阅读说明：本技术 回收方法 (Recovery method ) 是由 S.格兰维尔 P.F.平德 S.雅格布森 L.约翰森 C.雅格布森 K.B.安德森 S. 于 2018-04-03 设计创作，主要内容包括：披露了一种用于从发酵液中回收所需发酵产物的方法,其中该所需产物已在发酵期间发生沉淀。(A kind of method for tunning needed for recycling from fermentation liquid is disclosed, wherein the required product precipitates during fermentation.)

1. a kind of method for recycling required product from fermentation liquid, wherein the required product is present in the fermentation liquid with precipitation form In, method includes the following steps:

A) the first separating step of the fermentation liquid is separated in the first phase and the second phase, wherein first phase includes supernatant, is in The required product of soluble form and optional cell and cell fragment, and second phase includes the required production in precipitation form Object, cell and cell fragment；And

B) dissolving step, wherein dissolving the required product in precipitation form in second phase.

2. according to the method described in claim 1, this method further comprises the second separating step, wherein will be from step b's) The required product of the dissolution is separated with these cells and/or cell fragment.

3. method according to claim 1 or 2, wherein first phase include the liquid portion of the fermentation liquid at least 60%, for example, the fermentation liquid at least 70%, for example, at least 80%.

4. method according to any one of claim 1-3, wherein the second level subpackage contains required in this of solid form At least the 60% of product, preferably at least the 65% of the required product of solid form, for example, at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 85% and, for example, at least 99%.

5. method according to any of the preceding claims, wherein the required product includes one or more enzymes.

6. according to the method described in claim 5, wherein one or more enzymes are selected from the group for the enzymatic activity being made of following item: Aminopeptidase, amylase, amyloglucosidase, mannonase carbohydrase, carboxypeptidase, catalase, cellulase, chitin Enzyme, cutinase, cyclodextrin glycosyl transferases, deoxyribonuclease, esterase, galactosidase, beta galactosidase, glucose form sediment Powder enzyme, glucose oxidase, glucosidase, haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase, rouge Fat enzyme, lyases, mannosidase, oxidizing ferment, pectase, peroxidase, phytase, phenol oxidase, polyphenol oxidase, egg White enzyme, ribalgilase, transferase, transglutaminase, lysozyme, muramidase or zytase.

7. according to the method described in claim 6, wherein one or more enzymes are selected from and have extremely with one of SEQ ID NO:1-6 Few 80% sequence identity, such as at least 85% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence is same Property, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least protease of 99% sequence identity.

8. according to the method described in claim 6, wherein one or more enzymes are selected from and have extremely with one of SEQ ID NO:7-9 Few 80% sequence identity, such as at least 85% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence is same Property, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least amylase of 99% sequence identity.

9. method according to any of the preceding claims, wherein the required product is obtained from microorganism.

10. according to the method described in claim 9, wherein the microorganism is prokaryotes selected from the following: bacillus, shuttle Pseudomonas, enterococcus spp, ground bacillus category, lactobacillus, lactococcus, ocean bacillus, staphylococcus, streptococcus Category, streptomyces, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacterium, mud Bacillus, eisseria, Pseudomonas, Salmonella and Ureaplasma.

11. according to the method described in claim 10, wherein the microorganism is bacillus cell selected from the following: basophilic bud Spore bacillus, highland bacillus, bacillus amyloliquefaciens, plant bacillus amyloliquefaciens subspecies, bacillus brevis, cyclic annular gemma Bacillus, Bacillus clausii, bacillus coagulans, bacillus firmus, bacillus lautus, bacillus lentus, lichens Bacillus, bacillus megaterium, Methylotrophic bacillus, bacillus pumilus, husky good fortune bacillus, stearothermophilus bud Spore bacillus, bacillus subtilis and Bacillus thuringiensis cell.

12. according to the method described in claim 9, wherein the microorganism is eucaryote selected from the following: candida, the Chinese Inferior saccharomyces, Kluyveromyces, pichia, Blastocystis, Schizosaccharomyces, Ye Shi saccharomyces, acremonium, Aspergillus, Aureobasidium, smoke pipe are mould to be belonged to, intends the black powder of cured Pseudomonas, Chrysosporium, Coprinus, Coriolus Qu61, Cryptococcus, line Pseudomonas, Fusarium, Humicola, Magnaporthe grisea category, mucor, myceliophthora, new U.S. whip Pseudomonas, Neurospora, paecilomyces, blueness Mould category, penetrates arteries and veins Pseudomonas, pears capsule whip Pseudomonas, Pleurotus, Schizophyllum, Talaromyces, thermophilic ascomycete category, shuttle spore at flat lead fungi category Shell category, Tolypocladium, Trametes or trichoderma cell.

13. according to the method for claim 12, wherein the microorganism is selected from: Kluyveromyces lactis, saccharomyces carlsbergensis, wine brewing Yeast, saccharomyces diastaticus, Doug Laplace yeast, Saccharomyces kluyveri, promise yeast, ellipsoideus yeast or Yarrowia lipolytica, bubble Sheng Qu Mould, smelly aspergillus, aspergillus fumigatus, aspergillus japonicus, aspergillus nidulans, aspergillus niger, aspergillus oryzae, black thorn smoke pipe bacterium, dry plan wax bacterium, Ka Neiji are quasi- Wax bacterium, pale yellow quasi- wax pore fungi, Pernod wish tower intend wax bacterium, annulus intend wax bacterium, micro- red quasi- wax bacterium, worm intend wax bacterium, straight hem gold pityrosporion ovale, Chrysosporium keratinophilum, Lu Kenuo train of thought gold pityrosporion ovale, excrement shape gold pityrosporion ovale, rent pityrosporion ovale, queen Du Xiangjin pityrosporion ovale, heat The golden pityrosporion ovale of band, brown thin golden pityrosporion ovale, Coprinus cinereus, hairy fungus, bar spore shape fusarium, cereal fusarium, library prestige fusarium, machete sickle Spore, F.graminearum schw, red fusarium of standing grain, different spore fusarium, albizzia fusarium, fusarium oxysporum, racemosus fusarium, pink fusarium, elder sickle Spore, colour of skin fusarium, intend branch spore fusarium, sulphur color fusarium, circle fusarium, quasi- silk spore fusarium, empiecement fusarium, Humicola insolens, pubescence Humicola lanuginosa, thermophilic fungus destroyed wire, Neuraspora crassa, penicillium purpurogenum, Phanerochaete chrysosporium, penetrates arteries and veins bacterium, eryngo side at rice black wool mould Ear, autochthonal shuttle spore shell be mould, long domain Trametes trogii, Trametes versicolor, Trichoderma harzianum, trichodermaharzianum, long shoot trichoderma, trichoderma reesei or green Trichoderma cell.

14. method according to any of the preceding claims, wherein first separating step using centrifugation or filtering and It carries out.

15. method according to any of the preceding claims, wherein the dissolving step includes:

I. by second phase dilution 100%-2000% (w/w) in water or water-bearing media；

Ii. divalent salts are added；And

Iii., pH is adjusted to the pH value lower than 6.0.

16. according to the method for claim 15, wherein by second phase dilution 100%-2000% (w/w), preferably 100%-1500% (w/w), preferably 100%-1000% (w/w), and most preferably 200%-700% (w/w).

17. method described in any one of 5-16 according to claim 1, wherein the divalent salts are selected from comprising being selected from phosphate radical, sulphur Acid group, nitrate anion, acetate and chloride ion anion calcium salt, magnesium salts, ferrous salt and zinc salt, and based on this diluted the Two-phase is with 0.01%-5% (w/w), preferably 0.01%-1% (w/w), and the amount in more preferable 0.1%-0.5% (w/w) range adds Add.

18. method described in any one of 5-17 according to claim 1, wherein the pH is adjusted to lower than pH 6.0, it is preferably low In the pH value of pH 5.5, be especially adjusted to the pH value lower than 5.0, can prior to, concurrently with, or after adding the divalent salts into The row pH is adjusted, the pH value which can be adjusted between 2.0 and 5.5；The pH value being preferably adjusted between 2.0 and 5.0； The pH value being more preferably adjusted between 3.0 and 5.0, the pH value being especially adjusted between 4.0 and 5.0.

19. method described in any one of -14 according to claim 1, wherein the dissolving step carries out in the following manner: using water Or aqueous solution dilutes second phase, and the pH is adjusted to the pH value higher than 9.5, the pH being such as adjusted in 9.5 to 13 ranges Value, such as the pH being adjusted in 10 to 13 ranges.

20. method described in any one of -14 according to claim 1, wherein the dissolving step enhances the required production by addition The chemicals of the dissolution of object is completed.

21. according to the method for claim 20, wherein the chemicals of the dissolution of the enhancing required product is polyalcohol, such as Low molecular poly or at least two OH groups, preferably only there are two OH group C₂To C₈Alcohol；Particularly preferably Two OH groups are present in the polyalcohol on the adjacent carbon atom in chain, and the C₂-C₈Alcohol is aliphatic series and has straight chain carbon Chain.

22. method according to any of the preceding claims, wherein by first phase from first separating step Partially or completely it is added in the second phase of the dissolution.

23. method according to any of the preceding claims, this method comprises: pre- before first separating step Processing step, the pre-treatment step are selected from: dilution；It adjusts pH and/or temperature, the one or more stabilizers of addition, add one kind Or multiple protein enzyme inhibitor.

24. method according to any of the preceding claims, this method comprises: one after second separating step Or multiple downstream process, the one or more downstream process are selected from: ultrafiltration, concentration, stabilization, crystallization, is spray-dried and makes evaporation Grain.

Technical field

The present invention relates to a kind of for dissolving the improved method of protein crystal and/or protein precipitation in fermentation liquid.

Background technique

The fermentation production rate of industrial protein (such as enzyme, such as protease) has greatly improved in recent years.In many industrial mistakes Cheng Zhong, the concentration of protein is very high in fermentation liquid, so that finding most protein with crystalline substance at the end of fermentation process The form of body or solid precipitating exists.

WO 93/13125 discloses a kind of method for producing protease, wherein protease pass through during fermentation to Production medium is added precipitating reagent and is precipitated.It has been found that protease precipitate is made to avoid protease that egg occurs during fermentation White matter hydrolysis.

For many tunnings, it is expected that removing microorganism used in fermentation from product.Product recovery process One of first step be usually that cellular biomass is removed from product, such as by filtering or being centrifuged from comprising required product Solid cell biomass is removed in liquid.The step is commonly referred to as primary separating step, wherein by microorganism and liquid fraction point From, then can by the one or more downstream procedures processing of the liquid fraction, such as ultrafiltration, stabilisation, spray drying, granulation, Required product is finally transformed into form expected from the specific product and concentration.

However, if a part of required product exists in solid form after fermentation, must take adequate measures with The product is being dissolved before removing biomass in the liquid fraction comprising the product.

US 6,316,240 discloses a kind of method of enzyme for precipitating during being dissolved in zymotechnique, wherein will The pH of culture solution is adjusted to the pH between 9.5 and 13.0.This method is by taking starch enzyme fermentation as an example.

EP 2 125 865 discloses a kind of for dissolving the side of protease crystals and/or protease precipitate in fermentation liquid Method, this method comprises: fermentation liquid is diluted 100%-2000% (w/w)；Add divalent salts；And the pH value of fermentation liquid is adjusted To the pH value for being lower than 5.5.

EP 1 456 613 disclose it is a kind of for harvesting crystalline particle from fermentation liquid, especially crystallization alpha-amylase or The method of protease, wherein by separation of fermentative broth at biomass fraction, crystallization alpha-amylase or protease fraction and supernatant grade Point.

Summary of the invention

The present invention provides a kind of methods for recycling required product from fermentation liquid, and wherein required product is to precipitate shape Formula is present in fermentation liquid, this method comprises:

A) in the first phase and the second phase separation and fermentation liquid the first separating step, wherein the first phase include come from fermentation liquid Supernatant and its may include a part of cell and cell fragment from fermentation liquid, and the second phase includes precipitation form Required product, cell and cell fragment；And

B) dissolving step, wherein dissolving the required product of precipitation form.

In a preferred embodiment, this method further comprises the second separating step, wherein will be from step b's) The required product of dissolution is separated with cell and/or cell fragment.

The detailed description of sequence

SEQ ID NO:1: the amino acid sequence of slow bacillus (B.lentus) protease (Savinase)

SEQ ID NO:2: the amino acid sequence of bacillus amyloliquefaciens (B.amyloliquefaciens) protease (BPN ') Column

SEQ ID NO:3: subtilopeptidase A Carlsberg

SEQ ID NO:4: the protease TY145 of Bacillus spec (Bacillus sp.) is come from, in WO 92/ It is disclosed in 17577

SEQ ID NO:5: coming from the protease of nocardia species (Nocardiopsis sp.) NRRL 18262, It is disclosed in WO 88/03947

SEQ ID NO:6: pyrococcus furiosus (Pyrococcusfuriosus) protease drapes over one's shoulders in US 6,358,726 Dew.

SEQ ID NO:7: the alpha-amylase from Bacillus spec discloses in WO 2000/060060.

SEQ ID NO:8: the alphalise starch of bacillus stearothermophilus (Bacillus stearothermophilus) is come from Enzyme SEQ ID NO:9: the alpha-amylase of bacillus licheniformis (Bacillus licheniformis) is come from

Detailed description of the invention

Fig. 1 shows the dissolution of the muramidase from culture solution (CB) or from the second phase according to the present invention, reflection Experimental result in example 5.

Specific embodiment

The present invention relates to the primary separation of fermentation liquid, tunning needed for a portion exists in solid form, such as with Crystallization or amorphous form exist.For the present invention, required tunning be in crystallization or amorphous form or It is even not important in the mixture of these forms, it is important that most required product is in solid form, therefore first fraction It may be easy to carry out unlike simple solid/liquid separation process from step.The purpose of primary separating step is from required production Cell and cell fragment are removed in object.

The present invention relates to the methods of tunning needed for recycling from fermentation liquid, and wherein tunning is at least partly insoluble Form is present in fermentation liquid, such as exists with crystallization or amorphous form；This method comprises:

The first separating step of separation and fermentation liquid in the first phase and the second phase, wherein the first phase includes to come from fermentation liquid Liquid and soluble form required tunning；And the second phase include the required tunning of insoluble form, cell and/or Cell fragment；And

Dissolving step, wherein dissolving the required tunning in the second phase.

In some embodiments, the present invention relates to a kind of method, this method further comprises the second separating step, this second Separating step separates the liquid phase of the required tunning comprising dissolution with solid biomass, optionally in the first phase that will be dissolved After being mixed with part or all of the first phase from the first separating step.

The present invention is based on following observations: fermentation liquid seems containing the harmful component of some pairs of course of dissolution.Data seem The optimum condition for showing the high-dissolvability of protein (such as enzyme) is that have the condition of high enzyme concentration and low ion concns.However, answering The understanding, the present invention are not being bound by any particular theory.If having been found that hair can be not present in the dissolution of required product Occur in the case where at least part of the liquid portion containing these most of unfavorable components of zymotic fluid, is then beneficial.

According to the present invention, fermentation means such a process: one or more microorganisms include the one kind fermentor Or grown in the fermentation medium of all components needed for multiple-microorganism growth, so that generating includes one or more micro- lifes The fermentation liquid of object, used substrate component and one or more required tunnings.If the yield of required tunning is enough Height, then a part of tunning may precipitate in fermentation liquid, to form crystal or amorphous sediment.This is in this field In be well known, and this field has disclosed many fermentation process, microorganism, nutrient media components.The present invention is not by specific The limitation of fermentation process, as long as the fermentation process is provided in fermentation liquid comprising at least partly insoluble form (such as crystallization or without fixed Shape form) required tunning fermentation liquid.

The method for fermentative microorganism to generate required product has been described in this field, and wherein required product is being fermented Period is precipitated, such as US 6,316,240, WO 03/050274, WO 93/13125 and EP2125865, and for sending out Any of these methods of ferment microorganism can be used together with method of the invention.

Fermentation process can be to provide any conjunction of the fermentation liquid of the required tunning comprising at least partly insoluble form Suitable fermentation process, such as batch fermentation, fed-batch fermentation or continuously ferments.

The present invention can be used for plant-scale any fermentation, for example, for have at least 50 liters, such as at least 500 liters, such as extremely Few 5,000 liters, any fermentation of such as at least 50,000 liters of culture medium.

Tunning can be the spawn generated by microorganism accumulated in fermentation liquid.Tunning can be just Grade metabolin, secondary metabolites, protein, vitamin, hormone and carbohydrate.Tunning is preferably chosen from protein (such as enzyme).Tunning can even is that the mixture of two or more required enzymes, for example, comprising by cellulose degradation at low The mixture of all enzymes necessary to molecular weight sugar (such as glucose and cellobiose).

In one embodiment, tunning includes selected from by oxidoreducing enzyme (EC 1), transferase (EC2), hydrolase The enzyme of the group of the enzyme of (EC 3), lyases (EC 4), isomerase (EC 5) and ligase (EC 6) composition.

In another embodiment, enzyme is the active enzyme with the group selected from the enzymatic activity being made of following item: ammonia peptide Enzyme, amylase, amyloglucosidase, mannonase carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, angle Matter enzyme, cyclodextrin glycosyl transferases, deoxyribonuclease, esterase, galactosidase, beta galactosidase, glucoamylase, Glucose oxidase, glucosidase, haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase, lipase, Lyases, mannosidase, oxidizing ferment, pectase, peroxidase, phytase, phenol oxidase, polyphenol oxidase, protease, Ribalgilase, transferase, transglutaminase, lysozyme, muramidase or zytase.

Preferably, tunning is protease or alpha-amylase.

Suitable protease includes those of bacterium, fungi, plant, virus or animal origin, such as plant or microorganism Source.It is preferably microbe-derived.Mutant or protein engineered mutant including chemical modification.It can be alkali Property protease, such as serine protease or metalloproteinases.Serine protease may, for example, be (such as tryptose of S1 family Enzyme) or S8 family (such as subtilopeptidase A).Metalloproteinases may, for example, be the Thermophilic Bacteria egg from such as M4 family White enzyme or other metalloproteinases, such as from those of M5, M7 or M8 family.

Term " novel subtilases " refers to according to Siezen et al., Protein Engng. [protein engineering] 4 (1991) 719-737 and Siezen et al., the serine protease of 6 (1997) 501-523 of Protein Science [protein science] Subgroup.Serine protease is the protease for being characterized in that having the serine for forming covalent adduct with substrate in active site Subgroup.Novel subtilases can be divided into 6 subclass, that is, subtilopeptidase A family, thermophilic protein enzyme family, egg Bai Mei K family, lantibiotic peptidase families, Kexin family and Pyrolysin family.

The example of novel subtilases is derived from those of bacillus, is such as described in US 7262042 and WO 09/ Bacillus lentus (Bacillus lentus), Alkaliphilic bacillus (B.alkalophilus), withered grass gemma in 021867 Bacillus (B.subtilis), bacillus amyloliquefaciens (B.amyloliquefaciens), bacillus pumilus (Bacillus ) and bacillus gibsonii (Bacillus gibsonii) pumilus；And it is described in the hay bacillus egg in WO 89/06279 White enzyme lentus, subtilopeptidase A Novo, subtilopeptidase A Carlsberg, bacillus licheniformis (Bacillus Licheniformis), subtilopeptidase A BPN ', subtilopeptidase A 309, subtilopeptidase A 147 and withered grass bar Mycoproteinase 168 and the protease P D138 being described in (WO 93/18140).Other useful protease can be description Those of in WO 92/175177, WO 01/016285, WO02/026024 and WO 02/016547.Trypsin-like protease The example of enzyme is that trypsase (such as pig or Niu Laiyuan) and Fusarium (Fusarium) protease (are described in WO 89/ 06270, in WO 94/25583 and WO 05/040372), and derive from the pancreas curdled milk egg of cellulomonas cartae (Cellumonas) White enzyme (being described in WO 05/052161 and WO 05/052146).

In addition preferred protease is that the alkali protease from bacillus lentus DSM 5483 (is such as described in such as WO In 95/23221) and its variant (be described in WO 92/21760, WO 95/23221, EP 1921147 and EP 1921148 In).

The example of metalloproteinases is as being described in (international corporation, the Jie Neng section (Genencor of WO 07/044993 Int. the metalloprotease in)), as from bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Those.

In some embodiments, tunning according to the present invention includes protease, such as Savinase, BPN', withered grass bar Mycoproteinase Carlsberg, TY145,10R and these one of variant, these variants are 1,2,3,4,5,6,7,8,9 Or have in 10 amino acid positions and change, such as the substitution, insertion or missing of amino acid.

In some embodiments, tunning according to the present invention is and SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 sequence there is at least 60% sequence identity, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence is same The protease of property.

Tunning can be alpha-amylase or glucoamylase and can be bacterium or originated from fungus.Including albumen The mutant of matter engineering.Amylase includes for example from bacillus, such as bacillus licheniformis (Bacillus Licheniformis the alpha-amylase that specific bacterial strain (being described in greater detail in GB 1,296,839)) obtains.

Suitable amylase includes the amylase or itself and SEQ ID with the SEQ ID NO:2 in WO 95/10603 NO:3 has the variant of 90% sequence identity.Preferred variant is described in WO94/02597, WO 94/18314, WO 97/ In 43424 and in the SEQ ID NO:4 of WO 99/019467, there is the change replaced such as below one or more in position Body: 15,23,105,106,124,128,133,154,156,178,179,181,188,190,197,201,202,207,208, 209,211,243,264,304,305,391,408 and 444.

Different suitable amylase include have the SEQ ID NO:6 in WO 02/010355 amylase or its with SEQ ID NO:6 has the variant of 90% sequence identity.The preferred variants of SEQ ID NO:6 are that have in position 181 and 182 There is missing and there is those of substitution in position 193.

Other suitable amylase be include be shown in the SEQ ID NO:6 of WO 2006/066594 from solution starch The residue 1-33 of the alpha-amylase of bacillus and the bacillus licheniformis being shown in the SEQ ID NO:4 of WO 2006/066594 The hybrid alpha-amylases of the residue 36-483 of alpha-amylase or its variant with 90% sequence identity.This hybrid alpha-amylases Preferred variants be in one or more of following position have replace, missing or insertion those of: G48, T49, G107, H156, A181, N190, M197, I201, A209 and Q264.Comprising being shown in the SEQ ID NO:6 of WO 2006/066594 Derived from the hybrid alpha-amylases of the residue 36-483 of the residue 1-33 and SEQ ID NO:4 of the alpha-amylase of bacillus amyloliquefaciens Most preferably variant be have it is following replace those of:

M197T；

H156Y+A181T+N190F+A209V+Q264S；Or

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

In addition suitable amylase is the amylase or itself and SEQ with the SEQ ID NO:6 in WO 99/019467 ID NO:6 has the variant of 90% sequence identity.The preferred variants of SEQ ID NO:6 are one or more in following position Have in a and those of replace, lack or be inserted into: R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylase is that have those of missing in position R181 and G182 or position H183 and G184.

The other amylase that can be used is SEQ ID NO:1, SEQ ID NO:3, the SEQ with WO 96/023873 Those of ID NO:2 or SEQ ID NO:7 or itself and SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:7 has the variant of 90% sequence identity.SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:7 Preferred variants be have in one or more of following position replace, those of missing or insertion: 140,181,182, 183,184,195,206,212,243,260,269,304 and 476, the SEQ ID 2 using WO 96/023873 is for numbering. Preferred variant is to be selected from 181,182,183 and 184 two positions (such as 181 and 182,182 and 183 or position 183 With 184) in have missing those of.The most preferred amylase change of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:7 Body be in position 183 and 184 have missing and one in position 140,195,206,243,260,304 and 476 or There is those of substitution in multiple.

Other amylase that can be used are the SEQ having in SEQ ID NO:2, the WO01/66712 of WO 08/153815 The SEQ ID NO:2 of the amylase of ID NO:10 or itself and WO 08/153815 have 90% sequence identity or with WO 01/ SEQ ID NO:10 in 66712 has the variant of 90% sequence identity.SEQ ID NO:10's in WO 01/66712 is excellent Select variant be have in one or more of following position replace, those of missing or insertion: 176,177,178,179, 190,201,207,211 and 264.

In addition suitable amylase is the amylase or itself and SEQ ID with the SEQ ID NO:2 of WO 09/061380 NO:2 has the variant of 90% sequence identity.The preferred variants of SEQ ID NO:2 are in one or more of following position In have the end C- truncate and/or replace, missing or insertion those of: Q87, Q98, S125, N128, T131, T165, K178, R180、S181、T182、G183、M201、F202、N225、S243、N272、N282、Y305、R309、D319、Q320、Q359、 K444 and G475.The more preferable variant of SEQ ID NO:2 is that have to replace in one or more of following position: Q87E, R、Q98R、S125A、N128C、T131I、T165I、K178L、T182G、M201L、F202Y、N225E,R、N272E,R、S243Q, A, E, D, Y305R, R309A, Q320R, Q359E, K444E and G475K, and/or position R180 and/or S181 or T182 and/ Or there is those of missing in G183.The most preferred amylase variant of SEQ ID NO:2 is that have those of following substitution:

N128C+K178L+T182G+Y305R+G475K；

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

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

S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein these variants are the ends C- It is truncated, and optionally it is further contained in the substitution at position 243 and/or lacking at position 180 and/or position 181 It loses.

In addition suitable amylase is the amylase or itself and SEQ ID with the SEQ ID NO:1 of WO 13184577 NO:1 has the variant of 90% sequence identity.The preferred variants of SEQ ID NO:1 are in one or more of following position In have replace, missing or insertion those of: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476 and G477.The more preferable variant of SEQ ID NO:1 be one in following position or In multiple have replace: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K and G477K, and/or there is those of missing in position R178 and/or S179 or T180 and/or G181.SEQ ID The most preferred amylase variant of NO:1 is that have those of following substitution:

E187P+I203Y+G476K

E187P+I203Y+R458N+T459S+D460T+G476K

Wherein these variants are optionally further contained in substitution at position 241 and/or in position 178 and/or positions Missing at 179.

In addition suitable amylase is the amylase or itself and SEQ ID with the SEQ ID NO:1 of WO10104675 NO:1 has the variant of 90% sequence identity.The preferred variants of SEQ ID NO:1 are in one or more of following position In have replace, missing or insertion those of: N21, D97, V128, K177, R179, S180, I181, G182, M200, L204, E242, G477 and G478.The more preferable variant of SEQ ID NO:1 is that have to replace in one or more of following position: N21D, D97N, V128I, K177L, M200L, L204YF, E242QA, G477K and G478K, and/or in position R179 and/or There is those of missing in S180 or I181 and/or G182.The most preferred amylase variant of SEQ ID NO:1 be have it is following Those of replace:

N21D+D97N+V128I

Wherein these variants are optionally further contained in substitution at position 200 and/or in position 180 and/or positions Missing at 181.

Other suitable amylase be have the SEQ ID NO:12 in WO 01/66712 alpha-amylase or with SEQ ID NO:12 has the variant of at least 90% sequence identity.Preferred amylase variant is the SEQ ID in WO 01/66712 Have in one or more of following position in NO:12 and those of replace, lack or be inserted into: R28, R118, N174； R181,G182,D183,G184,G186,W189,N195,M202,Y298,N299,K302,S303,N306,R310,N314； R320,H324,E345,Y396,R400,W439,R444,N445,K446,Q449,R458,N471,N484.Particularly preferred shallow lake Powder enzyme includes having the missing of D183 and G184 and having the variant for replacing R118K, N195F, R320K and R458K, Yi Jiling In external one or more position selected from the group below have replace variant: M9, G149, G182, G186, M202, T257, In addition Y295, N299, M323, E345 and A339 most preferably have the variant replaced in all these positions.

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

In some embodiments, tunning according to the present invention is and SEQ ID NO:7, SEQ ID NO:8 or SEQ The sequence of ID NO:9 have at least 60% sequence identity, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, the amylase of at least 97%, at least 98% or at least 99% sequence identity.

Tunning can be obtained from microorganism.Microorganism can be naturally-produced tunning biology or it can To use recombinant DNA technology to generate, wherein making the gene for encoding required tunning and being suitable for expressing in expected host organism The appropriate control sequences (such as promoter, terminator) of the gene are operably connected, and are inserted into suitable host organism.

Microorganism can be prokaryotes or eucaryote.

Prokaryotic host cell can be any Gram-positive or gramnegative bacterium.Gram-positive bacterium include but It is not limited to: bacillus (Bacillus), fusobacterium (Clostridium), enterococcus spp (Enterococcus), native gemma Bacillus (Geobacillus), lactobacillus (Lactobacillus), lactococcus (Lactococcus), ocean gemma bar Pseudomonas (Oceanobacillus), staphylococcus (Staphylococcus), streptococcus (Streptococcus) and strepto- Pseudomonas (Streptomyces).Gramnegative bacterium includes but is not limited to campylobacter (Campylobacter), large intestine bar Bacterium (E.coli), Flavobacterium (Flavobacterium), Fusobacterium (Fusobacterium), Helicobacterium (Helicobacter), mud Bacillus (Ilyobacter), eisseria (Neisseria), Pseudomonas (Pseudomonas), Salmonella (Salmonella) and Ureaplasma (Ureaplasma).

Bacterial host cell can be any bacillus cell, including but not limited to: Alkaliphilic bacillus (Bacillus alkalophilus), highland bacillus (Bacillus altitudinis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), plant bacillus amyloliquefaciens (B.amyloliquefaciens Plantarum) subspecies, bacillus brevis (Bacillus brevis), Bacillus circulans (Bacillus circulans), Bacillus clausii (Bacillus clausii), bacillus coagulans (Bacillus coagulans), strong gemma bar Bacterium (Bacillus firmus), bacillus lautus (Bacillus lautus), bacillus lentus (Bacillus Lentus), bacillus licheniformis (Bacillus licheniformis), bacillus megaterium (Bacillus Megaterium), Methylotrophic bacillus (Bacillus methylotrophicus), bacillus pumilus (Bacillus pumilus), husky good fortune bacillus (Bacillus safensis), bacillus stearothermophilus (Bacillus Stearothermophilus), bacillus subtilis (Bacillus subtilis) and bacillus thuringiensis (Bacillus Thuringiensis) cell.

Bacterial host cell can also be any streptococcus cell, including but not limited to streptococcus equisimilis (Streptococcus equisimilis), streptococcus pyogenes (Streptococcus pyogenes), streptococcus uberis (Streptococcus uberis) and Malian drainage (Streptococcus equi Subsp.Zooepidemicus) cell.

Bacterial host cell can also be any Streptomyces cell, including but not limited to: not streptomyces chromogenes (Streptomyces achromogenes), deinsectization streptomycete (Streptomyces avermitilis), streptomyces coelicolor (Streptomyces coelicolor), streptomyces griseus (Streptomyces griseus) and shallow Streptomyces glaucoviolaceus (Streptomyces lividans) cell.

DNA, which is introduced into bacillus cell, to be accomplished by the following way: protoplast transformation is (referring to example Such as, Chang and Cohen, 1979, Mol.Gen.Genet. [molecular genetics and genomics] 168:111-115), competence Cell transformation is (see, for example, Young and Spizizen, 1961, J.Bacteriol. [Bacteriology] 81:823-829；Or Dubnau and Davidoff-Abelson, 1971, J.Mol.Biol. [J. Mol. BioL] 56:209-221), electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques [biotechnology] 6:742-751) or engagement (referring to For example, Koehler and Thorne, 1987, J.Bacteriol. [Bacteriology] 169:5271-5278).DNA is introduced big It can be accomplished by the following way in coli cell: protoplast transformation (see, for example, Hanahan, 1983, J.Mol.Biol. [J. Mol. BioL] 166:557-580) or electroporation (see, for example, Dower et al., 1988, Nucleic Acids Res. [nucleic acids research] 16:6127-6145).DNA is introduced into Streptomyces cell can be by following Mode is realized: protoplast transformation, electroporation be (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) [the linear microbiology of leaf (Prague)] 49:399-405), engagement (see, for example, Mazodier et al., 1989, J.Bacteriol. [Bacteriology] 171:3583-3585) or transduction (see, for example, Burke et al., 2001, Proc.Natl.Acad.Sci.USA [National Academy of Sciences proceeding] 98:6289-6294).It is thin that DNA is introduced into Pseudomonas It can be accomplished by the following way in born of the same parents: electroporation (see, for example, Choi et al., 2006, J.Microbiol.Methods [micro-biological process magazine] 64:391-397) or engagement (see, for example, Pinedo and Smets, 2005, Appl.Environ.Microbiol. [application and environmental microbiology] 71:51-57).DNA is introduced into streptococcus cell Can be accomplished by the following way: natural competence (natural competence) (see, for example, Perry and Kuramitsu, 1981, Infect.Immun. [infection and immune] 32:1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991, Microbios [microbiology] 68:189-207), electroporation is (see, for example, Buckley etc. People, 1999, Appl.Environ.Microbiol. [applying and environmental microbiology] 65:3800-3804) or engage (referring to For example, Clewell, 1981, Microbiol.Rev. [Microbi] 45:409-436).However, it is possible to use this field The known any method being introduced into DNA in host cell.

Host cell can also be eucaryote, such as mammal, insect, plant or fungal cell.

Host cell can be fungal cell." fungi " includes Ascomycota (Ascomycota), load as used herein Daughter bacteria door (Basidiomycota), chytridiomycota (Chytridiomycota) and Zygomycota (Zygomycota) and oomycota (Oomycota) and all mitosporic fungis (as defined in the following documents by Hawksworth et al.: Ainsworth and Bisby ' s Dictionary of The Fungi [Ainsworth and BisbyShi fungi dictionary], the 8th Version, 1995, International Applied Biosciences Center (CAB International), publishing house, univ cambridge uk (University Press,Cambridge,UK))。

Fungal host cells can be yeast cells." yeast " includes ascosporogenous yeast as used herein (ascosporogenous yeast) (Endomycetale (Endomycetales)), basidiosporogenous yeast (basidiosporogenous yeast) and belong to Fungi Imperfecti (Fungi Imperfecti) (gemma guiding principle (Blastomycetes)) yeast.Since the classification of yeast may change in future, for purposes of the present invention, yeast should As the Biology and Activities of Yeast biology of yeast [and activity] (Skinner, Passmore and Davenport is compiled, Soc.App.Bacteriol.Symposium Series No.9 [Applied Bacteriology Society's symposium Serial 9], defined as described in 1980).

Yeast host cell can be candida (Candida), Hansenula (Hansenula), Crewe not ferment Mother belongs to (Kluyveromyces), pichia (Pichia), Blastocystis (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces) or Ye Shi saccharomyces (Yarrowia) cell, such as Kluyveromyces lactis (Kluyveromyces lactis), saccharomyces carlsbergensis (Saccharomyces carlsbergensis), saccharomyces cerevisiae (Saccharomyces cerevisiae), saccharomyces diastaticus (Saccharomyces diastaticus), Doug Laplace yeast (Saccharomyces douglasii), Saccharomyces kluyveri (Saccharomyces kluyveri), promise ground yeast (Saccharomyces norbensis), ellipsoideus yeast (Saccharomyces oviformis) or Yarrowia lipolytica (Yarrowialipolytica) cell.

Fungal host cells can be filamentous fungal cells." filamentous fungi " includes Eumycota (Eumycota) and oomycota (Oomycota) all filamentous forms of subphylum (such as by Hawksworth et al., 1995 (seeing above) are defined).It is Filamentous Fungi, which is generally characterized by, to be made of chitin, cellulose, glucan, chitin, mannosan and other complicated polysaccharide Mycelia body wall.Nutrient growth is to be carried out by mycelia extension, and carbon catabolism is obligate aerobic.On the contrary, yeast is (such as Saccharomyces cerevisiae) nutrient growth be budding (budding) Lai Jinhang by unicellular thallus, and carbon catabolism can be It is fermentable.

Filamentous fungal host cell can be acremonium (Acremonium), aspergillus (Aspergillus), short stalk Mould category (Aureobasidium), intends cured Pseudomonas (Ceriporiopsis), Chrysosporium at the mould category (Bjerkandera) of smoke pipe (Chrysosporium), Coprinus (Coprinus), Coriolus Qu61 (Coriolus), Cryptococcus (Cryptococcus), line Ustilago (Filibasidium), Fusarium (Fusarium), Humicola (Humicola), Magnaporthe grisea category (Magnaporthe), mucor (Mucor), myceliophthora (Myceliophthora), new U.S. whip Pseudomonas (Neocallimastix), Neurospora (Neurospora), paecilomyces (Paecilomyces), Penicillium (Penicillium), flat lead fungi category (Phanerochaete), penetrate arteries and veins Pseudomonas (Phlebia), pears capsule whip Pseudomonas (Piromyces), Pleurotus (Pleurotus), Schizophyllum (Schizophyllum), Talaromyces (Talaromyces), Thermophilic ascomycete category (Thermoascus), Thielavia (Thielavia), Tolypocladium (Tolypocladium), Trametes (Trametes) or trichoderma (Trichoderma) cell.

For example, filamentous fungal host cell can be aspergillus awamori (Aspergillus awamori), smelly aspergillus (Aspergillus foetidus), aspergillus fumigatus (Aspergillus fumigatus), aspergillus japonicus (Aspergillus Japonicus), aspergillus nidulans (Aspergillus nidulans), aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), black thorn smoke pipe bacterium (Bjerkanderaadusta), dry plan wax bacterium (Ceriporiopsisaneirina), Ka Neiji intends wax bacterium (Ceriporiopsiscaregiea), pale yellow quasi- wax pore fungi (Ceriporiopsisgilvescens), Pernod wishes tower quasi- wax bacterium (Ceriporiopsispannocinta), annulus intends wax bacterium (Ceriporiopsisrivulosa), micro- red quasi- wax bacterium (Ceriporiopsissubrufa), worm intend wax bacterium (Ceriporiopsissubvermispora), straight hem gold pityrosporion ovale (Chrysosporiuminops), chrysosporium keratinophilum (Chrysosporiumkeratinophilum), Lu Kenuo train of thought gold pityrosporion ovale (Chrysosporiumlucknowense), excrement Shape gold pityrosporion ovale (Chrysosporiummerdarium), rent pityrosporion ovale (Chrysosporiumpannicola), queen Du Fragrant gold pityrosporion ovale (Chrysosporiumqueenslandicum), chrysosporium tropicum (Chrysosporiumtropicum), Brown thin golden pityrosporion ovale (Chrysosporiumzonatum), Coprinus cinereus (Coprinus cinereus), hairy fungus (Coriolushirsutus), bar spore shape fusarium (Fusarium bactridioides), cereal fusarium (Fusarium Cerealis), library prestige fusarium (Fusarium crookwellense), machete fusarium (Fusarium culmorum), cereal sickle Spore (Fusarium graminearum), red fusarium of standing grain (Fusarium graminum), different spore fusarium (Fusarium Heterosporum), albizzia fusarium (Fusarium negundi), fusarium oxysporum (Fusarium oxysporum), racemosus Fusarium (Fusarium reticulatum), pink fusarium (Fusarium roseum), elder fusarium (Fusarium Sambucinum), colour of skin fusarium (Fusarium sarcochroum), intend branch spore fusarium (Fusarium Sporotrichioides), sulphur color fusarium (Fusarium sulphureum), circle fusarium (Fusarium torulosum), quasi- Silk spore fusarium (Fusarium trichothecioides), empiecement fusarium (Fusarium venenatum), Humicola insolens (Humicolainsolens), Humicola lanuginosa (Humicolalanuginosa), rice black wool mould (Mucor miehei), thermophilic Ruin silk mould (Myceliophthorathermophila), Neuraspora crassa (Neurospora crassa), penicillium purpurogenum (Penicillium purpurogenum), Phanerochaete chrysosporium (Phanerochaetechrysosporium), arteries and veins bacterium is penetrated (Phlebia radiata), pleurotus eryngii (Pleurotuseryngii), autochthonal shuttle spore shell are mould (Thielaviaterrestris), long domain Trametes trogii (Trametesvillosa), Trametes versicolor (Trametes Versicolor), Trichoderma harzianum (Trichoderma harzianum), trichodermaharzianum (Trichoderma koningii), length Branch trichoderma (Trichoderma longibrachiatum), trichoderma reesei (Trichoderma reesei) or Trichoderma viride (Trichoderma viride) cell.

Fungal cell can be converted by following procedure, the process be related to protoplast formed, protoplast transformation and Regenerative cell's wall in a way known.Suitable procedure for converting aspergillus and pyr-trichoderma host cell is described in following In document: EP 238023, Yelton et al., 1984, Proc.Natl.Acad.Sci.USA [National Academy of Sciences proceedings] 81:1470-1474 and Christensen et al., 1988, Bio/Technology [biology/technology] 6:1419-1422.With In conversion Fusarium sp appropriate method by Malardier et al., 1989, Gene [gene] 78:147-156 and WO 96/ 00787 description.The program transformed yeast described by following documents: Becker and Guarente, Abelson, J.N. can be used And Simon, M.I. are edited, Guide to Yeast Genetics and Molecular Biology [yeast genetics with point Sub- biology guide], Methods in Enzymology [Enzymology method], volume 194, the 182-187 pages, new york academic goes out Ban She Co., Ltd (Academic Press, Inc., New York)；Ito et al., 1983, J.Bacteriol. [bacteriology is miscellaneous Will] 153:163；And Hinnen et al., 1978, Proc.Natl.Acad.Sci.USA [National Academy of Sciences proceedings] 75: 1920。

Pretreatment

Before separation method according to the present invention, one or more pre-treatment steps can be carried out to fermentation liquid, it is such as dilute It releases, adjust pH and/or temperature, addition can prevent the stabilizer of microorganism further growth, and addition can reduce protease activity Property and thus limit the degradation as caused by proteolytic activity inhibitor.

Pretreatment can also include cleavage step, for example, with chemical cleavage agents (such as one or more cell wall degrading enzymes (such as one or more lysozymes)) processing fermentation liquid.Preferred cleavage method includes addition lysozyme and/or hydrolase with molten Solution is as cell or the suspended material of remaining fermentation raw material.

First separating step

It according to the present invention, will be comprising at least partly being walked in the fermentation liquid of the required tunning of solid form in the first separation It is separated at least following two fraction: the first fraction in rapid, the liquid portion of the first order subpackage containing fermentation liquid, the liquid portion Comprising the required product of soluble form and other soluble matter, such as salt and remaining soluble nutrients, and it may include Cell and cell fragment；And second fraction, the second level subpackage contain the required product in solid form, cell, cell fragment With other insoluble substances.The first phase generated in the first separating step is alternatively referred to as primary concentrate or filtrate.Second phase Alternatively referred to as slurries or sludge phase.

First fraction and the second fraction may include liquid portion, cell and the cell fragment of fermentation liquid and be in solid shape The required product of formula, but ratio is entirely different.

At least the 60% of liquid portion of the first order subpackage containing fermentation liquid, for example, fermentation liquid at least 70%, for example, at least 80%, and second level subpackage contains rest part.

Second level subpackage contain in solid form required product at least 60%, preferably at least 65%, preferably at least 70%, Preferably at least 75%, preferably at least 80%, preferably at least 85% and most preferably at least 90%.

Cell and cell fragment distribute between the first phase and the second phase, and ratio depends in the first separating step making Particular separation technology.

The separating step that first separating step substantially completely or partially separates solid with liquid, therefore first point Any technology known in the art for separating solid from liquid phase can be used from step to carry out.First separating step can be with It is carried out using any combination of filtering, decantation or centrifugation or these technologies, and separates and can carry out in batches or continuously.

Depending on the selected isolation technics used in the first separating step, one or more chemistry are added before separation Product may be beneficial to help to separate.For example, as known in the art, can be added before the first separation process it is a kind of or A variety of flocculants.The type and amount of flocculant are selected based on the property of specific selected fermentation liquid, and will typically be used Simple routine experiment determines.This is known in the art, and the type and amount that select one or more flocculants are to promote Into separation completely within the scope of the technical ability of technical staff.For example, can be to be disclosed in WO 2004/001054 used according to the invention Method.

Dissolving step。

Method of the dissolving step for dissolving the required product in the second phase in any of dissolved solid product Middle progress typically relates to the chemicals dissolved with water or water-bearing media dilution, addition enhancing and/or adjusts pH.