic peracids or precursors thereof.

(6) Metal-containing bleach catalysts-the bleach component may be provided by a catalytic metal complex. One type of metal-containing bleach catalyst is a catalytic system comprising a transition metal cation having a defined bleach catalytic activity (e.g., a copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation), an auxiliary metal cation having little or no bleach catalytic activity (e.g., a zinc or aluminum cation), and a separator having defined stability constants for the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra (methylenephosphonic acid), and water-soluble salts thereof. Such catalysts are disclosed in US 4430243. Preferred catalysts are described in WO 09/839406, US 6218351 and WO 00/012667. Particularly preferred are transition metal catalysts or ligands which therefore act as cross-bridged multidentate N-donor ligands.

If desired, the compositions herein may be catalyzed by means of a manganese compound. Such compounds and levels of use are well known in the art and include, for example, the manganese-based catalysts disclosed in US 5576282.

Cobalt bleach catalysts useful herein are known and are described, for example, in US 5597936; in US 5595967. Such cobalt catalysts can be easily prepared by known procedures like for example the procedures taught in US 5597936 and US 5595967.

The compositions herein may also suitably comprise transition metal complexes of ligands, such as bispidones (bispidones) (US 7501389) and/or macropolycyclic rigid ligands-abbreviated as "MRL". As a practical matter, and not by way of limitation, the compositions and methods herein can be adjusted to provide about at least one part per billion of the active MRL species in the aqueous wash medium, and will typically provide from 0.005ppm to 25ppm, from 0.05ppm to 10ppm, or from 0.1ppm to 5ppm of MRL in the wash liquor.

Suitable transition metals in the transition metal bleach catalyst of the present invention include, for example, manganese, iron and chromium. Suitable MRLs include 5, 12-diethyl-1, 5,8, 12-tetraazabicyclo [6.6.2] hexadecane. Suitable transition metal MRLs can be readily prepared by known procedures, such as, for example, the procedures taught in US 6225464 and WO 00/32601.

(7) Photobleaches-suitable photobleaches include, for example, sulfonated zinc phthalocyanines, sulfonated aluminum phthalocyanines, xanthene dyes, and mixtures thereof. Preferred bleaching components for use in the compositions of the present invention comprise a source of hydrogen peroxide, a bleach activator and/or an organic peroxyacid, optionally generated in situ by reaction of the source of hydrogen peroxide and the bleach activator in combination with a bleach catalyst. Preferred bleaching components comprise a bleach catalyst, preferably an organic bleach catalyst as described above.

Particularly preferred bleach components are bleach catalysts, especially organic bleach catalysts.

Exemplary bleaching systems are also described in, for example, WO 2007/087258, WO 2007/087244, WO 2007/087259 and WO 2007/087242.

Fabric tonerThe composition may comprise a fabric hueing agent. Suitable fabric hueing agents include dyes, dye-clay conjugates, and pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes belonging to the following 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.

In another aspect, suitable small molecule dyes include small molecule dyes selected from the group consisting of: color index (Society of Dyers and Colorists, bradford, uk) numbered direct violet 9, direct violet 35, direct violet 48, direct violet 51, direct violet 66, direct violet 99, direct blue 1, direct blue 71, direct blue 80, direct blue 279, acid red 17, acid red 73, acid red 88, acid red 150, acid violet 15, acid violet 17, acid violet 24, acid violet 43, acid red 52, acid violet 49, acid violet 50, acid blue 15, acid blue 17, acid blue 25, acid blue 29, acid blue 40, acid blue 45, acid blue 75, acid blue 80, acid blue 83, acid blue 90 and acid blue 113, acid black 1, basic violet 3, basic violet 4, basic violet 10, basic violet 35, basic blue 3, basic blue 16, basic blue 22, basic blue 47, basic blue 66, basic blue 75, basic blue 159 and mixtures thereof. In another aspect, suitable small molecule dyes include small molecule dyes selected from the group consisting of: color index (division of dyers and colorists, bradford, uk) number acid violet 17, acid violet 43, acid red 52, acid red 73, acid red 88, acid red 150, acid blue 25, acid blue 29, acid blue 45, acid blue 113, acid black 1, direct blue 71, direct violet 51 and mixtures thereof. In another aspect, suitable small molecule dyes include small molecule dyes selected from the group consisting of: color index (division of dyers and colorists, bradford, uk) number acid violet 17, direct blue 71, direct violet 51, direct blue 1, acid red 88, acid red 150, acid blue 29, acid blue 113 or mixtures thereof.

Suitable polymeric dyes include polymeric dyes selected from the group consisting of: polymers containing conjugated chromogens (dye-polymer conjugates) and polymers in which chromogens are copolymerized into the polymer backbone, and mixtures thereof.

In another aspect, suitable polymeric dyes include polymeric dyes selected from the group consisting of: in that(Milliken) a fabric substantive colorant sold under the name, a dye-polymer conjugate formed from at least one reactive dye and a polymer selected from the group consisting of: comprising a hydroxyl moiety selected from,Primary amine moieties, secondary amine moieties, thiol moieties, and mixtures thereof. In yet another aspect, suitable polymeric dyes include polymeric dyes selected from the group consisting of:

violet CT, carboxymethyl CELLULOSE (CMC) conjugated with reactive blue, reactive violet or reactive red dyes, CMC conjugated with c.i. reactive blue 19 (sold under the product name AZO-CM-CELLULOSE product code S-ACMC by magazyme, wecker, ireland), alkoxylated triphenyl-methane polymeric colorants, alkoxylated thiophene polymeric colorants, and mixtures thereof.

Preferred hueing dyes include the brighteners found in WO 08/87497. These brighteners can be characterized by the following structure (I):

wherein R is₁And R₂May be independently selected from:

a)[(CH₂CR'HO)_x(CH₂CR"HO)_yH]

wherein R' is selected from the group consisting of: H. CH (CH)₃、CH₂O(CH₂CH₂O)_zH. And mixtures thereof; wherein R "is selected from the group consisting of: H. CH (CH)₂O(CH₂CH₂O)_zH. And mixtures thereof; wherein x + y is less than or equal to 5; wherein y is more than or equal to 1; and wherein z is 0 to 5;

b)R₁is alkyl, aryl or arylalkyl, and R₂＝[(CH₂CR'HO)_x(CH₂CR"HO)_yH]

Wherein R' is selected from the group consisting of: H. CH (CH)₃、CH₂O(CH₂CH₂O)_zH. And mixtures thereof; wherein R "is selected from the group consisting of: H. CH (CH)₂O(CH₂CH₂O)_zH. And mixtures thereof; wherein x + y is less than or equal to 10;wherein y is more than or equal to 1; and wherein z is 0 to 5;

c)R₁＝[CH₂CH₂(OR₃)CH₂OR₄]and R is₂＝[CH₂CH₂(O R₃)CH₂O R₄]

Wherein R is₃Selected from the group consisting of: H. (CH)₂CH₂O)_zH and mixtures thereof; and wherein z is 0 to 10;

wherein R is₄Selected from the group consisting of: (C)₁-C₁₆) Alkyl, aryl groups, and mixtures thereof; and is

d) Wherein R1 and R2 can be independently selected from the amino addition products of styrene oxide, glycidyl methyl ether, isobutyl glycidyl ether, isopropyl glycidyl ether, tert-butyl glycidyl ether, 2-ethylhexyl glycidyl ether, and glycidyl cetyl ether, followed by addition of from 1 to 10 alkylene oxide units.

Preferred whitening agents of the present invention can be characterized by the following structure (II):

Further preferred whitening agents of the present invention can be characterized by the following structure (III):

typically comprising a mixture having a total of 5 EO groups. Suitable preferred molecules are those in structure I having the following pendant groups in "part a" above.

R’

R”

R’

R”

c＝b

d＝a

Additional whitening agents used include those described in US 2008/34511 (Unilever). The preferred agent is "purple 13".

Suitable dye clay conjugates include dye clay conjugates selected from the group consisting of at least one cationic/basic dye and smectite clays, and mixtures thereof. In another aspect, suitable dye clay conjugates include dye clay conjugates selected from the group consisting of: a cationic/basic dye selected from the group consisting of: c.i. basic yellow 1 to 108, c.i. basic orange 1 to 69, c.i. basic red 1 to 118, c.i. basic violet 1 to 51, c.i. basic blue 1 to 164, c.i. basic green 1 to 14, c.i. basic brown 1 to 23, CI basic black 1 to 11, and one clay selected from the group consisting of: smectite clays, hectorite clays, saponite clays, and mixtures thereof. In yet another aspect, suitable dye clay conjugates include dye clay conjugates selected from the group consisting of: montmorillonite basic blue B7c.i.42595 conjugate, montmorillonite basic blue B9c.i.52015 conjugate, montmorillonite basic violet V3 c.i.42555 conjugate, montmorillonite basic green G1 c.i.42040 conjugate, montmorillonite basic red R1c.i.45160 conjugate, montmorillonite c.i. basic black 2 conjugate, hectorite basic blue B7c.i.42595 conjugate, hectorite basic blue B9c.i.52015 conjugate, hectorite basic violet V3 c.i.42555 conjugate, hectorite basic green G1 c.i.42040 conjugate, hectorite basic red R1c.i.45160 conjugate, hectorite basic c.i. basic black 2 conjugate, saponite basic blue B7c.i.42595 conjugate, saponite basic blue B9c.i.52015 conjugate, saponite basic blue B15 c.i. 3 c.i.15 conjugate, saponite basic red R yellow R5 c.i.i.42160 conjugate, saponite basic red r.i.i.4240 conjugate, saponite basic red r.i.42555 conjugate, saponite basic red r.15 c.i.15 conjugate, saponite yellow 15 c.15 conjugate, saponite yellow r.15 c.i.15 conjugate, saponite basic red yellow r.15 conjugate, and mixtures thereof.

Suitable pigments include pigments selected from the group consisting of: xanthenone, indanthrone, chloroindanthrone containing from 1 to 4 chlorine atoms, pyranthrone, dichloropyranthrone, monobromoachloropyranthrone, dibromodichloropyranthrone, tetrabromobisphene, perylene-3, 4,9, 10-tetracarboxylic acid diimide (wherein the imide groups may be unsubstituted or substituted with C1-C3-alkyl or phenyl or heterocyclic groups, and wherein the phenyl and heterocyclic groups may additionally carry substituents that do not impart solubility in water), anthrapyrimidine carboxylic acid amides, anthrone violet, isoanthrone violet, dioxazine pigments, copper phthalocyanines that may contain up to 2 chlorine atoms per molecule, polychlorinated copper phthalocyanines, or polybromochloro-copper phthalocyanines that contain up to 14 bromine atoms per molecule, and mixtures thereof.

In another aspect, suitable pigments include pigments selected from the group consisting of: ultramarine blue (c.i. pigment blue 29), ultramarine violet (c.i. pigment violet 15) and mixtures thereof.

The above-described fabric hueing agents may be used in combination (any mixture of fabric hueing agents may be used). Suitable toners are described in more detail in US 7208459. Preferred levels of dye in the compositions of the invention are from 0.00001 wt% to 0.5 wt%, or from 0.0001 wt% to 0.25 wt%. Preferably the concentration of the dye used in the treatment and/or cleaning step in the water is from 1ppb to 5ppm, 10ppb to 5ppm or 20ppb to 5 ppm. In preferred compositions, the concentration of surfactant will be from 0.2 to 3 g/l.

Encapsulated productThe composition may comprise an encapsulate. In one aspect, an encapsulate comprises a core, a shell having an inner surface and an outer surface, the shell encapsulating the core.

In one aspect of the encapsulate, the core may comprise a material selected from the group consisting of: a perfume brightener; a dye; an insect repellent; a silicone; a wax; a flavoring agent; a vitamin; a fabric softener; a skin care agent; in one aspect, paraffin wax; an enzyme; an antibacterial agent; a bleaching agent; sensates (sendate); and mixtures thereof; and the envelope may comprise a material selected from the group consisting of: polyethylene; a polyamide; polyvinyl alcohol, optionally containing other comonomers; polystyrene; a polyisoprene; a polycarbonate; a polyester; a polyacrylate; an aminoplast, which in one aspect may comprise a polyurea, polyurethane and/or polyurea-urethane, in one aspect, the polyurea may comprise a polyoxymethyleneurea and/or melamine formaldehyde; a polyolefin; a polysaccharide, which in one aspect may comprise alginate and/or chitosan; gelatin; shellac; an epoxy resin; vinyl polymer water-insoluble inorganic substance; a silicone; and mixtures thereof.

In one aspect of the encapsulate, the core may comprise a perfume.

In one aspect of the encapsulate, the shell may comprise melamine formaldehyde and/or cross-linked melamine formaldehyde.

In one aspect, suitable encapsulates may comprise a core material and a shell at least partially surrounding the core material. At least 75%, 85% or 90% of the encapsulates may have a breaking strength of from 0.2MPa to 10MPa, from 0.4MPa to 5MPa, from 0.6MPa to 3.5MPa or from 0.7MPa to 3 MPa; and has from 0% to 30%, from 0% to 20%, or from 0% to 5% leakage of benefit agent.

In one aspect, at least 75%, 85% or 90% of the encapsulates may have a particle size from 1 micron to 80 microns, from 5 microns to 60 microns, from 10 microns to 50 microns, or from 15 microns to 40 microns.

In one aspect, at least 75%, 85% or 90% of the encapsulates may have a particle wall thickness of from 30 to 250nm, from 80 to 180nm, or from 100 to 160 nm.

In one aspect, the core material of the encapsulate may comprise a material selected from the group consisting of perfume raw materials, and/or optionally a material selected from the group consisting of: vegetable oils, including neat vegetable oils and/or blended vegetable oils, including castor oil, coconut oil, cottonseed oil, grapeseed oil, rapeseed oil, soybean oil, corn oil, palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconut oil, palm kernel oil, castor oil, lemon oil, and mixtures thereof; esters of vegetable oils, esters including dibutyl adipate, dibutyl phthalate, butyl benzyl adipate, octyl benzyl adipate, tricresyl phosphate, trioctyl phosphate, and mixtures thereof; linear or branched hydrocarbons, including those having a boiling point above about 80 ℃; partially hydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls (including monoisopropyl biphenyls), alkylated naphthalenes (including dipropyl naphthalenes), petroleum spirits (including kerosene), mineral oils, and mixtures thereof; aromatic solvents including benzene, toluene and mixtures thereof; silicone oil; and mixtures thereof.

In one aspect, the wall material of the encapsulate may comprise a suitable resin comprising the reaction product of an aldehyde and an amine, with a suitable aldehyde comprising formaldehyde. Suitable amines include melamine, urea, benzoguanamine, glycoluril and mixtures thereof. Suitable melamines include methylolmelamine, methylated methylolmelamine, iminomelamine, and mixtures thereof. Suitable ureas include dimethylol urea, methylated dimethylol urea, urea-resorcinol, and mixtures thereof.

In one aspect, a suitable formaldehyde scavenger may be used with the encapsulate, e.g., in a capsule slurry, and/or added to such a composition before, during, or after the addition of the encapsulate to the composition. Suitable capsules may be made by the following teachings of US 2008/0305982, and/or US 2009/0247449.

In a preferred aspect, the composition may further comprise a deposition aid, preferably consisting of a group comprising cationic or nonionic polymers. Suitable polymers include cationic starch, cationic hydroxyethyl cellulose, polyvinyl formaldehyde, locust bean gum, mannan, xyloglucan, tamarind gum, polyethylene glycol terephthalate, and polymers containing dimethylaminoethyl methacrylate, optionally with one or more monomers selected from the group comprising acrylic acid and acrylamide.

PerfumeIn one aspect, the composition comprises a fragrance comprising one or more fragrance raw materials selected from the group consisting of 1,1' -oxybis-2-propanol, 1, 4-cyclohexanedicarboxylic acid, diethyl ester, (ethoxymethoxy) cyclododecane, 1, 3-nonanediol, monoacetate, (3-methylbutoxy) acetic acid, 2-propenyl ester, β -methylcyclododecaneethanol, 2-methyl-3- [ (1,7, 7-trimethylbicyclo [2.2.1 ] methyl]Hept-2-yl) oxy]-1-propanol, oxacyclohexadec-2-one, α -methyl-benzyl alcohol acetate, trans-3-ethoxy1,1, 5-trimethylcyclohexane; 4- (1, 1-dimethylethyl) cyclohexanol acetate; dodecahydro-3 a,6,6,9 a-tetramethylnaphtho [2,1-b ]]Furan, β -methylpropanal, β -methyl-3- (1-methylethyl) phenylpropanal, 4-phenyl-2-butanone, 2-methylbutyric acid, ethyl ester, benzaldehyde, 2-methylbutyric acid, 1-methylethyl ester, dihydro-5-pentyl-2 (3H) furanone, (2E) -1- (2,6, 6-trimethyl-2-cyclohexen-1-yl) -2-buten-1-one, dodecanal, undecanal, 2-ethyl- α -dimethylphenylpropanal, decanal, α -dimethylphenylethanol acetate, 2- (phenylmethylene) octanal, 2- [ [3- [4- (1, 1-dimethylethyl) phenyl ] propanal]-2-methylpropylidene]Amino group]Benzoic acid, methyl ester, 1- (2,6, 6-trimethyl-3-cyclohexen-1-yl) -2-buten-1-one, 2-pentylcyclopentanone, 3-oxo-2-pentylcyclopentaneacetic acid, methyl ester, 4-hydroxy-3-methoxybenzaldehyde, 3-ethoxy-4-hydroxybenzaldehyde, 2-heptylcyclopentanone, 1- (4-methylphenyl) ethanone, (3E) -4- (2,6, 6-trimethyl-1-cyclohexen-1-yl) -3-buten-2-one, (3E) -4- (2,6, 6-trimethyl-2-cyclohexen-1-yl) -3-buten-2-one, phenethyl alcohol, 2H-1-benzopyran-2-one, 4-methoxybenzaldehyde, 10-undecenal, propionic acid, phenylmethyl ester, β -methylphenylpentanol, 1-diethoxy-3, 7-dimethyl-2, 6-octadien-E, α -dimethylbenzyl alcohol, (2E) -1- (2, 6-trimethyl-1-cyclohexen-1-yl) -2-propenyl-2, 5-dimethoxycyclohexaneacetic acid, 2-propenyl, 2-1, 2-dimethoxycyclohexaneacetic acid, 2-propenyl, 2-1-propenyl, 2-1, 6-trimethylpentanoic acid, 2-propenyl, 2-1, 2]Octan-8-ketoxime; 4- (4-hydroxy-4-methylpentyl) -3-cyclohexene-1-carbaldehyde; 3-buten-2-ol; 2- [ [ [2,4 (or 3,5) -dimethyl-3-cyclohexenyl-1-yl ] amino]Methylene group]Amino group]Benzoic acid, methyl ester; 8-cyclohexadecan-1-one; methyl ionone; 2, 6-dimethyl-7-octen-2-ol; 2-methoxy-4- (2-propenyl) phenol; (2E) -3, 7-dimethyl-2, 6-octadien-1-ol; 2-hydroxy-benzoic acid, (3Z) -3-hexenyl ester; 2-tridecenenitrile; 4- (2, 2-dimethyl-6-methylenecyclohexyl) -3-methyl-3-buten-2-one; tetrahydro-4-methyl-2- (2-methyl-1-propenyl) -2H-pyran; acetic acid, (2-methylbutoxy) -, 2-propenyl ester; benzoic acid, 2-hydroxy-, 3-methylbutyl ester; 2-buten-1-one, 1- (2,6, 6-trimethyl-1-cyclohexen-1-yl) -, (Z) -; cyclopentanecarboxylic acid, 2-hexyl-3-oxo-, methyl ester, phenylpropylaldehyde, 4-ethyl- α -dimethyl-; 3-cyclohexene-1-carbaldehyde, 3- (4-hydroxy-4-methylpentyl) -, ethanone, 1- (2,3,4,7,8,8 a-hexahydro-3, 6,8, 8-tetramethyl-1H-3 a, 7-methanocamomile blue-5-yl-, - [3R- (3.α,3a. β,7.β,8a. α.)]2-methyl-2H-pyran-2-one, 6-butyltetrahydro-undecanal, phenylpropylaldehyde, 4- (1, 1-dimethylethyl) -. α -methyl-, 2(3H) -furanone, 5-heptyldihydro-benzoic acid, 2- [ (7-hydroxy-3, 7-dimethyloctylidene) amino]-, methyl; benzoic acid, 2-hydroxy-, phenylmethyl ester; naphthalene, 2-methoxy-; 2-cyclopenten-1-one, 2-hexyl-; 2(3H) -furanone, 5-hexyldihydro-; oxirane carboxylic acid, 3-methyl-3-phenyl-, ethyl ester; 2-oxabicyclo [2.2.2]Octane, 1,3, 3-trimethyl-, benzenepentanol,. gamma. -methyl-, 3-octanol, 3, 7-dimethyl-2, 6-octadienenitrile, 3, 7-dimethyl-6-octen-1-ol, terpineol acetate, 2-methyl-6-methylene-7-octen-2-ol, dihydro derivatives, 3a,4,5,6,7,7 a-hexahydro-4, 7-methano-1H-indene-6-ol propionate, 3-methyl-2-buten-1-ol acetate, (Z) -3-hexen-1-ol acetate, 2-ethyl-4- (2,2, 3-trimethyl-3-cyclopenten-1-yl) -2-buten-1-ol, 4- (octahydro-4, 7-methano-5H-indene-5-yl) -butyraldehyde, 3-2, 4-dimethyl-cyclohexene-1-carboxaldehyde, 1- (1,2,3,5, 7-dimethyl-3, 7-octadienyl) -1-hydroxy-1-ethyl-4- (3, 7, 7-dimethyl-cyclohexenyl-1-carbaldehyde, 7, 7-dimethyl-1-hydroxy-5-hexenyl) -butyraldehyde, 3, 4-dimethyl-cyclohexenyl-1-yl-oxo-2, 7, 7-6-1-methyl-1-2-hexahydro-1-2-1-hydroxy-1, 7, 7-1-methyl-1-hexenyl-1-naphthalenyl-5-oxo-2-methyl-1-methyl-hexahydro-1-2-ethyl-1-hexenyl-2-hexenyl-hexahydro-ethyl-6-1-oxo-ethyl-methyl-1-5-1-ethyl-5-yl-ethyl-5-1-naphthalenyl-oxoethyl-methyl-naphthalenyl-5-oxohexanoaldehyde, 2-2, 7, 7-2-methyl-6-ethyl-naphthalenyl-1-naphthalenyl-oxoethyl-naphthalenyl-2-naphthalenyl-2-oxo-2-carboxylic acid, 2-]And 1-methyl-4- (1-methylvinyl) cyclohexene and mixtures thereof.

In one aspect, the composition may comprise encapsulated perfume particles comprising a water-soluble hydroxyl compound or melamine-formaldehyde or modified polyvinyl alcohol. In one aspect, the encapsulates comprise (a) an at least partially water-soluble solid matrix comprising one or more water-soluble hydroxy compounds, preferably starch; and (b) a perfume oil encapsulated by the solid matrix.

In another aspect, the perfume may be pre-complexed with a polyamine, preferably a polyethyleneimine, to form a Schiff base (Schiff base).

The polymer-composition may comprise one or more polymers. Examples are carboxymethylcellulose, poly (vinyl-pyrrolidone), poly (ethylene glycol), poly (vinyl alcohol), poly (vinylpyridine-N-oxide), poly (vinylimidazole), polycarboxylates (e.g. polyacrylates), maleic/acrylic acid copolymers, and lauryl methacrylate/acrylic acid copolymers.

The composition may comprise one or more amphiphilic cleaning polymers, for example compounds having the following general structure: bis ((C)₂H₅O)(C₂H₄O)n)(CH₃)-N⁺-C_xH_2x-N⁺-(CH₃)-bis((C₂H₅O)(C₂H₄O) n), wherein n is from 20 to 30 and x is from 3 to 8, or sulfated or sulfonated variants thereof.

The compositions may comprise amphiphilic alkoxylated grease cleaning polymers having balanced hydrophilic and hydrophobic properties such that they remove grease particles from fabrics and surfaces. Particular embodiments of the amphiphilic alkoxylated grease cleaning polymers of the present invention comprise a core structure and a plurality of alkoxylated groups attached to that core structure. These may comprise alkoxylated polyalkyleneimines (polyalkenylimines), preferably having an inner polyethylene oxide block and an outer polypropylene oxide block.

Alkoxylated polycarboxylates (such as those prepared from polyacrylates) can be used herein to provide additional grease removal performance. Such materials are described in WO 91/08281 and PCT 90/01815. Chemically, these materials include a moiety having one ethylene group per 7-8 acrylate unitsPolyacrylates with oxo side chains. The side chain has the formula- (CH)₂CH₂O)_m(CH₂)_nCH₃Wherein m is 2 to 3 and n is 6 to 12. The side chains are ester-linked to the polyacrylate "backbone" to provide a "comb" polymer type structure. The molecular weight may vary, but is typically in the range of 2000 to 50,000. Such alkoxylated polycarboxylates can comprise from 0.05 wt.% to 10 wt.% of the compositions herein.

The isoprenoid-derived surfactants of the present invention, as well as mixtures formed with other co-surfactants and other adjuvant ingredients, are particularly suitable for use with amphiphilic graft copolymers, preferably comprising (i) a polyethylene glycol backbone; and (ii) at least one pendant moiety selected from the group consisting of polyvinyl acetate, polyvinyl alcohol, and mixtures thereof. A preferred amphiphilic graft copolymer is Sokalan HP22 supplied by BASF. Suitable polymers include random graft copolymers, preferably polyvinyl acetate grafted polyethylene oxide copolymers, having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is preferably 6000 and the weight ratio of polyethylene oxide to polyvinyl acetate is 40 to 60 with no more than 1 graft point per 50 ethylene oxide units.

Carboxylate polymerThe composition of the invention may also comprise one or more carboxylate polymers, such as a maleate/acrylate random copolymer or a polyacrylate homopolymer. In one aspect, the carboxylate polymer is a polyacrylate homopolymer having a molecular weight of from 4,000Da to 9,000Da or from 6,000Da to 9,000 Da.

Soil release polymersThe composition of the invention may also comprise one or more soil release polymers having a structure as defined by one of the following structures (I), (II) or (III):

(I)-[(OCHR¹-CHR²)_a-O-OC-Ar-CO-]_d

(II)-[(OCHR³-CHR⁴)_b-O-OC-sAr-CO-]_e

(III)-[(OCHR⁵-CHR⁶)_c-OR⁷]_f

wherein:

a. b and c are from 1 to 200;

d. e and f are from 1 to 50;

ar is 1, 4-substituted phenylene;

sAr is a1, 3-substituted phenylene radical which is SO-substituted in the 5-position₃Me substitution;

me is Li, K, Mg/2, Ca/2, Al/3, ammonium, monoalkylammonium, dialkylammonium, trialkylammonium or tetraalkylammonium, where the alkyl radical is C₁-C₁₈Alkyl or C₂-C₁₀Hydroxyalkyl, or mixtures thereof;

R¹、R²、R³、R⁴、R⁵and R⁶Independently selected from H or C₁-C₁₈N-alkyl or iso-alkyl; and is

R⁷Is straight-chain or branched C₁-C₁₈Alkyl, or straight or branched C₂-C₃₀Alkenyl, or cycloalkyl having 5 to 9 carbon atoms, or C₈-C₃₀Aryl, or C₆-C₃₀An arylalkyl group.

Suitable soil release polymers are polyester soil release polymers, such as the Rebel-o-tex polymers, including Rebel-o-tex, SF-2 and SRP6, available from Rhodia. Other suitable soil release polymers include Texcare polymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325, supplied by Clariant. Other suitable soil release polymers are Marloquest polymers, such as Marloquest SL, available from Sasol corporation (Sasol).

Cellulose polymersThe composition of the invention may also comprise one or more cellulose polymers, including those selected from alkylcelluloses, alkylalkoxyalkylcelluloses, carboxyalkylcelluloses, alkylcarboxyalkylcelluloses. In one aspect, the cellulosic polymer is selected from the group comprising: carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, methylCarboxymethyl cellulose and mixtures thereof. In one aspect, the carboxymethyl cellulose has a degree of carboxymethyl substitution of from 0.5 to 0.9 and a molecular weight of from 100,000Da to 300,000 Da.

The composition may be prepared according to methods known in the art, and may be in the form of a liquid or dry composition. The composition may be stabilized according to methods known in the art.

Methods of Using the variant alpha-amylases described herein-Industrial applications

The variant alpha-amylases described in the present invention have valuable properties allowing various industrial applications. In particular, the alpha-amylase may be used in ethanol production as well as in starch conversion processes.

Furthermore, the alpha-amylases of the present invention are particularly useful for the production of sweeteners/syrups and ethanol from starch or whole grains (see, e.g., U.S. Pat. No. 5,231,017, which is incorporated herein by reference), such as fuel ethanol, potable ethanol, and industrial ethanol.

In one embodiment, the present invention relates to the use of an alpha-amylase according to the invention in a liquefaction process. The resulting liquefact can be further processed into a syrup and/or a fermentation product.

Starch processing

Native starch consists of microscopic particles that are insoluble in water at room temperature. When the aqueous starch slurry is heated, the particles expand and eventually break, dispersing the starch molecules into solution. The expansion may be reversible at temperatures up to about 50 ℃ to 75 ℃. However, at higher temperatures, irreversible expansion, referred to as "gelatinization," begins. In this "pasting" process, there is a significant increase in viscosity. The granular starch to be processed may have a highly refined starch quality, preferably at least 90%, at least 95%, at least 97% or at least 99.5% pure, or it may be a coarser starch-containing material comprising (e.g. milled) whole grain including non-starch fractions such as germ residue and fiber. The raw material (e.g. whole grain) may be reduced in particle size, e.g. by milling, in order to unfold the structure and allow further processing. In dry milling, whole grain is milled and used. Wet milling provides good separation of germ from meal (starch particles and protein) and is often used in locations where starch hydrolysates are used, for example, in the production of syrups. Both dry and wet milling are starch processing methods well known in the art and may be used in the process of the present invention. Methods for reducing the particle size of the starch-containing material are well known to those skilled in the art.

Since the solids level in a typical industrial process is 30% -40%, the starch has to be diluted or "liquefied" so that it can be processed properly. In current commercial practice, this reduction in viscosity is achieved primarily through enzymatic degradation.

The liquefaction is carried out in the presence of an alpha-amylase, preferably a bacterial alpha-amylase and/or an acid fungal alpha-amylase. In one embodiment, a protease is also present during liquefaction. In one embodiment, phytase is also present during liquefaction. In one embodiment, a viscosity-reducing enzyme such as xylanase and/or beta-glucanase is also present during liquefaction.

During liquefaction, long-chain starch is degraded into branched and linear shorter units (maltodextrins) by alpha-amylase. Liquefaction may be carried out as a three-step hot slurry process. The slurry is heated to between 60-95 ℃ (e.g., 70-90 ℃, e.g., 77-86 ℃, 80-85 ℃, 83-85 ℃) and alpha-amylase is added to start liquefaction (dilution).

In one embodiment, the slurry can be jet cooked between 95 ℃ and 140 ℃ (e.g., 105 ℃ to 125 ℃) for about 1 to 15 minutes, e.g., about 3 to 10 minutes, and particularly about 5 minutes. The slurry is then cooled to 60-95 ℃ and more alpha-amylase is added to obtain the final hydrolysis (secondary liquefaction). The jet cooking process is carried out at a pH of 4.5-6.5, typically at a pH between 5 and 6. The alpha-amylase may be added in a single dose, for example, prior to jet cooking.

The liquefaction process is carried out at a temperature between 70 ℃ and 95 ℃, such as 80 ℃ to 90 ℃, such as about 85 ℃, for a period of time between about 10 minutes and 5 hours, typically 1 to 2 hours. The pH is between 4 and 7, for example between 5.5 and 6.2. To ensure optimal enzyme stability under these conditions, calcium may optionally be added (to provide 1-60ppm free calcium ion, e.g., about 40ppm free calcium ion). After such treatment, the liquefied starch will typically have a "dextrose equivalent" (DE) of 10-16.

Generally, liquefaction and liquefaction conditions are well known in the art.

Saccharification can be carried out using conditions well known in the art with a carbohydrate-source generating enzyme, particularly a glucoamylase or a beta-amylase, and optionally a debranching enzyme (e.g., isoamylase or pullulanase). For example, the entire saccharification step may last from about 24 to about 72 hours. However, it is common to perform pre-saccharification at a temperature between 30 ℃ and 65 ℃ (typically about 60 ℃) for typically 40-90 minutes, followed by complete saccharification during fermentation in a Simultaneous Saccharification and Fermentation (SSF) process. Typically at a temperature in the range of 20 ℃ to 75 ℃ (e.g., 25 ℃ to 65 ℃ and 40 ℃ to 70 ℃, typically about 60 ℃) and at a pH between about 4 and 5, typically at about pH 4.5.

The saccharification step and fermentation step may be performed sequentially or simultaneously. In one embodiment, saccharification and fermentation are performed simultaneously (referred to as "SSF"). However, typically, a pre-saccharification step is performed at a temperature of 30 ℃ to 65 ℃, typically about 60 ℃, for about 30 minutes to 2 hours (e.g., 30 to 90 minutes), followed by complete saccharification during what is known as Simultaneous Saccharification and Fermentation (SSF). The pH is typically between 4.2 and 4.8, e.g., pH 4.5. In a Simultaneous Saccharification and Fermentation (SSF) process, there is no holding stage for saccharification, but in fact, yeast and enzyme are added together.

In a typical saccharification process, the maltodextrin produced during liquefaction is converted to dextrose by the addition of a glucoamylase and optionally a debranching enzyme, such as isoamylase (U.S. Pat. No. 4,335,208) or pullulanase. The temperature was lowered to 60 ℃ before adding glucoamylase and debranching enzyme. The saccharification process is carried out for 24-72 hours. The pH is lowered to below 4.5 while maintaining a high temperature (above 95 ℃) prior to addition of the saccharifying enzymes, in order to inactivate the liquefying α -amylase. This process reduces the formation of short oligosaccharides called "panose precursors" which cannot be properly hydrolyzed by the debranching enzyme. Normally, about 0.2% to 0.5% of the saccharification product is the branched trisaccharide panose (Glc p α 1-6Glc p α 1-4Glc), which is not degraded by pullulanase. If active amylase from the liquefaction step is still present during saccharification (i.e. not denatured), the amount of panose can be as high as 1% -2%, which is highly undesirable as it significantly reduces saccharification yield.

Other fermentation products may be fermented under conditions and temperatures well known to those skilled in the art and appropriate for the fermenting organism in question.

The fermentation product may be recovered by methods well known in the art (e.g., by distillation).

In one embodiment, the process of the present invention further comprises the following steps prior to converting the starch-containing material to sugar/dextrin:

(x) Reducing the particle size of the starch-containing material; and

(y) forming a slurry comprising the starch-containing material and water.

In one embodiment, the starch-containing material is milled to reduce particle size. In one embodiment, the particle size is reduced to between 0.05-3.0mm, preferably 0.1-0.5mm, or such that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fits through a sieve having a 0.05-3.0mm screen, preferably a 0.1-0.5mm screen.

The aqueous slurry may contain 10-55 wt.% Dry Solids (DS), preferably 25-45 wt.% Dry Solids (DS), more preferably 30-40 wt.% Dry Solids (DS) of the starch-containing material.

Conventional starch conversion processes such as liquefaction and saccharification processes are described, for example, in U.S. Pat. nos. 3,912,590, EP252730, and EP 063909, which are incorporated herein by reference.

In one embodiment, the conversion process degrades starch into lower molecular weight carbohydrate components, such as sugars or fat replacers, the process including a debranching step.

When converting starch into sugars, the starch is depolymerized. Such depolymerization processes consist for example of a pretreatment step and two or three successive process steps, i.e. a liquefaction process, a saccharification process and, depending on the desired end product, an optional isomerization process.

When the desired final sugar product is, for example, high fructose syrup, dextrose syrup can be converted to fructose. After the saccharification process, the pH is increased to a value in the range of 6-8, e.g. pH 7.5, and calcium is removed by ion exchange. The dextrose syrup is then converted to a high fructose syrup using, for example, immobilized glucose isomerase.

Production of fermentation products

Fermentable sugars (e.g., dextrins, monosaccharides, particularly glucose) are produced by enzymatic saccharification. These fermentable sugars can be further purified and/or converted into useful sugar products. In addition, these sugars can be used as fermentation feeds in microbial fermentation processes to produce end products, such as alcohols (e.g., ethanol and butanol), organic acids (e.g., succinic acid, 3-HP and lactic acid), sugar alcohols (e.g., glycerol), ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate, 2, 5-diketo-D-gluconate and 2-keto-L-gulonic acid), amino acids (e.g., lysine), proteins (e.g., antibodies and fragments thereof).

In one embodiment, fermentable sugars obtained during the liquefaction process step are used to produce alcohol, and in particular ethanol. In ethanol production, an SSF process is typically used, in which saccharifying enzymes are added together with fermenting organisms (e.g., yeast), and then performed at temperatures of 30-40 ℃.

The organism used in the fermentation depends on the desired end product. Typically, yeast is used as the fermenting organism if ethanol is the desired end product. In some preferred embodiments, the ethanologenic microorganism is a yeast, and in particular a saccharomyces, such as a saccharomyces cerevisiae strain (U.S. Pat. No. 4,316,956). Various Saccharomyces cerevisiae are commercially available and these include, but are not limited to, FALI (Fleischmann' S Yeast), SUPERSTART (Altech), FERMOL (food ingredient division of Inssman (DSM S), Inc.)pecialites), RED STAR (lesofre) and angelol Yeast (Angel Yeast Company (Angel Yeast Company, china), Ethanol RED (lesofre), Innova Drive (Novozymes A/S), Innova Lift (Novesson). The starting yeast amount used in these methods is an amount effective to produce a commercially effective amount of ethanol in a suitable time (e.g., to produce at least 10% ethanol from a substrate having a DS of between 25% and 40% in less than 72 hours). Yeast cells are generally present at about 10⁴To about 10¹²And preferably from about 10⁷To about 10¹⁰Viable yeast counts per mL volume of fermentation broth. After the yeast is added to the mash, it is typically subjected to fermentation for about 24-96 hours, e.g., 35-60 hours. The temperature is between about 26 ℃ and 34 ℃, typically about 32 ℃, and the pH is from pH 3 to 6, e.g., around pH4 to 5.

In addition to fermenting microorganisms (e.g., yeast), the fermentation may also include nutrients as well as additional enzymes, including phytases. The use of yeast in fermentation is well known in the art.

In further embodiments, the use of an appropriate fermenting microorganism can produce a fermentation end product, as known in the art, including, for example, glycerol, 1, 3-propanediol, gluconate, 2-keto-D-gluconate, 2, 5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lactic acid, amino acids, and derivatives thereof. More specifically, when lactic acid is the desired end product, the species lactobacillus (lactobacillus casei) may be used; when glycerol or 1, 3-propanediol is the desired end product, E.coli can be used; and Pantoea citrosum can be used as a fermenting microorganism when 2-keto-D-gluconate, 2, 5-diketo-D-gluconate and 2-keto-L-gulonic acid are desired end products. The list listed above is only an example and the person skilled in the art knows many fermenting microorganisms that can be used to obtain the desired end product.

Method for producing a fermentation product from a material containing gelatinized starch

In this aspect, the invention relates to processes for producing a fermentation product, particularly ethanol, from starch-containing material, the processes comprising: a liquefaction step, and a saccharification and fermentation step performed sequentially or simultaneously. Accordingly, the present invention relates to processes for producing a fermentation product from starch-containing material, the processes comprising the steps of:

(a) liquefying a starch-containing material in the presence of a variant alpha-amylase of the invention;

(b) saccharifying the liquefied material obtained in step (a) with glucoamylase;

In one embodiment, a protease, such as an acid fungal protease or a metalloprotease, is added before, during, and/or after liquefaction. In one embodiment, the metalloprotease is derived from a strain of Thermoascus, e.g., Thermoascus aurantiacus, especially Thermoascus aurantiacus cgmccno. 0670. In another embodiment, the protease is a bacterial protease, in particular a serine protease, more in particular an S8 protease, in particular a protease derived from a strain of the genus pyrococcus, more in particular from pyrococcus furiosus disclosed in US6,358,726.

Glucoamylase is added/present during the saccharification step. The glucoamylase may be derived from a strain of aspergillus, e.g., aspergillus niger or aspergillus awamori; strains of the genus talaromyces, in particular talaromyces emersonii; or a strain of athelia, in particular athelia rolfsii; a strain of trametes, e.g., trametes annulata; a strain of the genus mucorales, in particular mucorales densatus or mucorales fragilis; or mixtures thereof. Other suitable glucoamylases may also be used, see section "Present during saccharification and/or fermentation And/or added glucoamylase”。

The saccharification step (b) and fermentation step (c) may be performed sequentially or simultaneously. When the process is carried out as a sequential saccharification and fermentation process, pullulanase and/or protease may be added during saccharification and/or fermentation, and when steps (b) and (c) are carried out simultaneously (SSF process), before or during fermentation. The pullulanase and/or protease may also advantageously be added before liquefaction (pre-liquefaction treatment), i.e. before or during step (a), and/or after liquefaction (post-liquefaction treatment), i.e. after step (a). The pullulanase is most advantageously added before or during liquefaction, i.e. before or during step (a). The fermentation product, such as especially ethanol, may optionally be recovered after fermentation, for example by distillation. The fermenting organism is preferably a yeast, preferably a strain of saccharomyces cerevisiae. In a preferred embodiment, the yeast expresses a variant glucoamylase of the invention. In a particular embodiment, the method of the present invention further comprises, before step (a), the steps of:

x) reducing the particle size of the starch-containing material, preferably by milling (e.g., using a hammer mill);

y) forming a slurry comprising the starch-containing material and water.

In one embodiment, the particle size is less than a No. 7 mesh, such as a No. 6 mesh. Mesh No. 7 is commonly used in conventional prior art methods. The aqueous slurry may contain from 10 to 55, for example 25 to 45 and 30 to 40 w/w% Dry Solids (DS) of the starch-containing material. The slurry is heated above the gelatinization temperature and the alpha-amylase variant may be added to start liquefaction (thinning). In one embodiment, the slurry may be jet cooked to further gelatinize the slurry prior to being subjected to the alpha-amylase in step (a). In one embodiment, liquefaction may be performed as a three-step hot slurry process. The slurry is heated to between 60-95 ℃, preferably between 70-90 ℃, e.g. preferably between 80-85 ℃ at pH 4-6, preferably 4.5-5.5 and optionally the alpha-amylase variant is added along with pullulanase and/or protease, preferably metalloprotease, to start liquefaction (dilution). In one embodiment, the slurry may then be jet cooked at a temperature between 95 ℃ and 140 ℃, preferably 100 ℃ to 135 ℃, e.g. 105 ℃ to 125 ℃ for about 1 to 15 minutes, preferably about 3 to 10 minutes, especially about 5 minutes. The slurry is cooled to 60-95 ℃ and more alpha-amylase and optionally pullulanase and/or protease are added to complete the hydrolysis (secondary liquefaction). The liquefaction process is usually carried out at a pH of 4.0-6, in particular at a pH of from 4.5 to 5.5. The saccharification step (b) may be performed using conditions well known in the art. For example, the complete saccharification process may last from about 24 to about 72 hours, however, typically only a pre-saccharification of typically 40-90 minutes is performed at a temperature between 30 ℃ and 65 ℃, typically about 60 ℃, followed by a complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). Saccharification is typically carried out at a temperature of from 20 ℃ to 75 ℃, preferably from 40 ℃ to 70 ℃, typically about 60 ℃ and at a pH between 4 and 5, generally at about pH 4.5. The most widely used process in the production of fermentation products, especially ethanol, is the Simultaneous Saccharification and Fermentation (SSF) process, in which process the saccharification is absent a holding stage, meaning that a fermenting organism (e.g. yeast) and an enzyme can be added together. SSF may typically be performed at a temperature of from 25 ℃ to 40 ℃, e.g. from 28 ℃ to 35 ℃, e.g. from 30 ℃ to 34 ℃, preferably about 32 ℃. In one embodiment, the fermentation is carried out for 6 to 120 hours, in particular 24 to 96 hours.

Method for producing syrup from material containing gelatinized starch

In this regard, the fermentation step is omitted, generally as described above for the "process for producing a fermentation product from a material comprising gelatinized starch". Accordingly, in this aspect, the present invention relates to a process for producing a syrup from starch-containing material, the process comprising the steps of:

a) liquefying the starch-containing material at a temperature above the initial gelatinization temperature in the presence of a variant alpha-amylase of the invention or a composition of the invention; and

b) saccharifying the product of step a) in the presence of glucoamylase.

In one embodiment, step b) is performed using a glucoamylase and:

i) a fungal alpha-amylase;

ii) an isoamylase;

iii) fungal alpha-amylase and isoamylase.

In a particular embodiment, a pullulanase is present in step a) and/or b).

Proteases present and/or added during liquefaction

In one embodiment according to the invention, the thermostable protease, together with the alpha-amylase, e.g. thermostable alpha-amylase, and optionally the carbohydrate source producing enzyme, in particular a thermostable glucoamylase, or thermostable pullulanase may be present and/or added during liquefaction.

Proteases are classified into the following groups according to their catalytic mechanism: serine proteases (S), cysteine proteases (C), aspartic proteases (a), metalloproteinases (M) and also proteases (U) of unknown or not yet classified, see Handbook of proteolytic Enzymes, a.j.barrett, n.d.rawlings, j.f.Woessner (ed.), Academic Press [ Academic Press ] (1998), in particular summary section.

In a preferred embodiment, the thermostable protease used according to the invention is a "metalloprotease", defined as belonging to EC 3.4.24 (metalloendopeptidase); EC 3.4.24.39 (acid metalloprotease) is preferred.

To determine whether a given protease is a metalloprotease, reference is made to the above-mentioned "Handbook of proteolytic enzymes" and the guidelines indicated therein. Such a determination can be made for all types of proteases, whether they are naturally occurring or wild-type proteases; or a genetically engineered or synthetic protease.

Protease activity may be measured using any suitable assay, wherein a substrate is employed which comprises peptide bonds relevant to the specificity of the protease in question. The determination of the pH value and the determination of the temperature likewise apply to the protease in question. Examples of measuring the pH value are pH 6,7,8, 9,10 or 11. Examples of measurement temperatures are 30 ℃,35 ℃, 37 ℃,40 ℃, 45 ℃, 50 ℃, 55 ℃,60 ℃, 65 ℃, 70 ℃ or 80 ℃.

Examples of protease substrates are caseins, such as Azurine-Crosslinked Casein, AZCL-Casein. Two protease assays are described below in the materials and methods section, with the so-called "AZCL-casein assay" being the preferred assay.

There is no limitation on the source of the protease used in the method of the present invention as long as it satisfies the thermostability characteristics defined below.

The protease may be, for example, a variant of a wild-type protease, provided that the protease has the thermostability characteristics defined herein.

In one embodiment, the protease has a thermostability at 85 ℃ of greater than 60%, e.g., greater than 90%, e.g., greater than 100%, e.g., greater than 110%, as determined using a Zein-BCA assay.

In one embodiment, the protease has a thermal stability at 85 ℃ of between 60% -120%, such as between 70% -120%, such as between 80% -120%, such as between 90% -120%, such as between 100% -120%, such as 110% -120%, as determined using the Zein-BCA assay.

In one embodiment, the thermostable protease is a variant of a metalloprotease as defined above. In one embodiment, the thermostable protease used in the method of the invention is of fungal origin, e.g. a fungal metalloprotease, e.g. derived from a strain of thermoascus, preferably a strain of thermoascus aurantiacus, especially thermoascus aurantiacus cgmccno.0670 (classified as EC 3.4.24.39).

In one embodiment, the thermostable protease is a variant disclosed in: the mature part of the metalloprotease shown in SEQ ID NO:2 or the mature part of SEQ ID NO:1 in W2010/008841 disclosed in WO2003/048353 and shown herein as SEQ ID NO:20, further having mutations selected from the list of:

-S5*+D79L+S87P+A112P+D142L；

-D79L+S87P+A112P+T124V+D142L；

-S5*+N26R+D79L+S87P+A112P+D142L；

-N26R+T46R+D79L+S87P+A112P+D142L；

-T46R+D79L+S87P+T116V+D142L；

-D79L+P81R+S87P+A112P+D142L；

-A27K+D79L+S87P+A112P+T124V+D142L；

-D79L+Y82F+S87P+A112P+T124V+D142L；

-D79L+S87P+A112P+T124V+A126V+D142L；

-D79L+S87P+A112P+D142L；

-D79L+Y82F+S87P+A112P+D142L；

-S38T+D79L+S87P+A112P+A126V+D142L；

-D79L+Y82F+S87P+A112P+A126V+D142L；

-A27K+D79L+S87P+A112P+A126V+D142L；

-D79L+S87P+N98C+A112P+G135C+D142L；

-D79L+S87P+A112P+D142L+T141C+M161C；

-S36P+D79L+S87P+A112P+D142L；

-A37P+D79L+S87P+A112P+D142L；

-S49P+D79L+S87P+A112P+D142L；

-S50P+D79L+S87P+A112P+D142L；

-D79L+S87P+D104P+A112P+D142L；

-D79L+Y82F+S87G+A112P+D142L；

-S70V+D79L+Y82F+S87G+Y97W+A112P+D142L；

-D79L+Y82F+S87G+Y97W+D104P+A112P+D142L；

-S70V+D79L+Y82F+S87G+A112P+D142L；

-D79L+Y82F+S87G+D104P+A112P+D142L；

-D79L+Y82F+S87G+A112P+A126V+D142L；

-Y82F+S87G+S70V+D79L+D104P+A112P+D142L；

-Y82F+S87G+D79L+D104P+A112P+A126V+D142L；

-A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L；

-A27K+Y82F+S87G+D104P+A112P+A126V+D142L；

-A27K+D79L+Y82F+D104P+A112P+A126V+D142L；

-A27K+Y82F+D104P+A112P+A126V+D142L；

-A27K+D79L+S87P+A112P+D142L；

-D79L+S87P+D142L。

in a preferred embodiment, the thermostable protease is a variant disclosed in: the mature part of the metalloprotease shown in SEQ ID NO:2 disclosed in WO2003/048353 or the mature part of SEQ ID NO:1 in WO2010/008841 and shown in SEQ ID NO:20 herein, further has mutations selected from the list of:

D79L+S87P+A112P+D142L；

D79L + S87P + D142L; or

A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L。

In one embodiment, the protease variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% but less than 100% identity to the mature part of the polypeptide of SEQ ID No. 2 disclosed in WO2003/048353 or the mature part of SEQ ID No. 1 disclosed in WO2010/008841 or SEQ ID No. 20 herein.

Thermostable proteases may also be derived from bacteria, in particular the S8 protease, more in particular the S8 protease from a pyrococcus species or a thermophilic coccus species.

In one embodiment, the thermostable protease is derived from a strain of the bacterium pyrococcus, such as a strain of pyrococcus furiosus (pfu protease).

In one embodiment, the protease is one as shown in U.S. Pat. No. 6,358,726-B1 (Takara Shuzo Company) SEQ ID NO:1, and herein SEQ ID NO: 19.

In another embodiment, the thermostable protease is a protease disclosed in SEQ ID No. 19 herein or a protease having at least 80% identity, e.g., at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID No. 1 in U.S. patent No. 6,358,726-B1 or SEQ ID No. 19 herein.

Glucoamylase present and/or added in liquefaction

In one embodiment, in the liquefaction step a) in the process of the invention (i.e. the oil recovery process and the fermentation product production process), a glucoamylase is present and/or added.

In a preferred embodiment, the glucoamylase present and/or added in the liquefaction step a) is derived from a penicillium strain, in particular a penicillium oxalicum strain as disclosed in SEQ ID No. 2 or SEQ ID No. 21 herein as in WO 2011/127802.

In one embodiment, the glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the mature polypeptide shown in SEQ ID No. 2 in WO 2011/127802 or SEQ ID No. 21 herein.

In a preferred embodiment, the glucoamylase is a variant of the penicillium oxalicum glucoamylase shown in SEQ ID No.2 of WO 2011/127802 or SEQ ID No. 21 herein having a K79V substitution (numbering using the mature sequence shown in SEQ ID No. 21), such as the variant disclosed in WO 2013/053801.

In one embodiment, the penicillium oxalicum glucoamylase has the K79V substitution (numbering using SEQ ID NO: 21), and preferably further has one of the following substitutions:

T65A; or

Q327F; or

E501V; or

Y504T; or

Y504 —; or

T65A + Q327F; or

T65A + E501V; or

T65A + Y504T; or

T65A + Y504; or

Q327F + E501V; or

Q327F + Y504T; or

Q327F + Y504; or

E501V + Y504T; or

E501V + Y504; or

T65A + Q327F + E501V; or

T65A + Q327F + Y504T; or

T65A + E501V + Y504T; or

Q327F + E501V + Y504T; or

T65A + Q327F + Y504; or

T65A + E501V + Y504; or

Q327F + E501V + Y504; or

T65A + Q327F + E501V + Y504T; or

T65A+Q327F+E501V+Y504*；

E501V + Y504T; or

T65A + K161S; or

T65A + Q405T; or

T65A + Q327W; or

T65A + Q327F; or

T65A + Q327Y; or

P11F + T65A + Q327F; or

R1K + D3W + K5Q + G7V + N8S + T10K + P11S + T65A + Q327F; or

P2N + P4S + P11F + T65A + Q327F; or

P11F + D26C + K33C + T65A + Q327F; or

P2N + P4S + P11F + T65A + Q327W + E501V + Y504T; or

R1E + D3N + P4G + G6R + G7A + N8A + T10D + P11D + T65A + Q327F; or

P11F + T65A + Q327W; or

P2N + P4S + P11F + T65A + Q327F + E501V + Y504T; or

P11F + T65A + Q327W + E501V + Y504T; or

T65A + Q327F + E501V + Y504T; or

T65A + S105P + Q327W; or

T65A + S105P + Q327F; or

T65A + Q327W + S364P; or

T65A + Q327F + S364P; or

T65A + S103N + Q327F; or

P2N + P4S + P11F + K34Y + T65A + Q327F; or

P2N + P4S + P11F + T65A + Q327F + D445N + V447S; or

P2N + P4S + P11F + T65A + I172V + Q327F; or

P2N + P4S + P11F + T65A + Q327F + N502; or

P2N + P4S + P11F + T65A + Q327F + N502T + P563S + K571E; or

P2N + P4S + P11F + R31S + K33V + T65A + Q327F + N564D + K571S; or

P2N + P4S + P11F + T65A + Q327F + S377T; or

P2N + P4S + P11F + T65A + V325T + Q327W; or

P2N + P4S + P11F + T65A + Q327F + D445N + V447S + E501V + Y504T; or

P2N + P4S + P11F + T65A + I172V + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + Q327F + S377T + E501V + Y504T; or

P2N + P4S + P11F + D26N + K34Y + T65A + Q327F; or

P2N + P4S + P11F + T65A + Q327F + I375A + E501V + Y504T; or

P2N + P4S + P11F + T65A + K218A + K221D + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + S103N + Q327F + E501V + Y504T; or

P2N + P4S + T10D + T65A + Q327F + E501V + Y504T; or

P2N + P4S + F12Y + T65A + Q327F + E501V + Y504T; or

K5A + P11F + T65A + Q327F + E501V + Y504T; or

P2N + P4S + T10E + E18N + T65A + Q327F + E501V + Y504T; or

P2N + T10E + E18N + T65A + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + Q327F + E501V + Y504T + T568N; or

P2N + P4S + P11F + T65A + Q327F + E501V + Y504T + K524T + G526A; or

P2N + P4S + P11F + K34Y + T65A + Q327F + D445N + V447S + E501V + Y504T; or

P2N + P4S + P11F + R31S + K33V + T65A + Q327F + D445N + V447S + E501V + Y504T; or

P2N + P4S + P11F + D26N + K34Y + T65A + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + F80 + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + K112S + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + Q327F + E501V + Y504T + T516P + K524T + G526A; or

P2N + P4S + P11F + T65A + Q327F + E501V + N502T + Y504; or

P2N + P4S + P11F + T65A + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + S103N + Q327F + E501V + Y504T; or

K5A + P11F + T65A + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + Q327F + E501V + Y504T + T516P + K524T + G526A; or

P2N + P4S + P11F + T65A + K79A + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + K79G + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + K79I + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + K79L + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + K79S + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + L72V + Q327F + E501V + Y504T; or

S255N + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + E74N + V79K + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + G220N + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + Y245N + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + Q253N + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + D279N + Q327F + E501V + Y504T; or

P2N + P4S + P11F + T65A + Q327F + S359N + E501V + Y504T; or

P2N + P4S + P11F + T65A + Q327F + D370N + E501V + Y504T; or

P2N + P4S + P11F + T65A + Q327F + V460S + E501V + Y504T; or

P2N + P4S + P11F + T65A + Q327F + V460T + P468T + E501V + Y504T; or

P2N + P4S + P11F + T65A + Q327F + T463N + E501V + Y504T; or

P2N + P4S + P11F + T65A + Q327F + S465N + E501V + Y504T; or

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T。

In a preferred embodiment, the glucoamylase present and/or added in the liquefaction is a penicillium oxalicum glucoamylase having a K79V substitution and preferably further having one of the following substitutions:

-P11F+T65A+Q327F；

P2N + P4S + P11F + T65A + Q327F (numbering using SEQ ID NO: 21).

In one embodiment, the glucoamylase variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the polypeptide of SEQ ID No. 21 herein.

The glucoamylase may be added in an amount of from 0.1 to 100 micrograms EP/g, such as from 0.5 to 50 micrograms EP/g, such as from 1 to 25 micrograms EP/g, such as from 2 to 12 micrograms EP/g DS.

Glucoamylases present and/or added in saccharification and/or fermentation

In the processes of the present invention (i.e., oil recovery processes and fermentation product production processes), glucoamylase is present and/or added during saccharification and/or fermentation, preferably Simultaneous Saccharification and Fermentation (SSF).

In one embodiment, the glucoamylase present and/or added in the saccharification and/or fermentation is of fungal origin, preferably from a strain of aspergillus, preferably aspergillus niger, aspergillus awamori (a.awamori) or aspergillus oryzae; or a strain of Trichoderma, preferably Trichoderma reesei; or a strain of the genus Talaromyces, preferably Talaromyces emersonii; or a strain of trametes, preferably trametes annulata (t. cingulata); or a strain of the genus diplopodia; or a strain of the genus mucorales, such as mucorales fragilis or mucorales densatus; or a strain of the genus nigrostriata (Nigrofomes).

In one embodiment, the glucoamylase is derived from a strain of the genus Talaromyces, e.g., Talaromyces emersonii, e.g., the strain set forth in SEQ ID NO:22 herein,

in one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising a polypeptide of SEQ ID NO 22 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, 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%, or at least 99% identity with a polypeptide of SEQ ID No. 22 herein.

In one embodiment, the glucoamylase is derived from a strain of the genus Millettia, in particular a strain of Millettia haemolytica (SEQ ID NO 2, 4 or 6) as described in WO2011/066576, for example the strain shown as SEQ ID NO:4 in WO2011/066576 or SEQ ID NO:23 herein.

In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO 23 herein;

In one embodiment, the glucoamylase is derived from a strain of the genus Myxophycus, e.g.a strain of Myxophycus fragilis or Myxophycus densus, in particular a strain of the genus Myxophycus as described in WO 2011/068803 (SEQ ID NO:2, 4,6, 8, 10, 12, 14 or 16). In a preferred embodiment, the glucoamylase is Gloeophyllum fragrans shown in SEQ ID No.2 of WO 2011/068803 or SEQ ID No. 24 herein.

In a preferred embodiment, the glucoamylase is derived from Gloeophyllum fragrans, such as Gloeophyllum fragrans shown in SEQ ID NO:24 herein. In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising a polypeptide of SEQ ID NO 24 herein;

In another embodiment, the glucoamylase is derived from Pleurotus densatus, such as shown in SEQ ID NO:25 herein. In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising a polypeptide of SEQ ID NO 25 herein;

In one embodiment, the glucoamylase is derived from a strain of the genus leptinotarsa, in particular a strain of the species leptinotarsa as disclosed in WO 2012/064351.

In one embodiment, the glucoamylase may be added to the saccharification and/or fermentation in the following amounts: 0.0001 to 20AGU/g DS, preferably 0.001 to 10AGU/g DS, in particular between 0.01 and 5AGU/g DS, for example 0.1 to 2AGU/g DS.

Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300L; SAN^TMSUPER，SAN^TMEXTRA L，SPIRIZYME^TMPLUS，SPIRIZYME^TMFUEL，SPIRIZYME^TMB4U，SPIRIZYME^TMULTRA，SPIRIZYME^TMEXCEL and AMG^TME (from novicent corporation); OPTIDEX^TM300, GC480, GC417 (from DuPont); AMIGASE^TMAnd AMIGASE^TMPLUS (from DSM); G-ZYME^TMG900，G-ZYME^TMAnd G990 ZR (from dupont).

According to a preferred embodiment of the invention, the glucoamylase is present and/or added in combination with the alpha-amylase during saccharification and/or fermentation. Examples of suitable alpha-amylases are described below.

α -amylase present and/or added during saccharification and/or fermentation

In one embodiment, in the process of the invention, an alpha-amylase is present and/or added during saccharification and/or fermentation. In a preferred embodiment, the alpha-amylase is of fungal or bacterial origin. In a preferred embodiment, the alpha-amylase is a fungal acid stable alpha-amylase. Fungal acid stable alpha-amylases are alpha-amylases having activity in the pH range of 3.0 to 7.0 and preferably in the pH range of 3.5 to 6.5, including activity at pH of about 4.0, 4.5, 5.0, 5.5, and 6.0.

In a preferred embodiment, the alpha-amylase present and/or added during saccharification and/or fermentation is derived from a strain of Rhizomucor, preferably the strain Rhizomucor pusillus, e.g. the strain shown in SEQ ID NO. 3 in WO 2013/006756, e.g. a Rhizomucor pusillus alpha-amylase hybrid having an Aspergillus niger linker and a starch binding domain, e.g. the hybrid shown in SEQ ID NO. 26 herein or a variant thereof.

In one embodiment, the alpha-amylase present and/or added in the saccharification and/or fermentation is selected from the group consisting of:

(i) an alpha-amylase comprising a polypeptide of SEQ ID No. 26 herein;

(ii) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, 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%, or at least 99% identity to a polypeptide of SEQ ID No. 26 herein.

In a preferred embodiment, the alpha-amylase is a variant of the alpha-amylase shown in SEQ ID No. 26 having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H + Y141W; G20S + Y141W; a76G + Y141W; G128D + Y141W; G128D + D143N; P219C + Y141W; N142D + D143N; Y141W + K192R; Y141W + D143N; Y141W + N383R; Y141W + P219C + a 265C; Y141W + N142D + D143N; Y141W + K192RV 410A; G128D + Y141W + D143N; Y141W + D143N + P219C; Y141W + D143N + K192R; G128D + D143N + K192R; Y141W + D143N + K192R + P219C; G128D + Y141W + D143N + K192R; or G128D + Y141W + D143N + K192R + P219C (numbering using SEQ ID NO: 26).

In one embodiment, the alpha-amylase is derived from Rhizomucor miehei having an Aspergillus niger glucoamylase linker and Starch Binding Domain (SBD), preferably disclosed herein as SEQ ID NO:26, preferably with one or more of the following substitutions: G128D, D143N, preferably G128D + D143N (numbering using SEQ ID NO: 19).

In one embodiment, the alpha-amylase variant present and/or added in the saccharification and/or fermentation has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the polypeptide of SEQ ID No. 26 herein.

In a preferred embodiment, the ratio between glucoamylase present and/or added during saccharification and/or fermentation and alpha-amylase may preferably be in the range of 500:1 to 1:1, e.g. from 250:1 to 1:1, such as from 100:2 to 100:50, such as from 100:3 to 100: 70.

Pullulanase present and/or added during liquefaction and/or saccharification and/or fermentation.

Pullulanase may be present and/or added during the liquefaction step a) and/or the saccharification step b) or the fermentation step c) or the simultaneous saccharification and fermentation.

Pullulanase (e.c3.2.1.41, pullulan 6-glucanohydrolase) is a debranching enzyme (debranching enzyme) characterized by its ability to hydrolyze, for example, the α -1, 6-glucosidic bonds in amylopectin (amylopectin) and amylopectin (pullulan).

Pullulanases contemplated according to the present invention include pullulanase from Bacillus amyloliquefaciens (Bacillus amyloderamificans) disclosed in U.S. Pat. No.4,560,651 (hereby incorporated by reference), pullulanase from Bacillus amyloderamificans (SEQ ID NO:2) disclosed in WO 01/51620 (hereby incorporated by reference), pullulanase from Bacillus amyloliquefaciens (Bacillus deramificans) disclosed in WO 01/51620 (hereby incorporated by reference) as SEQ ID NO:4, pullulanase from Bacillus pullulans disclosed in WO 01/51620 (hereby incorporated by reference) as SEQ ID NO:6, and pullulanase described in FEMS microbiology letters (FEMS Mic.Let.) (1994)115, 97-106.

According to the invention, the pullulanase may be added in an effective amount, including preferred amounts of about 0.0001-10mg enzyme protein per gram DS, preferably 0.0001-0.10mg enzyme protein per gram DS, more preferably 0.0001-0.010mg enzyme protein per gram DS. The pullulanase activity can be determined as NPUN. The assay for determining NPUN is described in the materials and methods section below.

Suitable commercially available pullulanase products include PROMOZYME D, PROMOZYME D^TMD2 (Novexin, Denmark), OPTIMAX L-300 (Jenenco Int., USA), and AMANO 8 (Amano, Japan).

The fermentation product, such as especially ethanol, may optionally be recovered after fermentation, for example by distillation. A variety of suitable starchy starting materials are listed in the "starchy materials" section below. In one embodiment, the starch-containing material is corn or wheat.

The fermenting organism is preferably a yeast, preferably a strain of Saccharomyces, especially a strain of Saccharomyces cerevisiae. A variety of suitable fermenting organisms are listed in the "fermenting organisms" section above. In a preferred embodiment, steps ii) and iii) are performed sequentially or simultaneously (i.e. as an SSF process). The aqueous slurry may contain from 10 to 55 wt.% dry solids, preferably 25 to 45 wt.% dry solids, more preferably 30 to 40 wt.% dry solids of the starch-containing material. Heating the slurry above an initial gelatinization temperature. An alpha-amylase, preferably a bacterial alpha-amylase, may be added to the slurry. In one embodiment, the slurry is also jet cooked to further gelatinize the slurry prior to being subjected to alpha-amylase in liquefaction step i).

The temperature during step (i) is above the initial gelatinization temperature, e.g. between 80 ℃ and 90 ℃, e.g. around 85 ℃.

In one embodiment, the liquefaction is performed in a three-step hot slurry process. The slurry is heated to between 60 ℃ to 95 ℃, preferably 80 ℃ to 90 ℃, and alpha-amylase is added to start liquefaction (dilution). The slurry is then jet cooked at a temperature between 95 ℃ and 140 ℃, preferably 105 ℃ to 125 ℃ for 1-15 minutes, preferably 3-10 minutes, especially about 5 minutes. The slurry is cooled to 60-95 ℃, preferably 80-90 ℃, and further alpha-amylase is added to end the hydrolysis (secondary liquefaction). The liquefaction process is typically carried out at a pH of 4.5 to 6.5, such as about 4.8, or 5.0 to 6.2, such as 5.0 to 6.0, such as 5.0 to 5.5, such as about 5.2, such as about 5.4, such as about 5.6, such as about 5.8. The milled and liquefied starch is called "mash (mash)".

The saccharification in step ii) may be performed using conditions well known in the art. For example, the entire saccharification process may last from about 24 hours to about 72 hours. In one embodiment, the pre-saccharification step is completed at a temperature between 30 ℃ and 65 ℃, typically about 60 ℃, in 40-90 minutes, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation step (SSF). Saccharification is typically carried out at a temperature of from 30 ℃ to 70 ℃, e.g. 55 ℃ to 65 ℃, typically around 60 ℃, and at a pH between 4 and 5, generally at about pH 4.5.

The most widely used process in the production of fermentation products, particularly ethanol, is the Simultaneous Saccharification and Fermentation (SSF) process, in which there is no holding stage for saccharification.

When the fermenting organism is a strain of yeast, such as saccharomyces cerevisiae, and the desired fermentation product is ethanol, SSF may typically be carried out at a temperature between 25 ℃ and 40 ℃, such as between 28 ℃ and 36 ℃, such as between 30 ℃ and 34 ℃, for example about 32 ℃. In one embodiment, the fermentation is carried out for 6 to 120 hours, in particular 24 to 96 hours.

Other fermentation products may be fermented under conditions and temperatures well known to those skilled in the art and appropriate to the fermenting organism in question.

Fermentation medium

The environment in which fermentation is carried out is often referred to as a "fermentation medium". The fermentation medium includes a fermentation substrate, i.e., a source of carbohydrates that are metabolized by the fermenting organism. According to the invention, the fermentation medium may comprise one or more nutrients and growth stimulators for the one or more fermenting organisms. Nutrients and growth stimulants are widely used in the field of fermentation, and include nitrogen sources such as ammonia; urea, vitamins and minerals or combinations thereof.

Fermenting organisms

The term "fermenting organism" refers to any organism suitable for use in a fermentation process and capable of producing a desired fermentation product, including bacterial and fungal organisms, especially yeast. Particularly suitable fermenting organisms are capable of fermenting (i.e., converting) a sugar (e.g., glucose or maltose) directly or indirectly into a desired fermentation product (e.g., ethanol). Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeasts include strains of Saccharomyces species, in particular Saccharomyces cerevisiae.

Suitable concentrations of viable fermenting organisms during fermentation (e.g., SSF) are well known in the art or can be readily determined by one skilled in the art. In one embodiment, a fermenting organism, such as an ethanol fermenting yeast (e.g., saccharomyces cerevisiae), is added to the fermentation medium such that viable fermenting organisms, such as yeast, are counted per mL of fermentation mediumAt 10 from⁵To 10¹²Preferably from 10⁷To 10¹⁰In particular about 5x10⁷And (4) respectively.

Examples of commercially available yeasts include, for example, RED STAR^TMAnd ETHANOL RED^TMYeast (available from Fungiase Tech/Lesfure, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC^TMFresh yeast (available from Ethanol Technology, Wisconsin, USA), BIOFERM AFT and XR (available from NABC-North American Bioproducts Corporation, Georgia, USA), GERT STRAND (available from Gert Strand AB, Sweden), FERMIL IOL (available from Disemann specialty products, DSM Specialties)),

drive (Novixin Co.),Lift (Novit Corp.).

Starch-containing material

Any suitable starch-containing material may be used in accordance with the present invention. The starting materials are generally selected based on the desired fermentation product. Examples of starch-containing materials suitable for use in the process of the invention include whole grains, corn, wheat, barley, rye, milo, sago, tapioca, sorghum, rice, peas, beans or sweet potatoes or mixtures thereof or starches derived therefrom, or cereals. Corn and barley of waxy (waxy type) and non-waxy (non-waxy type) types are also contemplated. In a preferred embodiment, the starch-containing material used for ethanol production according to the invention is corn or wheat.

Fermentation product

The term "fermentation product" means a product produced by a process that includes a fermentation step performed using a fermenting organism. Fermentation products contemplated according to the present invention include alcohols (e.g., ethanol,Methanol, butanol; polyols such as glycerol, sorbitol and inositol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g. H)₂And CO₂) (ii) a Antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g. riboflavin, B)₁₂In a preferred embodiment, the fermentation product is ethanol, e.g., fuel ethanol, potable ethanol, i.e., neutral potable ethanol, or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry.

Recovery of fermentation products

After fermentation or SSF, the fermentation product may be separated from the fermentation medium. The slurry may be distilled to extract the desired fermentation product (e.g., ethanol). Alternatively, the desired fermentation product may be extracted from the fermentation medium by microfiltration or membrane filtration techniques. The fermentation product may also be recovered by steam stripping or other methods well known in the art.

The invention is further illustrated in the following numbered examples.

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Alpha-amylase variants and polynucleotides encoding same

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