CN101560509A - Alkaline proteinase - Google Patents

Abstract

The invention provide an alkaline protease, a gene for coding the same, a recombinant vector containing the gene, a transformant containing the recombinant vector, and an abluent composition containing the alkaline protease. The invention provides the alkaline protease having the activity, high specific activity and detergency even in the condition with the high fatty acid and capable of adding into the abluent.

Description

Sumizyme MP

The application's dividing an application that be the denomination of invention submitted November 22 calendar year 2001 for the application 01130397.2 of " Sumizyme MP "

Technical field

The present invention relates to stronger and as the Sumizyme MP that joins the enzyme that has good detergency in the washing composition than height alive and scale resistance.

Background technology

Proteolytic enzyme is usually used in various fields, for example comprises the various washing composition, makeup, bath shampoo, food improver of cloth-washing detergent and the medicine of digester and anti-inflammatory agent for example.Wherein, detergent use proteolytic enzyme is at industrial mass production and have very big market scale.Many proteolytic enzyme are now put on market.

In most of the cases, the dirt on the clothes not only contains protein, and contains for example various ingredients of lipid and solids.Therefore require washing composition to have sufficiently high detergency, so that remove the compound dirt of these reality.Find from this angle, Sumizyme MP even under the situation that high density lipid acid is arranged, can keep the casein hydrolysis activity, even and dirt is not only to be grouped into by protein group, but can present good washing when forming by the various ingredients of for example protein and lipid, and its molecular weight is about 43,000, the inventor has applied for a patent (WO99/18218) to it.

Yet, need aspect work, scale resistance and detergency, be better than above-mentioned Sumizyme MP and can be used for the Sumizyme MP of the washing composition of extensive composition.

Summary of the invention

The inventor has retrieved these mainly from the Sumizyme MP of enzyme variants.Yet above-mentioned Sumizyme MP is different fully with the serine protease that with the subtilisin is representative at the zymetology aspect of performance, so that the modification position of subtilisin does not provide useful information.The inventor finds after further research, for obtain better than work, resistance to oxidation is stable and detergency, the novel alkali proteinase that keeps the performance of above-mentioned Sumizyme MP simultaneously, they must have a specified amino acid residues in the predetermined position of its aminoacid sequence.

Therefore, one aspect of the present invention provides a kind of Sumizyme MP, wherein 256 of 104 of (a) 84, (b), (c) of SEQ ID NO:1 or (d) 369 or its corresponding position the amino-acid residue disappearance or be selected from:

In the position (a): arginine residues,

In the position (b): proline residue,

In the position (c): L-Ala, Serine, glutamine, Xie Ansuan, leucine, l-asparagine, L-glutamic acid or asparagicacid residue and

In the position (d): asparagicacid residue.

The present invention also provides a kind of Sumizyme MP on the other hand, wherein SEQ ID NO:1 (e) 66 or 264, (f) 57,101-106 everybody, 136,193 or 342, (g) 46 or 205, (h) 54,119,138,148 or 195, (i) 247, (j) 124, (k) 107 or (l) 257 or in the amino-acid residue disappearance of its corresponding position or be selected from:

In the position (e): glutamine, aspartic acid, Serine, L-glutamic acid, L-Ala, Threonine, leucine, methionine(Met), halfcystine, Xie Ansuan, glycine or Isoleucine residue,

In the position (f): Methionin, Serine, glutamine, phenylalanine, Xie Ansuan, arginine, tyrosine, leucine, Isoleucine, Threonine, methionine(Met), halfcystine, tryptophane, aspartic acid, L-glutamic acid, Histidine, proline(Pro) or alanine residue

In the position (g): tyrosine, tryptophane, L-Ala, l-asparagine, L-glutamic acid, Threonine, Xie Ansuan, leucine, Isoleucine, Histidine, Serine, Methionin, glutamine, methionine(Met) or cysteine residues,

In the position (h): tryptophane, phenylalanine, L-Ala, l-asparagine, L-glutamic acid, Threonine, Xie Ansuan, Histidine, Serine, Methionin, glutamine, methionine(Met), glycine, aspartic acid, proline(Pro), arginine or cysteine residues

In the position (i): tryptophane, phenylalanine, L-Ala, l-asparagine, L-glutamic acid, Threonine, Xie Ansuan, leucine, Isoleucine, Histidine, Serine, glutamine, methionine(Met) or cysteine residues,

In the position (j): L-Ala or lysine residue,

In the position (k): Methionin, arginine, L-Ala or serine residue and

In the position (l): Xie Ansuan or Isoleucine residue.

Another aspect of the invention also provide a kind of this Sumizyme MP of encoding gene, contain the recombinant vectors of this gene and contain the transformant of this carrier.

Further aspect of the present invention also provides a kind of detergent composition that contains this Sumizyme MP.

The accompanying drawing summary

Fig. 1 is the figure of the detergency of description basic protein enzyme variants; Fig. 2 is for describing the figure alive that compares of each basic protein enzyme variants; Be the relative residual active figure of description with Fig. 3 with each basic protein enzyme variants after the oxidizer treatment.

Implement best mode of the present invention

As mentioned above, in Sumizyme MP of the present invention, 256 of 104 of (a) 84, (b), (c) of SEQ ID NO:1 or (d) 369 or its corresponding position the amino-acid residue disappearance or be selected from: in the position (a): arginine residues, in the position (b): proline residue, in the position (c): L-Ala, Serine, glutamine, Xie Ansuan, leucine, l-asparagine, L-glutamic acid or asparagicacid residue and in the position (d): asparagicacid residue; Perhaps at (e) 66 or 264 of SEQ ID NO:1, (f) 57,101-106 is every, 136,193 or 342, (g) 46 or 205, (h) 54,119,138,148 or 195, (i) 247, (j) 124, (k) 107 or (l) 257 or in the amino-acid residue of its corresponding position disappearance or be selected from: in the position (e): glutamine, aspartic acid, Serine, L-glutamic acid, L-Ala, Threonine, leucine, methionine(Met), halfcystine, Xie Ansuan, glycine or Isoleucine residue, in the position (f): Methionin, Serine, glutamine, phenylalanine, Xie Ansuan, arginine, tyrosine, leucine, Isoleucine, Threonine, methionine(Met), halfcystine, tryptophane, aspartic acid, L-glutamic acid, Histidine, proline(Pro) or alanine residue, in the position (g): tyrosine, tryptophane, L-Ala, l-asparagine, L-glutamic acid, Threonine, Xie Ansuan, leucine, Isoleucine, Histidine, Serine, Methionin, glutamine, methionine(Met) or cysteine residues, in the position (h): tryptophane, phenylalanine, L-Ala, l-asparagine, L-glutamic acid, Threonine, Xie Ansuan, Histidine, Serine, Methionin, glutamine, methionine(Met), glycine, aspartic acid, proline(Pro), arginine or cysteine residues, in the position (i): tryptophane, phenylalanine, L-Ala, l-asparagine, L-glutamic acid, Threonine, Xie Ansuan, leucine, Isoleucine, Histidine, Serine, glutamine, methionine(Met) or cysteine residues, in the position (j): L-Ala or lysine residue, in the position (k): Methionin, arginine, L-Ala or serine residue and in the position (l): Xie Ansuan or Isoleucine residue.

Especially, the Sumizyme MP meaning of the present invention is the Sumizyme MP with aminoacid sequence of SEQ ID NO:1 representative, wherein lacked or be scheduled at the amino-acid residue that is selected from above-mentioned (a)-(d) and position (e)-(l), perhaps the meaning is another Sumizyme MP, and wherein the amino-acid residue in its corresponding position has lacked or be predetermined.They can be wild-type enzyme, wild-type variant or artificial variant.

" another Sumizyme MP " can be wild-type enzyme or wild-type variant.Preferably have scale resistance and by the molecular weight that SDS-PAGE measures be 43,000 ± 2,000 the sort of, wherein more preferably have aminoacid sequence the sort of that aminoacid sequence with SEQ ID NO:1 has at least 60% homology.Particularly preferably be have with the aminoacid sequence of SEQ ID NO:1 have at least 60% homology aminoacid sequence, have the sort of of scale resistance, work at alkaline range (pH 8 or bigger), stably has at least 80% residual activity pH 10 times even when handling 10 minutes for 50 ℃, suppressed by diisopropyl fluorophosphate (DFP) and phenylmethane sulfonic acid fluoride (PMSF), and the molecular weight of measuring by SDS-PAGE is 43,000 ± 2,000.The meaning that term used herein " has scale resistance " is to have at least 50% residual activity (synthesizing substrate tests) in 50mM superoxol (containing 5mM calcium chloride) when pH 10 (20mMBritton-Robinson damping fluid) and 20 ℃ are handled 20 minutes down when it.

The example of " Sumizyme MP with aminoacid sequence of SEQ ID NO:1 representative " comprises that KP43[is derived from Bacillus strain KSM-KP43 (FERM BP-6532), WO99/18218], and the example of " having the Sumizyme MP that the aminoacid sequence of at least 60% homology is arranged with the aminoacid sequence of SEQ ID NO:1 " comprises that the Proteinase K P9860[of the aminoacid sequence with SEQ ID NO:2 representative is derived from Bacillus strain KSM-KP9860 (FERM BP-6534), WO99/18218], proteolytic enzyme E-1[with aminoacid sequence of SEQ ID NO:3 representative is derived from Bacillus strain D-6 (FERM P-1592), JP740710], proteolytic enzyme Ya[with aminoacid sequence of SEQ ID NO:4 representative is derived from Bacillus strain Y (FERM BP-1029), JP861210), proteolytic enzyme SD521[with aminoacid sequence of SEQ ID NO:5 representative is derived from Bacillus strain SD-521 (FERM BP-11162), JP910821], protease A-1 with aminoacid sequence of SEQ IDNO:6 representative (is derived from NCIB12289, WO8801293) and the protease A-2 with aminoacid sequence of SEQ ID NO:7 representative (be derived from NCIB12513, WO8801293).Wherein, be preferably selected from the aminoacid sequence of SEQ ID NOS.2-7 or have at least 80% with it, more preferably at least 90%, the Sumizyme MP of at least 95% homology particularly.

The homology of aminoacid sequence is calculated (Science, 227,1435 (1985)) by the Lipman-Pearson method.

" amino-acid residue in the corresponding position " can be by using known algorithm, and for example Lipman-Pearson algorithm comparing amino acid sequence is identified.The position of " amino-acid residue of corresponding position " can be determined by the aminoacid sequence that contrasts this proteolytic enzyme by this way in the sequence of each proteolytic enzyme.Suppose that this corresponding position is present in the three-dimensional same position in the aminoacid sequence of SEQ ID NO:1, and the amino-acid residue that is present in this same position brings similar effect to the specific function of this proteolytic enzyme.

Specifically, (a) be lysine residue at 84 the amino-acid residue of SEQ ID NO:1.By using aforesaid method, the amino-acid residue in its corresponding position can be defined as 83 the lysine residue of SEQ ID NO:3.This amino-acid residue is preferably arginine.

(b) although 104 the amino-acid residue of SEQID NO:1 is a leucine residue, this amino-acid residue or its corresponding amino-acid residue are preferably proline residue.

(c) although 256 the amino-acid residue of SEQ ID NO:1 is a methionine residues, preferred especially this amino-acid residue is L-Ala, Serine, glutamine, Xie Ansuan, leucine, l-asparagine, L-glutamic acid or asparagicacid residue.

(d) although 369 the amino-acid residue of SEQ ID NO:1 is an asparagicacid residue, this amino-acid residue or its corresponding amino-acid residue are preferably asparagine residue.

(e) although 66 or 264 the amino-acid residue of SEQ ID NO:1 is an asparagine residue, but this amino-acid residue is preferably glutamine, aspartic acid, Serine, L-glutamic acid, L-Ala, Threonine, leucine, methionine(Met), halfcystine, Xie Ansuan, glycine or Isoleucine residue, and preferred especially aspartic acid, Serine or glutaminic acid residue.Preferred situation is to be asparagicacid residue and to be serine residue at 264 amino-acid residue at 66 amino-acid residue.

(f) although SEQ ID NO:1 57, the amino-acid residue of 101-106,136,193 and 342 position separately is glycine residue, this amino-acid residue is preferably Methionin, Serine, glutamine, phenylalanine, Xie Ansuan, arginine, tyrosine, leucine, Isoleucine, Threonine, methionine(Met), halfcystine, tryptophane, aspartic acid, L-glutamic acid, Histidine, proline(Pro) or alanine residue.Particularly preferred situation is that the amino-acid residue in 57,136,193 or 342 positions is an alanine residue, is arginine residues at 103 amino-acid residue perhaps.

(g) although be phenylalanine residue at 46 or 205 the amino-acid residue of SEQ ID NO:1, this amino-acid residue is preferably tyrosine, tryptophane, L-Ala, l-asparagine, L-glutamic acid, Threonine, Xie Ansuan, leucine, Isoleucine, Histidine, Serine, Methionin, glutamine, methionine(Met) or cysteine residues.Particularly preferred situation is to be leucine residue at 46 amino acid.

(h) although be tyrosine residues at 54,119,138,148 or 195 the amino-acid residue of SEQ ID NO:1, this amino-acid residue is preferably tryptophane, phenylalanine, L-Ala, l-asparagine, L-glutamic acid, Threonine, Xie Ansuan, Histidine, Serine, glutamine, methionine(Met), glycine, aspartic acid, proline(Pro), Methionin, arginine or cysteine residues.Particularly preferred situation is to be L-Ala, aspartic acid, L-glutamic acid, glutamine, Xie Ansuan, tryptophane, glycine, Methionin, Threonine, methionine(Met), halfcystine, phenylalanine, proline(Pro), Serine, arginine, l-asparagine or histidine residues at 195 amino-acid residue.

(i) although be lysine residue at 247 the amino-acid residue of SEQ ID NO:1, this amino-acid residue is preferably tryptophane, phenylalanine, L-Ala, l-asparagine, L-glutamic acid, Threonine, Xie Ansuan, leucine, Isoleucine, Histidine, Serine, glutamine, methionine(Met) or cysteine residues.As this amino-acid residue of 247, preferred especially arginine or threonine residues.

(j) although be arginine residues at 124 the amino-acid residue of SEQ ID NO:1, this amino-acid residue is preferably L-Ala or lysine residue.

(k) although be leucine residue at 107 the amino-acid residue of SEQ ID NO:1, this amino-acid residue is preferably Methionin, arginine, L-Ala or serine residue, and preferred especially lysine residue.

(l) although be alanine residue at 257 the amino-acid residue of SEQ ID NO:1, this amino-acid residue is preferably Xie Ansuan or Isoleucine residue, and is preferably the Xie Ansuan residue especially.

About preferred " another Sumizyme MP " in the top illustration, shown the specific examples (table 1-a, table 1-b) of (a)-(d) of aminoacid sequence (SEQ ID NO:1) and position (e)-(l) and amino-acid residue below corresponding to Proteinase K P43.

Table 1-a

Table 1-b

In Sumizyme MP of the present invention, can carry out simultaneously (a)-(d) or the disappearance or the selection of amino-acid residue (e)-(l) in two or more positions.

When Sumizyme MP of the present invention was a variant, " proteolytic enzyme with aminoacid sequence of SEQ ID NO:1 representative " or top " another Sumizyme MP " that exemplifies were used as the Sumizyme MP (it can being referred to as " parent Sumizyme MP ") before the sudden change.Introduce sudden change by desired location, can obtain Sumizyme MP of the present invention at this parent Sumizyme MP.For example, it can or substitute (a)-(d) and (e)-(l) the position or the corresponding position of another basic protein enzyme amino acid sequence of the aminoacid sequence of the SEQ ID NO:1 be selected from above-mentioned Proteinase K P43 by disappearance with another amino-acid residue, the amino-acid residue of the corresponding position of the aminoacid sequence of the SEQ IDNOS:2-7 representative of more specifically saying so obtains.

Sumizyme MP of the present invention for example can use the gained mutator gene to transform suitable host by introducing sudden change to the parent Sumizyme MP of clone gene coding, cultivates this recombinant host then and obtains.Can use conventional gene recombination technology, for example according to the encode clone of the gene that the parent Sumizyme MP uses of the method described in WO99/18218, JP901128 or the WO98/56927.

For the transgenation of the parent Sumizyme MP of encoding, can adopt now popular random mutation or location specific the sudden change in any one.More particularly, can use for example TakaraShuzo Co., Ltd. " Site-Directed Mutagenesis System Mutan-SuperExpress Kit " is by using as " PCR protocols " (Academic Press, NewYork, 1990) recombinant PCR described in (polymerase chain reaction) suddenlys change, and the required sequence of gene can use the sequence corresponding to another gene of required sequence to replace.

For by using the gained mutator gene to produce ease variants of the present invention, for example can use following method.By with this mutator gene with can be stably the dna vector of its amplification combined or stably increase by the DNA that this mutator gene is incorporated into the ease variants of the present invention of will encoding on the chromosomal DNA that can stably keep it, then this gene is introduced and to be stablized and express effectively among the host of this gene, produce this misfolded proteins enzyme thus.The host who satisfies above-mentioned condition comprises the microorganism that belongs to genus bacillus, intestinal bacteria, mould, yeast and actinomyces.

Thus obtained Sumizyme MP of the present invention has stable protease activity in alkaline zone, the casein hydrolysis activity is not suppressed by higher lipid acid, and the molecular weight of measuring by SDS-PAGE is 43,000 ± 2,000.For example, the ease variants that can obtain from the proteolytic enzyme as the aminoacid sequence with SEQID NO:1 of parent bacterial strain has following physical and chemical activity.

(i) the pH scope that works

It works in the wide pH scope of 4-13 and have at least 80% best pH activity value under pH 6-12.

(ii) stable p H scope

It can stablized 30 minutes during for 6-11 at pH under 40 ℃ the treatment condition.

The (iii) influence of lipid acid

Its casein hydrolysis activity is not suppressed by oleic acid.

These proteolytic enzyme of the present invention have good ratio work, scale resistance and detergency, can be used as the enzyme that adds in the various detergent composition thus.Particularly the amino-acid residue in the position of SEQ ID NO:1 (a)-(d) or its corresponding position has lacked or specified proteolytic enzyme has good washing.Wherein, the amino-acid residue in (c) 256 or its corresponding position is that those proteolytic enzyme of L-Ala, Serine, glutamine, Xie Ansuan, leucine, l-asparagine, L-glutamic acid or asparagicacid residue had not only had high ratio and live but also have strong scale resistance.Amino-acid residue in the position of SEQ ID NO:1 (e)-(l) or its corresponding position has lacked or specified proteolytic enzyme has good especially ratio work.

Above-mentioned proteolytic enzyme can enough present its active amount and join in the detergent composition of the present invention.Although can add 0.1-5000P.U. in every 1kg detergent composition, consider to add 1000P.U. or still less, preferred 500P.U. from economic aspect.

Various enzymes and Sumizyme MP of the present invention combination can be used for detergent composition of the present invention.Example comprises lytic enzyme, oxydase, reductase enzyme, transferring enzyme, lyase, isomerase, ligase enzyme and synthetic enzyme.Wherein proteolytic enzyme, cellulase, lipase, M-Zyme, esterase, at, amylase, Starch debranching enzyme, polygalacturonase, mannase, glucosaccharase, dextranase, rCO, peroxidase, laccase and the proteolytic enzyme except being used for this Sumizyme MP of the present invention are preferred.

Proteolytic enzyme comprises Alcalase, Esperase, Savinase and Everlase (product of the Novo Nordisk that respectively does for oneself), Properase and the Purafect (product of the Genencor International Inc. that respectively does for oneself) and the KAP (Kao Corp) of commercially available acquisition.Cellulase comprises Cellzyme and Carezyme (product of the Novo Nordisk that respectively does for oneself), KAC (KaoCorp.) and pass through the alkali cellulose enzyme that Bacillus strain KSM-S237 produces described in the flat 10-313859 of Japanese Patent Application Laid-Open.Amylase comprises Termamyl and Duramyl (product of the Novo Nordiek that respectively does for oneself), Purastar (Genencor International Inc.) and KAM (Kao Corp.).Lipase comprises Lipolase and Liporase Ultra (product of the Novo Nordisk that respectively does for oneself).The top enzyme that exemplifies can 0.001-10%, and the amount of preferred 0.03-5% adds.

Amount that can 0.5-60wt.% (this paper back will abbreviate " % " as) joins tensio-active agent in the detergent composition.Preferably in powdered detergent composition and liquid detergent composition, add 10-45% and 20-50%. respectively when detergent composition of the present invention is bleach detergent or automatic dishwasher washing composition, often can 1-10%, the preferably amount of 1-5% adding tensio-active agent.

Amount that can 0.01-50% adds the divalent-metal ion scavenging agent, is preferably 5-40%.

Can 0.01-80%, the preferably amount of 1-40% adding alkaline reagents and inorganic salt.

Can 0.001-10%, the amount of preferred 1-5% adds anti-soil thing deposition agent again.

Preferably the amount with 1-10% adds SYNTHETIC OPTICAL WHITNER (for example hydrogen peroxide or percarbonate), when using SYNTHETIC OPTICAL WHITNER, can add the bleach-activating agent of 0.01-10%.

As white dyes, can use biphenyl type (as " Tinopal CBS-X ") and Stilbene type (as the DM fluorescence dye).Preferred add-on is 0.001-2%.

Can prepare detergent composition of the present invention by Sumizyme MP and the above-mentioned known detergent component that uses aforesaid method to obtain in the usual way.Can select the washing composition form according to application target.Example comprises liquid, powder and particle.

When joining Sumizyme MP of the present invention in the powdered detergent composition; preferred preparing washing agent particle in advance; according to the method described in the clear 62-25790 of Japanese Patent Application Laid-Open the basic protein enzyme granulate is sneaked into wherein then, in order to avoid workman and end user are by preparation or use this washing composition and contact or prevent this enzyme thermal induction inactivation or decomposition with this enzyme.

Thus obtained detergent composition of the present invention can be used as detergent for washing clothes, bleach detergent, automatic dishwasher washing composition, pipeline cleaning agent and artificial teeth sanitising agent.Be preferably used as detergent for washing clothes, bleach detergent or automatic dishwasher washing composition especially.

Embodiment 1

Randomly sudden change is incorporated in the following manner in the proteolytic enzyme structure gene of the about 2.0kb that contains terminator codon.At first, can the increase primer of this 2.0kb of use carries out PCR.A kind of PCR master's mixture contains 5ng template DNA, 20pmoL phosphorylated primer, the various dNTP of 20nmoL, 1 μ moLTris/HCl (pH 8.3), 5imoL KCl, 0.15 μ moL MgCl ₂With 2.5U TaqDNA polysaccharase, and its total amount is adjusted to 100 μ L.By under 94 ℃, leave standstill this template changed in 5 minutes after, carry out round-robin PCR 30 times, each circulation by 94 ℃ handle down 1 minute, handled 1 minute down and handle down at 55 ℃ and formed in 1.5 minutes at 72 ℃.With this PCR purification of products, follow wash-out in 100 μ L sterilized waters by " PCR purification of products test kit " (product of Boeringer Manheim).Except carrying out the PCR second time with 1 this elutriant of μ L under the external condition similar of template DNA to PCR for the first time.After the second time, PCR finished, with to the similar mode of the PCR first time with this PCR purification of products, then wash-out in 100 μ L sterilized waters.

Use Takara Shuzo Co., the dna fragmentation that " LATaq " that Ltd. produces will increase by polymeric enzyme reaction is incorporated in the carrier.Specifically, in the elutriant that should purify to 35 μ L, add 5 μ L LATaq and afterwards, its total amount is adjusted to 50 μ L with a kind of dNTP solution of damping fluid (10 times of concentration), 8 μ L and 0.5 μ L LATaq archaeal dna polymerase and as the 20ng plasmid pHA64TS (having and expression vector pHA64 bonded proteolytic enzyme structure gene) of template.30 circulations are carried out in the PCR of gained liquid reaction, each circulation by 94 ℃ handle down 1 minute, handled 1 minute down and handle down at 55 ℃ and formed in 4 minutes at 72 ℃.By the ethanol sedimentation of following, collect this PCR product.This PCR product has the plasmid shape that has a kerf at 5 ' primer end of primer.Ligase enzyme reaction by T4 ligase enzyme (Takara Shuzo Co., the product of Ltd.) connects this notch portion.

By using this ligase enzyme reaction mixture of 10 μ L, carry out the conversion of Bacillus subtilus bacterial strain ISW1214, obtain about 4 * 10 thus ⁵Transformant.The transformant of this bacterial strain ISW1214 of gained is gone up the formation of cultivating and observing salt containing the substratum of skimming milk (containing 1% skimming milk, 1% bacto-tryptone, 1% sodium-chlor, 0.5% yeast extract, 1.5% agar and 7.5 μ g/ml tsiklomitsins), and the formation supposition of this salt has reflected the secretory volume of proteolytic enzyme.

Embodiment 2: the purifying of enzyme

Prepare the protease activity fraction in such a way.Under 30 ℃, the transformant that obtains among the embodiment 1 is gone up and cultivated 60 hours in culture medium A (3% poly-peptone S (product of Nippon Pharmaceutical), 0.5% yeast extract, 1% flesh of fish medicinal extract (Wako Pure Chemical Industries, the product of Ltd.), 0.15% dipotassium hydrogen phosphate, 0.02% magnesium sulfate 7 hydrate, 4% maltose and 7.5 μ g/mL tsiklomitsins).In the supernatant liquor of thus obtained substratum, add ammonium sulfate, obtain 90% saturatedly, protein is saltoutd.The sample that will obtain by saltouing be dissolved in the tris HCl damping fluid (pH 7.5) that 10mM contains 2mM calcium chloride.Use the relative same buffer of dialysis membrane that gained solution is dialysed a night.Part in the dialysis membrane is applied to 10mM contains on the DEAE Bio-Gel A (the breadboard product of Bio-Rad) that tris HCl damping fluid (pH 7.5) balance of 2mM calcium chloride crosses, thereby collect the protease activity part that is not adsorbed onto on this ion-exchanger.Further this active part is applied on " SP-Toyopearl 550W " (product of Tosoh Corp.) of crossing with the same buffer balance, then, obtains the protease activity part thus with 0-50mM sodium chloride solution wash-out.By SDS-PAGE electrophoretic analysis gained part, thereby this proteolytic enzyme that confirm to obtain is uniform protein basically.Use bovine serum albumin (the breadboard product of Bio-Rad) to measure protein concn according to people's such as Lowry method (J.Biol.Chem.193,265-275 (1981)) as standard.

Embodiment 3: the measuring method of protease activity

(1) synthetic substrate test

Use is measured decomposition rate by the synthetic peptide that Glt-Ala-Ala-Pro-Leu (A-A-P-L) makes as substrate.Specifically, borate/KCl the damping fluid (PH 10.5) and 3mM Glt-A-A-P-L-pNA (the Peptide Institute that 50mM are contained every kind of enzyme to be evaluated, the product of Inc) kept 10 minutes down at 30 ℃, then the absorbancy under the results of regular determination 420nm.Definite peptidolytic activity that gathers way by the absorbancy under the 420nm of per unit hour.Use breadboard this activity of proteins of protein determination kit measurement of Bio-Rad.

(2) natural substrate test

1.0mL is contained the caseic 50mM borate buffer solution of 1% (w/v) (pH 10) after 30 ℃ keep 5 minutes down, and the enzyme solution of adding 0.1mL also reacted 15 minutes.The reaction terminating liquid (0.11M Tricholroacetic Acid-0.22M sodium-acetate-0.33M acetic acid) that in this reaction mixture, adds 2.0mL.At room temperature the gained mixture was left standstill 30 minutes, filter then.By the acid-soluble protein matter in the quantitatively definite filtrate of the method for people such as Lowery modification.Specifically, in this filtrate, add 2.5mL alkaline copper solution [1% sodium-potassium tartrate: 1% copper sulfate: 1% yellow soda ash=1: 1: 100) afterwards, gained solution was at room temperature left standstill 10 minutes.Then, the rare phenol solution (with the phenol reagent (product of Kanto Kagaku) of 2 times of ion exchanged water dilutions) that adds 0.25mL.The gained mixture is measured the absorbancy under the 660nm after 30 ℃ keep 30 minutes down.1 unit of enzyme is appointed as in above-mentioned reaction the amount that in 1 minute, discharges corresponding to the enzyme of the acid-soluble protein matter hydrolysate of 1mmol tyrosine.

Embodiment 4

(1) preparation of granulated detergent

Detergency to the washing composition described in the embodiment 3 of WO99/29830 is estimated.Specifically, pour 465kg water into be equipped with agitating vane 1m ³In the mixing tank.Reach after 55 ℃ in its water temperature, add 50% (w/v) sodium dodecyl benzene sulfonate aqueous solution of 48kg and 40% (w/v) polyacrylic acid sodium water solution of 135kg.After stirring 15 minutes, add 120kg yellow soda ash, 60kg sodium sulfate, 9kg S-WAT and 3kg fluorescence dye.After stirring once more 15 minutes, add the 300kg zeolite.This mixture was stirred 30 minutes, thereby obtain a homogeneous slurry (moisture content of these slurries is 50wt.%).By from being installed near the pressure nozzle spraying drying cat head this slurries of spraying, obtain a basic granules (high-temperature gas delivered at the bottom of the tower spray-drying tower under 225 ℃ and under 105 ℃ from tower top discharge).

Then, the nonionogenic tenside of 15 weight parts, the 50wt.% sodium dodecyl benzene sulfonate aqueous solution of 15 weight parts and the polyoxyethylene glycol of 1 weight part are mixed being heated under 70 ℃, obtain a mixture thus.In Loedige mixing tank (volume: 20L is equipped with a chuck for Matsuzaka Giken Co., the product of Ltd.), pour the basic granules that 100 parts of weight obtain from above into, stir and start knife mill (4000rpm) by main shaft (150rpm).75 ℃ warm water is flowed with 10L/min in chuck, in 3 minutes, this mixture is poured into wherein, stirred then 5 minutes.With the crystal aluminosilicate of 10 parts of weight surface coverage, obtain the granulated detergent the finished product thus with detergent particles.

[raw materials used]

Sodium dodecyl benzene sulfonate aqueous solution: " Neopelex F65 " (product of Kao Corp.)

Nonionogenic tenside: " Emulgen 108KM " (product of Kao), the average ethylene oxide that 8.5mol is arranged that adds

The polyacrylic acid sodium water solution: molecular-weight average is 10000 (according to the preparations of the method described in the embodiment of the flat 2-24283 of Japanese patent application publication No.)

Yellow soda ash: heavy ash (Central Glass Co., the product of Ltd.)

Zeolite: " Zeolite 4A ", median size is 3.5im (product of Tosoh Corp)

Polyoxyethylene glycol: " K-PEG6000 " (molecular-weight average 8500, product of Kao Corp.)

Fluorescence dye: " Tinopal CBS-X " (product of Ciba Geigy)

(2) preparation of granulating proteolytic enzyme

By the purification goods of Sumizyme MP of the present invention and parent Sumizyme MP, serve as basis preparation granulating proteolytic enzyme (6P.U./g) with the method described in the Japanese Patent Application Laid-Open number clear 62-257990.

(3) mensuration of detergency

1L be adjusted to 20 ℃ the calcium aqueous solution (in the 71.2mg lime carbonate/1L), each detergent composition shown in the dissolving 0.67g table 2.Use gained solution, use Terg-O-tometer (product of Ueshima Seisakusho) under 20 ℃ and 100rpm, will cut into test cloth (" EMPA117 " (by Swiss Federal Laboratories for MaterialsTesting and Research preparation, blood/breast/carbon) washing 10 minutes of 6 * 6cm sheet.After rinsing and the drying, use its brightness of spectrophotometric determination (" CM3500d ", the product of MINOLTA).With following equation is the basic calculation detergency.The results are shown in table 2.

The measurement result of the detergency of the ease variants that obtains among the embodiment 1 is shown in Fig. 1.Each all demonstrates basic protein enzyme variants of the present invention than the high detergency of wild-type enzyme of not introducing sudden change.

Table 2

Embodiment 5

The result of the protease activity of the ease variants that obtains among the embodiment 1 according to synthetic substrate test or natural substrate test determination (be determined at 195 and the 256 amino acids residues proteolytic enzyme through modification respectively by back one test, and by the other proteolytic enzyme of last test determination) is shown in Fig. 2.Basic protein enzyme variants of the present invention presents high ratio and lives.

Embodiment 6

Contain in the 100mM borate buffer solution (pH 10.5) of 3% aqueous hydrogen peroxide solution at 2mL, add every kind of ease variants by purifying and obtaining among 50 μ L part embodiment 1.The gained mixture was left standstill under 30 ℃ 30 minutes.Add an amount of catalase (product of BoehringerManheim) and remove after the excess hydrogen peroxide, by synthetic substrate test determination residual protein enzymic activity.In Fig. 3, shown that with respect to activity before handling be residual activity after handling with aqueous hydrogen peroxide solution at 100% o'clock.

Basic protein enzyme variants of the present invention presents high scale resistance than parent Sumizyme MP.

Industrial applicibility

The present invention can provide in addition under high concentration aliphatic acid, have activity, have high specific activity, Oxidative resistance and washing performance, and can be used as the alkali protease that joins the enzyme in the washing agent.

Sequence table

<110>KAO?CORPORATION

＜120〉Sumizyme MP

<130>

<150>JP?P2000-355166

<151>2000-11-22

<150>JP?P2001-114048

<151>2001-4-12

<160>7

<170>PatentIn?Ver.2.1

<210>1

<211>434

<212>PRT

＜213〉genus bacillus (Bacillus sp.)

<400>1

Asn?Asp?Val?Ala?Arg?Gly?Ile?Val?Lys?Ala?Asp?Val?Ala?Gln?Ser?Ser

1 5 10 15

Tyr?Gly?Leu?Tyr?Gly?Gln?Gly?Gln?Ile?Val?Ala?Val?Ala?Asp?Thr?Gly

20 25 30

Leu?Asp?Thr?Gly?Arg?Asn?Asp?Ser?Ser?Met?His?Glu?Ala?Phe?Arg?Gly

35 40 45

Lys?Ile?Thr?Ala?Leu?Tyr?Ala?Leu?Gly?Arg?Thr?Asn?Asn?Ala?Asn?Asp

50 55 60

Thr?Asn?Gly?His?Gly?Thr?His?Val?Ala?Gly?Ser?Val?Leu?Gly?Asn?Gly

65 70 75 80

Ser?Thr?Asn?Lys?Gly?Met?Ala?Pro?Gln?Ala?Asn?Leu?Val?Phe?Gln?Ser

85 90 95

Ile?Met?Asp?Ser?Gly?Gly?Gly?Leu?Gly?Gly?Leu?Pro?Ser?Asn?Leu?Gln

100 105 110

Thr?Leu?Phe?Ser?Gln?Ala?Tyr?Ser?Ala?Gly?Ala?Arg?Ile?His?Thr?Asn

115 120 125

Ser?Trp?Gly?Ala?Ala?Val?Asn?Gly?Ala?Tyr?Thr?Thr?Asp?Ser?Arg?Asn

130 135 140

Val?Asp?Asp?Tyr?Val?Arg?Lys?Asn?Asp?Met?Thr?Ile?Leu?Phe?Ala?Ala

145 150 155 160

Gly?Asn?Glu?Gly?Pro?Asn?Gly?Gly?Thr?Ile?Ser?Ala?Pro?Gly?Thr?Ala

165 170 175

Lys?Asn?Ala?Ile?Thr?Val?Gly?Ala?Thr?Glu?Asn?Leu?Arg?Pro?Ser?Phe

180 185 190

Gly?Ser?Tyr?Ala?Asp?Asn?Ile?Asn?His?Val?Ala?Gln?Phe?Ser?Ser?Arg

195 200 205

Gly?Pro?Thr?Lys?Asp?Gly?Arg?Ile?Lys?Pro?Asp?Val?Met?Ala?Pro?Gly

210 215 220

Thr?Phe?Ile?Leu?Ser?Ala?Arg?Ser?Ser?Leu?Ala?Pro?Asp?Ser?Ser?Phe

225 230 235 240

Trp?Ala?Asn?His?Asp?Ser?Lys?Tyr?Ala?Tyr?Met?Gly?Gly?Thr?Ser?Met

245 250 255

Ala?Thr?Pro?Ile?Val?Ala?Gly?Asn?Val?Ala?Gln?Leu?Arg?Glu?His?Phe

260 265 270

Val?Lys?Asn?Arg?Gly?Ile?Thr?Pro?Lys?Pro?Ser?Leu?Leu?Lys?Ala?Ala

275 280 285

Leu?Ile?Ala?Gly?Ala?Ala?Asp?Ile?Gly?Leu?Gly?Tyr?Pro?Asn?Gly?Asn

290 295 300

Gln?Gly?Trp?Gly?Arg?Val?Thr?Leu?Asp?Lys?Ser?Leu?Asn?Val?Ala?Tyr

305 310 315 320

Val?Asn?Glu?Ser?Ser?Ser?Leu?Ser?Thr?Ser?Gln?Lys?Ala?Thr?Tyr?Ser

325 330 335

Phe?Thr?Ala?Thr?Ala?Gly?Lys?Pro?Leu?Lys?Ile?Ser?Leu?Val?Trp?Ser

340 345 350

Asp?Ala?Pro?Ala?Ser?Thr?Thr?Ala?Ser?Val?Thr?Leu?Val?Asn?Asp?Leu

355 360 365

Asp?Leu?Val?Ile?Thr?Ala?Pro?Asn?Gly?Thr?Gln?Tyr?Val?Gly?Asn?Asp

370 375 380

Phe?Thr?Ser?Pro?Tyr?Asn?Asp?Asn?Trp?Asp?Gly?Arg?Asn?Asn?Val?Glu

385 390 395 400

Asn?Val?Phe?Ile?Asn?Ala?Pro?Gln?Ser?Gly?Thr?Tyr?Thr?Ile?Glu?Val

405 410 415

Gln?Ala?Tyr?Asn?Val?Pro?Val?Gly?Pro?Gln?Thr?Phe?Ser?Leu?Ala?Ile

420 425 430

Val?Asn

<210>2

<211>434

<212>PRT

＜213〉genus bacillus (Bacillus sp.)

<400>2

Asn?Asp?Val?Ala?Arg?Gly?Ile?Val?Lys?Ala?Asp?Val?Ala?Gln?Ser?Ser

1 5 10 15

Tyr?Gly?Leu?Tyr?Gly?Gln?Gly?Gln?Ile?Val?Ala?Val?Ala?Asp?Thr?Gly

20 25 30

Leu?Asp?Thr?Gly?Arg?Asn?Asp?Ser?Ser?Met?His?Glu?Ala?Phe?Arg?Gly

35 40 45

Lys?Ile?Thr?Ala?Leu?Tyr?Ala?Leu?Gly?Arg?Thr?Asn?Asn?Ala?Asn?Asp

50 55 60

Thr?Asn?Gly?His?Gly?Thr?His?Val?Ala?Gly?Ser?Val?Leu?Gly?Asn?Gly

65 70 75 80

Ala?Thr?Asn?Lys?Gly?Met?Ala?Pro?Gln?Ala?Asn?Leu?Val?Phe?Gln?Ser

85 90 95

Ile?Met?Asp?Ser?Ser?Gly?Gly?Leu?Gly?Gly?Leu?Pro?Ser?Asn?Leu?Gln

100 105 110

Thr?Leu?Phe?Ser?Gln?Ala?Phe?Ser?Ala?Gly?Ala?Arg?Ile?His?Thr?Asn

115 120 125

Ser?Trp?Gly?Ala?Ala?Val?Asn?Gly?Ala?Tyr?Thr?Thr?Asp?Ser?Arg?Asn

130 135 140

Val?Asp?Asp?Tyr?Val?Arg?Lys?Asn?Asp?Met?Thr?Ile?Leu?Phe?Ala?Ala

145 150 155 160

Gly?Asn?Glu?Gly?Pro?Asn?Gly?Gly?Thr?Ile?Ser?Ala?Pro?Gly?Thr?Ala

165 170 175

Lys?Asn?Ala?Ile?Thr?Val?Gly?Ala?Thr?Glu?Asn?Leu?Arg?Pro?Ser?Phe

180 185 190

Gly?Ser?Tyr?Ala?Asp?Asn?Ile?Asn?His?Val?Ala?Gln?Phe?Ser?Ser?Arg

195 200 205

Gly?Pro?Thr?Lys?Asp?Gly?Arg?Ile?Lys?Pro?Asp?Val?Met?Ala?Pro?Gly

210 215 220

Thr?Tyr?Ile?Leu?Ser?Ala?Arg?Ser?Ser?Leu?Ala?Pro?Asp?Ser?Ser?Phe

225 230 235 240

Trp?Ala?Asn?His?Asp?Ser?Lys?Tyr?Ala?Tyr?Met?Gly?Gly?Thr?Ser?Met

245 250 255

Ala?Thr?Pro?Ile?Val?Ala?Gly?Asn?Val?Ala?Gln?Leu?Arg?Glu?His?Phe

260 265 270

Val?Lys?Asn?Arg?Gly?Ile?Thr?Pro?Lys?Pro?Ser?Leu?Leu?Lys?Ala?Ala

275 280 285

Leu?Ile?Ala?Gly?Ala?Ala?Asp?Val?Gly?Leu?Gly?Tyr?Pro?Asn?Gly?Asn

290 295 300

Gln?Gly?Trp?Gly?Arg?Val?Thr?Leu?Asp?Lys?Ser?Leu?Asn?Val?Ala?Tyr

305 310 315 320

Val?Asn?Glu?Ser?Ser?Ala?Leu?Ser?Thr?Ser?Gln?Lys?Ala?Thr?Tyr?Thr

325 330 335

Phe?Thr?Ala?Thr?Ala?Gly?Lys?Pro?Leu?Lys?Ile?Ser?Leu?Val?Trp?Ser

340 345 350

Asp?Ala?Pro?Ala?Ser?Thr?Thr?Ala?Ser?Val?Thr?Leu?Val?Asn?Asp?Leu

355 360 365

Asp?Leu?Val?Ile?Thr?Ala?Pro?Asn?Gly?Thr?Arg?Tyr?Val?Gly?Asn?Asp

370 375 380

Phe?Ser?Ala?Pro?Phe?Asp?Asn?Asn?Trp?Asp?Gly?Arg?Asn?Asn?Val?Glu

385 390 395 400

Asn?Val?Phe?Ile?Asn?Ser?Pro?Gln?Ser?Gly?Thr?Tyr?Thr?Ile?Glu?Val

405 410 415

Gln?Ala?Tyr?Asn?Val?Pro?Val?Gly?Pro?Gln?Asn?Phe?Ser?Leu?Ala?Ile

420 425 430

Val?Asn

<210>3

<211>433

<212>PRT

＜213〉genus bacillus (Bacillus sp.)

<400>3

Asn?Asp?Val?Ala?Arg?Gly?Ile?Val?Lys?Ala?Asp?Val?Ala?Gln?Asn?Asn

1 5 10 15

Tyr?Gly?Leu?Tyr?Gly?Gln?Gly?Gln?Val?Val?Ala?Val?Ala?Asp?Thr?Gly

20 25 30

Leu?Asp?Thr?Gly?Arg?Asn?Asp?Ser?Ser?Met?His?Glu?Ala?Phe?Arg?Gly

35 40 45

Lys?Ile?Thr?Ala?Leu?Tyr?Ala?Leu?Gly?Arg?Thr?Asn?Asn?Ala?Asn?Asp

50 55 60

Pro?Asn?Gly?His?Gly?Thr?His?Val?Ala?Gly?Ser?Val?Leu?Gly?Asn?Ala

65 70 75 80

Leu?Asn?Lys?Gly?Met?Ala?Pro?Gln?Ala?Asn?Leu?Val?Phe?Gln?Ser?Ile

85 90 95

Met?Asp?Ser?Ser?Gly?Gly?Leu?Gly?Gly?Leu?Pro?Ser?Asn?Leu?Asn?Thr

100 105 110

Leu?Phe?Ser?Gln?Ala?Trp?Asn?Ala?Gly?Ala?Arg?Ile?His?Thr?Asn?Ser

115 120 125

Trp?Gly?Ala?Pro?Val?Asn?Gly?Ala?Tyr?Thr?Ala?Asn?Ser?Arg?Gln?Val

130 135 140

Asp?Glu?Tyr?Val?Arg?Asn?Asn?Asp?Met?Thr?Val?Leu?Phe?Ala?Ala?Gly

145 150 155 160

Asn?Glu?Gly?Pro?Asn?Ser?Gly?Thr?Ile?Ser?Ala?Pro?Gly?Thr?Ala?Lys

165 170 175

Asn?Ala?Ile?Thr?Val?Gly?Ala?Thr?Glu?Asn?Tyr?Arg?Pro?Ser?Phe?Gly

180 185 190

Ser?Ile?Ala?Asp?Asn?Pro?Asn?His?Ile?Ala?Gln?Phe?Ser?Ser?Arg?Gly

195 200 205

Ala?Thr?Arg?Asp?Gly?Arg?Ile?Lys?Pro?Asp?ValThr?Ala?Pro?Gly?Thr

210 215 220

Phe?Ile?Leu?Ser?Ala?Arg?Ser?Ser?Leu?Ala?Pro?Asp?Ser?Ser?Phe?Trp

225 230 235 240

Ala?Asn?Tyr?Asn?Ser?Lys?Tyr?Ala?Tyr?Met?Gly?Gly?Thr?Ser?Met?Ala

245 250 255

Thr?Pro?Ile?Val?Ala?Gly?Asn?Val?Ala?Gln?Leu?Arg?Glu?His?Phe?Ile

260 265 270

Lys?Asn?Arg?Gly?Ile?Thr?Pro?Lys?Pro?Ser?Leu?Ile?Lys?Ala?Ala?Leu

275 280 285

Ile?Ala?Gly?Ala?Thr?Asp?Val?Gly?Leu?Gly?Tyr?Pro?Ser?Gly?Asp?Gln

290 295 300

Gly?Trp?Gly?Arg?Val?Thr?Leu?Asp?Lys?Ser?Leu?Asn?Val?Ala?Tyr?Val

305 310 315 320

Asn?Glu?Ala?Thr?Ala?Leu?Thr?Thr?Gly?Gln?Lys?Ala?Thr?Tyr?Ser?Phe

325 330 335

Gln?Thr?Gln?Ala?Gly?Lys?Pro?Leu?Lys?Ile?Ser?Leu?Val?Trp?Thr?Asp

340 345 350

Ala?Pro?Gly?Ser?Thr?Thr?Ala?Ser?Tyr?Thr?Leu?Val?Asn?Asp?Leu?Asp

355 360 365

Leu?Val?Ile?Thr?Ala?Pro?Asn?Gly?Gln?Lys?Tyr?Val?Gly?Asn?Asp?Phe

370 375 380

Ser?Tyr?Pro?Tyr?Asp?Asn?Asn?Trp?Asp?Gly?Arg?Asn?Asn?Val?Glu?Asn

385 390 395 400

Val?Phe?Ile?Asn?Ala?Pro?Gln?Ser?Gly?Thr?Tyr?Thr?Ile?Glu?Val?Gln

405 410 415

Ala?Tyr?Asn?Val?Pro?Ser?Gly?Pro?Gln?Arg?Phe?Ser?Leu?Ala?Ile?Val

420 425 430

His

<210>4

<211>433

<212>PRT

＜213〉genus bacillus (Bacillus sp.)

<400>4

Asn?Asp?Val?Ala?Arg?Gly?Ile?Val?Lys?Ala?Asp?Val?Ala?Gln?Asn?Asn

1 5 10 15

Tyr?Gly?Leu?Tyr?Gly?Gln?Gly?Gln?Val?Val?Ala?Val?Ala?Asp?Thr?Gly

20 25 30

Leu?Asp?Thr?Gly?Arg?Asn?Asp?Ser?Ser?Met?His?Glu?Ala?Phe?Arg?Gly

35 40 45

Lys?Ile?Thr?Ala?Leu?Tyr?Ala?Leu?Gly?Arg?Thr?Asn?Asn?Ala?Ser?Asp

50 55 60

Pro?Asn?Gly?His?Gly?Thr?His?Val?Ala?Gly?Ser?Val?Leu?Gly?Asn?Ala

65 70 75 80

Leu?Asn?Lys?Gly?Met?Ala?Pro?Gln?Ala?Asn?Leu?Val?Phe?Gln?Ser?Ile

85 90 95

Met?Asp?Ser?Ser?Gly?Gly?Leu?Gly?Gly?Leu?Pro?Ser?Asn?Leu?Asn?Thr

100 105 110

Leu?Phe?Ser?Gln?Ala?Trp?Asn?Ala?Gly?Ala?Arg?Ile?His?Thr?Asn?Ser

115 120 125

Trp?Gly?Ala?Pro?Val?Asn?Gly?Ala?Tyr?Thr?Ala?Asn?Ser?Arg?Gln?Val

130 135 140

Asp?Glu?Tyr?Val?Arg?Asn?Asn?Asp?Met?Thr?Val?Leu?Phe?Ala?Ala?Gly

145 150 155 160

Asn?Glu?Gly?Pro?Asn?Ser?Gly?Thr?Ile?Ser?Ala?Pro?Gly?Thr?Ala?Lys

165 170 175

Asn?Ala?Ile?Thr?Val?Gly?Ala?Thr?Glu?Asn?Tyr?Arg?Pro?Ser?Phe?Gly

180 185 190

Ser?Ile?Ala?Asp?Asn?Pro?Asn?His?Ile?Ala?Gln?Phe?Ser?Ser?Arg?Gly

195 200 205

Ala?Thr?Arg?Asp?Gly?Arg?Ile?Lys?Pro?Asp?Val?Thr?Ala?Pro?Gly?Thr

210 215 220

Phe?Ile?Leu?Ser?Ala?Arg?Ser?Ser?Leu?Ala?Pro?Asp?Ser?Ser?Phe?Trp

225 230 235 240

Ala?Asn?Tyr?Asn?Ser?Lys?Tyr?Ala?Tyr?Met?Gly?Gly?Thr?Ser?Met?Ala

245 250 255

Thr?Pro?Ile?Val?Ala?Gly?Asn?Val?Ala?Gln?Leu?Arg?Glu?His?Phe?Ile

260 265 270

Lys?Asn?Arg?Gly?Ile?Thr?Pro?Lys?Pro?Ser?Leu?Ile?Lys?Ala?Ala?Leu

275 280 285

Ile?Ala?Gly?Ala?Thr?Asp?Val?Gly?Leu?Gly?Tyr?Pro?Asn?Gly?Asp?Gln

290 295 300

Gly?Trp?Gly?Arg?Val?Thr?Leu?Asn?Lys?Ser?Leu?Asn?Val?Ala?Tyr?Val

305 310 315 320

Asn?Glu?Ala?Thr?Ala?Leu?Ala?Thr?Gly?Gln?Lys?Ala?Thr?Tyr?Ser?Phe

325 330 335

Gln?Ala?Gln?Ala?Gly?Lys?Pro?Leu?Lys?Ile?Ser?Leu?Val?Trp?Thr?Asp

340 345 350

Ala?Pro?Gly?Ser?Thr?Thr?Ala?Ser?Tyr?Thr?Leu?Val?Asn?Asp?Leu?Asp

355 360 365

Leu?Val?Ile?Thr?Ala?Pro?Asn?Gly?Gln?Lys?Tyr?Val?Gly?Asn?Asp?Phe

370 375 380

Ser?Tyr?Pro?Tyr?Asp?Asn?Asn?Trp?Asp?Gly?Arg?Asn?Asn?Val?Glu?Asn

385 390 395 400

Val?Phe?Ile?Asn?Ala?Pro?Gln?Ser?Gly?Thr?Tyr?Ile?Ile?Glu?Val?Gln

405 410 415

Ala?Tyr?Asn?Val?Pro?Ser?Gly?Pro?Gln?Arg?Phe?Ser?Leu?Ala?Ile?Val

420 425 430

His

<210>5

<211>433

<212>PRT

＜213〉genus bacillus (Bacillus sp.)

<400>5

Asn?Asp?Val?Ala?Arg?Gly?Ile?Val?Lys?Ala?Asp?Val?Ala?Gln?Asn?Asn

1 5 10 15

Tyr?Gly?Leu?Tyr?Gly?Gln?Gly?Gln?Val?Val?Ala?Val?Ala?Asp?Thr?Gly

20 25 30

Leu?Asp?Thr?Gly?Arg?Asn?Asp?Ser?Ser?Met?His?Glu?Ala?Phe?Arg?Gly

35 40 45

Lys?Ile?Thr?Ala?Leu?Tyr?Ala?Leu?Gly?Arg?Thr?Asn?Asn?Ala?Asn?Asp

50 55 60

Pro?Asn?Gly?His?Gly?Thr?His?Val?Ala?Gly?Ser?Val?Leu?Gly?Asn?Ala

65 70 75 80

Leu?Asn?Lys?Gly?Met?Ala?Pro?Gln?Ala?Asn?Leu?Val?Phe?Gln?Ser?Ile

85 90 95

Met?Asp?Ser?Ser?Gly?Gly?Leu?Gly?Gly?Leu?Pro?Ser?Asn?Leu?Asn?Thr

100 105 110

Leu?Phe?Ser?Gln?Ala?Trp?Asn?Ala?Gly?Ala?Arg?Ile?His?Thr?Asn?Ser

115 120 125

Trp?Gly?Ala?Pro?Val?Asn?Gly?Ala?Tyr?Thr?Ala?Asn?Ser?Arg?Gln?Val

130 135 140

Asp?Glu?Tyr?Val?Arg?Asn?Asn?Asp?Met?Thr?Val?Leu?Phe?Ala?Ala?Gly

145 150 155 160

Asn?Glu?Gly?Pro?Asn?Ser?Gly?Thr?Ile?Ser?Ala?Pro?Gly?Thr?Ala?Lys

165 170 175

Asn?Ala?Ile?Thr?Val?Gly?Ala?Thr?Glu?Asn?Tyr?Arg?Pro?Ser?Phe?Gly

180 185 190

Ser?Leu?Ala?Asp?Asn?Pro?Asn?His?Ile?Ala?Gln?Phe?Ser?Ser?Arg?Gly

195 200 205

Ala?Thr?Arg?Asp?Gly?Arg?Ile?Lys?Pro?Asp?Val?Thr?Ala?Pro?Gly?Thr

210 215 220

Phe?Ile?Leu?Ser?Ala?Arg?Ser?Ser?Leu?Ala?Pro?Asp?Ser?Ser?Phe?Trp

225 230 235 240

Ala?Asn?Tyr?Asn?Ser?Lys?Tyr?Ala?Tyr?Met?Gly?Gly?Thr?Ser?Met?Ala

245 250 255

Thr?Pro?Ile?Val?Ala?Gly?Asn?Val?Ala?Gln?Leu?Arg?Glu?His?Phe?Ile

260 265 270

Lys?Asn?Arg?Gly?Ile?Thr?Pro?Lys?Pro?Ser?Leu?Ile?Lys?Ala?Ala?Leu

275 280 285

Ile?Ala?Gly?Ala?Thr?Asp?Val?Gly?Leu?Gly?Tyr?Pro?Ser?Gly?Asp?Gln

290 295 300

Gly?Trp?Gly?Arg?Val?Thr?Leu?Asp?Lys?Ser?Leu?Asn?Val?Ala?Tyr?Val

305 310 315 320

Asn?Glu?Ala?Thr?Ala?Leu?Ala?Thr?Gly?Gln?Lys?Ala?Thr?Tyr?Ser?Phe

325 330 335

Gln?Ala?Gln?Ala?Gly?Lys?Pro?Leu?Lys?Ile?Ser?Leu?Val?Trp?Thr?Asp

340 345 350

Ala?Pro?Gly?Ser?Thr?Thr?Ala?Ser?Tyr?Thr?Leu?Val?Asn?Asp?Leu?Asp

355 360 365

Leu?Val?Ile?Thr?Ala?Pro?Asn?Gly?Gln?Lys?Tyr?Val?Gly?Asn?Asp?Phe

370 375 380

Ser?Tyr?Pro?Tyr?Asp?Asn?Asn?Trp?Asp?Gly?Arg?Asn?Asn?Val?Glu?Asn

385 390 395 400

Val?Phe?Ile?Asn?Ala?Pro?Gln?Ser?Gly?Thr?Tyr?Thr?Ile?Glu?Val?Gln

405 410 415

Ala?Tyr?Asn?Val?Pro?Ser?Gly?Pro?Gln?Arg?Phe?Ser?Leu?Ala?Ile?Val

420 425 430

His

<210>6

<211>434

<212>PRT

＜213〉genus bacillus (Bacillus sp.)

<400>6

Asn?Asp?Val?Ala?Arg?Gly?Ile?Val?Lys?Ala?Asp?Val?Ala?Gln?Ser?Ser

1 5 10 15

Tyr?Gly?Leu?Tyr?Gly?Gln?Gly?Gln?Val?Val?Ala?Val?Ala?Asp?Thr?Gly

20 25 30

Leu?Asp?Thr?Gly?Arg?Asn?Asp?Ser?Ser?Met?His?Glu?Ala?Phe?Arg?Gly

35 40 45

Lys?Ile?Thr?Ala?Ile?Tyr?Ala?Leu?Gly?Arg?Thr?Asn?Asn?Ala?Asn?Asp

50 55 60

Pro?Asn?Gly?His?Gly?Thr?His?Val?Ala?Gly?Ser?Val?Leu?Gly?Asn?Gly

65 70 75 80

Thr?Ser?Asn?Lys?Gly?Met?Ala?Pro?Gln?Ala?Asn?Leu?Val?Phe?Gln?Ser

85 90 95

Val?Met?Asp?Ser?Asn?Gly?Gly?Leu?Gly?Gly?Leu?Pro?Ser?Asn?Val?Ser

100 105 110

Thr?Leu?Phe?Ser?Gln?Ala?Tyr?Ser?Ala?Gly?Ala?Arg?Ile?His?Thr?Asn

115 120 125

Ser?Trp?Gly?Ala?Pro?Val?Asn?Gly?Ala?Tyr?Thr?Thr?Asp?Ser?Arg?Asn

130 135 140

Val?Asp?Asp?Tyr?Val?Arg?Lys?Asn?Asp?Met?Ala?Val?Leu?Phe?Ala?Ala

145 150 155 160

Gly?Asn?Glu?Gly?Pro?Asn?Gly?Gly?Thr?Ile?Ser?Ala?Pro?Gly?Thr?Ala

165 170 175

Lys?Asn?Ala?Ile?Thr?Val?Gly?Ala?Thr?Glu?Asn?Leu?Arg?Pro?Ser?Phe

180 185 190

Gly?Ser?Tyr?Ala?Asp?Asn?Ile?Asn?His?Val?Ala?Gln?Phe?Ser?Ser?Arg

195 200 205

Gly?Pro?Thr?Lys?Asp?Gly?Arg?Ile?Lys?Pro?Asp?Val?Met?Ala?Pro?Gly

210 215 220

Thr?Phe?Ile?Leu?Ser?Ala?Arg?Ser?Ser?Leu?Ala?Pro?Asp?Ser?Ser?Phe

225 230 235 240

Trp?Ala?Asn?His?Asp?Ser?Lys?Tyr?Ala?Tyr?Met?Gly?Gly?Thr?Ser?Met

245 250 255

Ala?Thr?Pro?Ile?Val?Ala?Gly?Asn?Val?Ala?Gln?Leu?Arg?Glu?His?Phe

260 265 270

Ile?Lys?Asn?Arg?Gly?Ile?Thr?Pro?Lys?Pro?Ser?Leu?Leu?Lys?Ala?Ala

275 280 285

Leu?Ile?Ala?Gly?Ala?Thr?Asp?Ile?Gly?Leu?Gly?Tyr?Pro?Ser?Gly?Asn

290 295 300

Gln?Gly?Trp?Gly?Arg?Val?Thr?Leu?Asp?Lys?Ser?Leu?Asn?Val?Ala?Phe

305 310 315 320

Val?Asn?Glu?Thr?Ser?Ser?Leu?Ser?Thr?Asn?Gln?Lys?Ala?Thr?Tyr?Ser

325 330 335

Phe?Thr?Ala?Gln?Ser?Gly?Lys?Pro?Leu?Lys?Ile?Ser?Leu?Val?Trp?Ser

340 345 350

Asp?Ala?Pro?Ala?Ser?Thr?Ser?Ala?Ser?Val?Thr?Leu?Val?Asn?Asp?Leu

355 360 365

Asp?Leu?Val?Ile?Thr?Ala?Pro?Asn?Gly?Thr?Lys?Tyr?Val?Gly?Asn?Asp

370 375 380

Phe?Thr?Ala?Pro?Tyr?Asp?Asn?Asn?Trp?Asp?Gly?Arg?Asn?Asn?Val?Glu

385 390 395 400

Asn?Val?Phe?Ile?Asn?Ala?Pro?Gln?Ser?Gly?Thr?Tyr?Thr?Val?Glu?Val

405 410 415

Gln?Ala?Tyr?Asn?Val?Pro?Gln?Gly?Pro?Gln?Ala?Phe?Ser?Leu?Ala?Ile

420 425 430

Val?Asn

<210>7

<211>433

<212>PRT

＜213〉genus bacillus (Bacillus sp.)

<400>7

Asn?Asp?Val?Ala?Arg?Gly?Ile?Val?Lys?Ala?Asp?Val?Ala?Gln?Asn?Asn

1 5 10 15

Phe?Gly?Leu?Tyr?Gly?Gln?Gly?Gln?Ile?Val?Ala?Val?Ala?Asp?Thr?Gly

20 25 30

Leu?Asp?Thr?Gly?Arg?Asn?Asp?Ser?Ser?Met?His?Glu?Ala?Phe?Arg?Gly

35 40 45

Lys?Ile?Thr?Ala?Leu?Tyr?Ala?Leu?Gly?Arg?Thr?Asn?Asn?Ala?Asn?Asp

50 55 60

Pro?Asn?Gly?His?Gly?Thr?His?Val?Ala?Gly?Ser?Val?Leu?Gly?Asn?Ala

65 70 75 80

Thr?Asn?Lys?Gly?Met?Ala?Pro?Gln?Ala?Asn?Leu?Val?Phe?Gln?Ser?Ile

85 90 95

Met?Asp?Ser?Gly?Gly?Gly?Leu?Gly?Gly?Leu?Pro?Ala?Asn?Leu?Gln?Thr

100 105 110

Leu?Phe?Ser?Gln?Ala?Tyr?Ser?Ala?Gly?Ala?Arg?Ile?His?Thr?Asn?Ser

115 120 125

Trp?Gly?Ala?Pro?Val?Asn?Gly?Ala?Tyr?Thr?Thr?Asp?Ser?Arg?Asn?Val

130 135 140

Asp?Asp?Tyr?Val?Arg?Lys?Asn?Asp?Met?Thr?Ile?Leu?Phe?Ala?Ala?Gly

145 150 155 160

Asn?Glu?Gly?Pro?Gly?Ser?Gly?Thr?Ile?Ser?Ala?Pro?Gly?Thr?Ala?Lys

165 170 175

Asn?Ala?Ile?Thr?Val?Gly?Ala?Thr?Glu?Asn?Leu?Arg?Pro?Ser?Phe?Gly

180 185 190

Ser?Tyr?Ala?Asp?Asn?Ile?Asn?His?Val?Ala?Gln?Phe?Ser?Ser?Arg?Gly

195 200 205

Pro?Thr?Arg?Asp?Gly?Arg?Ile?Lys?Pro?Asp?Val?Met?Ala?Pro?Gly?Thr

210 215 220

Tyr?Ile?Leu?Ser?Ala?Arg?Ser?Ser?Leu?Ala?Pro?Asp?Ser?Ser?Phe?Trp

225 230 235 240

Ala?Asn?His?Asp?Ser?Lys?Tyr?Ala?Tyr?Met?Gly?Gly?Thr?Ser?Met?Ala

245 250 255

Thr?Pro?Ile?Val?Ala?Gly?Asn?Val?Ala?Gln?Leu?Arg?Glu?His?Phe?Val

260 265 270

Lys?Asn?Arg?Gly?Val?Thr?Pro?Lys?Pro?Ser?Leu?Leu?Lys?Ala?Ala?Leu

275 280 285

Ile?Ala?Gly?Ala?Ala?Asp?Val?Gly?Leu?Gly?Phe?Pro?Asn?Gly?Asn?Gln

290 295 300

Gly?Trp?Gly?Arg?Val?Thr?Leu?Asp?Lys?Ser?Leu?Asn?Val?Ala?Phe?Val

305 310 315 320

Asn?Glu?Thr?Ser?Pro?Leu?Ser?Thr?Ser?Gln?Lys?Ala?Thr?Tyr?Ser?Phe

325 330 335

Thr?Ala?Gln?Ala?Gly?Lys?Pro?Leu?Lys?Ile?Ser?Leu?Val?Trp?Ser?Asp

340 345 350

Ala?Pro?Gly?Ser?Thr?Thr?Ala?Ser?Leu?Thr?Leu?Val?Asn?Asp?Leu?Asp

355 360 365

Leu?Val?Ile?Thr?Ala?Pro?Asn?Gly?Thr?Lys?Tyr?Val?Gly?Asn?Asp?Phe

370 375 380

Thr?Ala?Pro?Tyr?Asp?Asn?Asn?Trp?Asp?Gly?Arg?Asn?Asn?Val?Glu?Asn

385 390 395 400

Val?Phe?Ile?Asn?Ala?Pro?Gln?Ser?Gly?Thr?Tyr?Thr?Val?Glu?Val?Gln

405 410 415

Ala?Tyr?Asn?Val?Pro?Val?Ser?Pro?Gln?Thr?Phe?Ser?Leu?Ala?Ile?Val

420 425 430

His

Claims (6)

1. Sumizyme MP is wherein in 54,119,138,148 or 195 the amino-acid residue disappearance of SEQ ID NO:1 or be selected from: tryptophane, phenylalanine, L-Ala, l-asparagine, L-glutamic acid, Threonine, Xie Ansuan, Histidine, Serine, Methionin, glutamine, methionine(Met), glycine, aspartic acid, proline(Pro), arginine or cysteine residue;

Wherein, described Sumizyme MP has the higher scourability of enzyme than SEQ ID NO:1.

2. gene of the described Sumizyme MP of claim 1 of encoding.

3. recombinant vectors that contains gene described in the claim 2.

4. transformant that contains just like recombinant vectors described in the claim 3.

5. transformant as claimed in claim 4 wherein is used as the host with microorganism.

6. detergent composition, it contains just like the Sumizyme MP described in the claim 1.

Images