CN1302106C - Subtilase enzymes of the I-S1 I-S2 sub-groups having an additional amino acid residue in an active site loop region - Google Patents

Abstract

The present invention relates to subtilase enzymes of the I-S1 and I-S2 sub-groups having an additional amino acid in the active site loop (b) region from positions 95 to 103. The variant subtilases of the present invention exhibit improved wash performance in a detergent in comparison to its parent enzyme.

Description

The I-S1 and the I-S2 subgroup subtilase enzymes that in avtive spot ring district, have additional amino acid residue

Technical field

The present invention relates to a kind of new I-S1 and the subtilase enzymes (subtilase) of I-S2 subgroup, it has at least one extra amino-acid residue at 100 from avtive spot ring (b) district of 95-103 position.These proteolytic enzyme show scourability outstanding or improvement when being used for washing composition, cleaning and detergent composition.The invention still further relates to coding and being used for the gene of described expression of enzymes, when described gene inserts appropriate host cell or the biochron is expressed described enzyme.The invention still further relates to described gene transformation and host cell that can express described enzyme variants, and the method for preparing novel enzyme

Background of invention

In detergent industry, the application of enzyme in the washing prescription is above 30 years.The enzyme that is used for these preparations comprises: proteolytic enzyme, lipase, amylase, cellulase and other enzyme, perhaps their mixture.Commercial most important enzyme is a proteolytic enzyme.

The proteolytic enzyme of increasing commercial use is the protein engineering variant of naturally occurring wild-type protease, as DURAZYM ^(Novo Nordisk A/S), RELASE ^(NovoNordisk A/S), MAXAPEM ^(Gist-Brocades N.V.), PURAFECT ^(Genencor international corporation).

Also has the existing in the art description of many ease variants, as EP 130756 (GENENTECH) (issuing patent 34,606 (GENENCOR) again) corresponding to the U.S.; EP214435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050 (GENEX); EP 260105 (GENENCOR); Thomas, Russell and Fersht (1985) nature (Nature) 318375-376; Thomas, Russell and Fersht (1987) molecular biology magazine (J.Mol.Biol.) 193 803-813; The natural 328496-500 of Russell and Fersht (1987) (1987); WO 88/08028 (Genex); WO 88/08033 (AMGEN); WO95/27049 (SOLVAY S.A.); WO 95/30011 (PROCTER ﹠amp; GAMBLE company); WO 95/30010 (PROCTER ﹠amp; GAMBLE company); WO95/29979 (PROCTER ﹠amp; GAMBLE company); US 5.543.302 (SOLVAYS.A.); EP 251 446 (GENENCOR); WO 89/06279 (Novo Nordisk A/S); WO 91/00345 (Novo Nordisk A/S); EP 525 610 A1 (SOLVAY) and WO94/02618 (GIST-BROCADES N.V.).

Yet although described many useful ease variants, many industrial uses still need the proteolytic enzyme or the ease variants of new improvement.

Therefore, the purpose of this invention is to provide the proteolytic enzyme or the protein engineering ease variants of improvement, in particular for detergent industry.

Summary of the invention

The inventor has found such subtilase enzymes, those known length of at least one avtive spot chain rate wherein, and described enzyme shows improved scourability in detergent composition.Making up especially subtilisin 309 (BLSAVI or SAVINASE ^) subtilisin (subtilisin) variant in it is identified, demonstrate improved wash characteristics with respect to this enzyme of parent's wild-type enzyme.This point is described in we previous application DK1332/97.

Some subtilase enzymes in known I-S1 (true " subtilisin ") or I-S2 (high alkalinity subtilisin) subgroup or its variant have at least one extra amino-acid residue 100 (or between 100 and 101) from avtive spot ring (b) district of 95-103 position, with at present known those with in described application, describe those compare, described enzyme shows the scourability of beat all improvement.

The improved proteolytic enzyme of the present invention can be by separate obtaining from natural resources, or by at least one extra amino-acid residue of importing (insertion) acquisition in the avtive spot ring (b) between 100 and 101 of wild subtilase enzymes (for the avtive spot ring and position encodedly state as follows).

Although this discovery is at subtilisin 309, expectation may produce or separating analogous useful subtilase enzymes or subtilase variant.

In addition, may be by screening natural strain isolated specifically, to differentiate new wild-type subtilase enzymes, it contains at least one avtive spot ring (b) longer than the corresponding avtive spot ring of known wild-type subtilase enzymes, as subtilisin 309, this subtilase enzymes is considered to have the amino-acid residue of an insertion between 100 and 101, and compares with maximally related known subtilisin (as subtilisin 309) and to be presented at scourability outstanding in the washing composition.

Relevant sequence relatively reaches coding with reference to seeing following Fig. 1,1a, 2 and 2a, show subtilisin BPN ' (BASBPN) (a) and subtilisin 309 (BLSAVI) sequence between (b) relatively, and (BASBPN) (a) and subtilisin Carlsberg (g) the sequence comparison between (BLSCAR) of subtilisin BPN '.In Fig. 1 and 2, utilize the conventional GAP of the GCG software package sequence of carrying out as follows to compare, and the sequence of Fig. 1 a and 2a is relatively with shown in the WO 91/00345.These sequences are used as the reference of residue coding more in the present invention.

Defined seven avtive spot rings (a) to (g) (amino-acid residues of two ends shown in comprising) to contain amino-acid residue at this as lower curtate:

(a) zone between the amino-acid residue 33-43;

(b) zone between the amino-acid residue 95-103;

(c) zone between the amino-acid residue 125-132;

(d) zone between the amino-acid residue 153-173;

(e) zone between the amino-acid residue 181-195;

(f) zone between the amino-acid residue 202-204; With

(g) zone between the amino-acid residue 218-219.

Therefore, first aspect present invention relates to the I-S1 of a kind of isolating (that is: purity＞10%) and the subtilase enzymes of I-S2 subgroup, it has at least one extra amino-acid residue at 100 from avtive spot ring (b) district of 95-103 position, and described thus additional amino acid residue is corresponding to the insertion fragment of at least one amino-acid residue between the 100-101 position.

Second aspect present invention relates to the DNA isolation sequence of code book invention subtilase variant.

Third aspect present invention relates to the expression vector of the DNA isolation sequence that contains code book invention subtilase variant.

Fourth aspect present invention relates to the microbial host cell of the expression vector that has transformed fourth aspect.

The invention still further relates to the production of subtilisin of the present invention.

Enzyme of the present invention can usually be produced by the following method, and culturing micro-organisms is also therefrom separated and reclaimed enzyme with pure basically form; Perhaps be inserted in the suitable microorganism host, cultivate the subtilase enzymes that the host needs with expression, and reclaim enzyme product by expression vector with fourth aspect.

The invention still further relates to the composition that contains subtilase enzymes of the present invention or subtilase variant.

The present invention relates to enzyme of the present invention purposes in many industrial related application, the particularly purposes in cleaning compositions at last, and containing the cleaning compositions of mutant enzyme, especially contain the purposes in the detergent composition of mutant subtilisin.Definition

Before further discussing the present invention in detail, define following term earlier.

Amino acid whose name

The A=Ala=L-Ala

The V=Val=Xie Ansuan

The L=Leu=leucine

The I=Ile=Isoleucine

The P=Pro=proline(Pro)

The F=Phe=phenylalanine

The W=Trp=tryptophane

The M=Met=methionine(Met)

The G=Gly=glycine

The S=Ser=Serine

The T=Thr=Threonine

The C=Cys=halfcystine

Y=Tyr=tyrosine

The N=Asn=l-asparagine

The Q=Gln=glutamine

The D=Asp=aspartic acid

E=Glu=L-glutamic acid

K=Lys=Methionin

The R=Arg=arginine

The H=His=Histidine

Any amino acid of X=Xaa=

The nucleic acid name

The A=VITAMIN B4

The G=guanine

The C=cytosine(Cyt)

T=thymus pyrimidine (only being present among the DNA)

U=uridylic (only being present among the RNA)

The name of variant

When the various enzyme variants of describing the present invention's generation or comprising, adopt following nomenclature so that reference:

At first by isolating or parent's wild-type enzyme and subtilisin BPN ' arrangement comparison definition reference framework (BASBPN).

Utilize following parameter: interval produce penalize=8 and interval extend penalize=8 and every other parameter remain default value, the GAP routine analysis by GCG software package 9.1 editions can obtain to arrange contrast.

Another kind method is the arrangement contrast that utilizes between the known generally acknowledged subtilase enzymes, the arrangement contrast as shown in WO91/00345.As a rule, difference is unimportant.

Fig. 1,1a, 2 and 2a in shown respectively subtilisin BPN ' (BASBPN) and subtilisin 309 (BLSAVI) and subtilisin Carlsberg (BLSCAR)) between sequence relatively.Many disappearances and insertion have been defined with respect to BASBPN.In Fig. 1, than BASBPN, subtilisin 309 has 6 disappearances at 36,58,158,162,163 and 164, and in Fig. 1 a, than BASBPN, subtilisin 309 has same disappearance at 36,56,159,164,165 and 166.In Fig. 2, than BASBPN, subtilisin Carlsberg has 1 disappearance at 58, and in Fig. 2 a, than BASBPN, subtilisin Carlsberg has 1 disappearance at 56.These the disappearance Fig. 1,1a, 2 and 2a in show by asterisk (*).

The multiple modification of carrying out in wild-type enzyme usually uses following three kinds of key elements to show:

The amino acid that original amino acid sites is replaced

Therefore note G195E is meant that 195 glycine replaced by L-glutamic acid.

At original amino acid can be under the situation of any amino-acid residue, can use shorthand notation sometimes, only represents site and substituted amino acid:

The amino acid that replace in the site

During the modification of this symbol in expression homology subtilase enzymes especially suitable (seeing below).

Similarly, when replacing amino-acid residue clueless true, be expressed as:

Original amino acid sites

When original amino acid all may be any amino acid with replacement amino acid, then only represent the site, as: 170.

When original amino acid and/or replace that amino acid can comprise more than one amino acid but during non-all amino acid, then optionally amino acid is shown in the bracket { }:

Original amino acid sites { the amino acid of replacement ₁..., the amino acid of replacement _n}

For specific variant, use special trigram or one-letter symbol, comprise with Xaa and X and represent any amino-acid residue.

Replace:

195 replace glycine by L-glutamic acid and are done by note in the site:

Gly195Glu or G195E

195 replace glycine by any amino acid and are done by note in the site:

Gly195Xaa or G195X, perhaps

Gly195 or G195

Therefore 170 replace any amino-acid residue note by Serine and do in the site:

Xaa170Ser or X170S, perhaps

170Ser or 170S

During the modification of this symbol in expression homology subtilase enzymes especially suitable (seeing below).Therefore, 170Ser means and comprises that for example the interior Arg170Ser that Lys170Ser modifies and BLSAVI is interior of BASBPN modifies.Relevant these examples are referring to Fig. 1.

Can comprise more than one amino acid but non-all amino acid whose modifications for original amino acid and/or replacement amino acid, replace 170 arginine by glycine, L-Ala, Serine or Threonine and can remember work:

Arg170{Gly, Ala, Ser, Thr} or R170{G, A, S, T}

With the expression variant

R170G，R170A，R170S，R170T。

Deletion:

The deleted note of 195 glycine is done:

Gly195 ^*Or G195 ^*

Correspondingly, the deletion of an above amino-acid residue, do as 195 and 196 glycine and leucic deletion note:

Gly195 ^*+ Leu196 ^*Or G195 ^*+ L196 ^*

Insert:

Inserting the Methionin note behind the insertion of an extra amino-acid residue such as the G195 does:

Gly195GlyLys or G195GK; Perhaps

When inserting an above amino-acid residue,, can remember work as inserting Methionin, L-Ala and Serine behind the G195:

Gly195GlyLysAlaSer or G195GKAS

Under these circumstances, the amino-acid residue position period of the numbering of the amino-acid residue of insertion before with this insertion amino acid adds that lowercase represents.In above-mentioned example, sequence 194-196 can remember work:

194 195 196

BLSAVI A - G - L

194 195 195a 195b 195c 196

Modification A-G-K-A-S-L

If insert an amino-acid residue identical, obviously in name, several modes can be arranged with existing amino-acid residue.For example, in above-mentioned example, behind glycine, insert a glycine and can be expressed as G195GG.This identical change also can be expressed as A194AG, show by:

194 195 196

BLSAVI A-G-L changes into

194 195 195a 196

Modification A-G-G-L

194 194a 195 196

Such situation is that those skilled in the art are conspicuous, so the corresponding notation that inserts of the same type of G195GG and expression means and comprises such degeneracy notation that is equal to.

The filling of breach:

When a kind of enzyme was compared the existence deletion with the subtilisin sequence that is used to number, the insertion notation in this site was similar to usefulness:

^*36Asp or ^*36D

36 the insertion of expression aspartic acid in the site.

Multiple modification:

The variant that contains multiple modification separates with plus sige, as:

Arg170Tyr+Gly195Glu or R170Y+G195E

Be illustrated in the modification that arginine and glycine are replaced with tyrosine and L-glutamic acid respectively in site 170 and 195.

Perhaps Tyr167{Gly for example, Ala, Ser, Thr}+Arg170{Gly, Ala, Ser, Thr}

The expression variant

Tyr167Gly+Arg170Gly， Tyr167Gly+Arg170Ala，

Tyr167Gly+Arg170Ser， Tyr167Gly+Arg170Thr，

Tyr167Ala+Arg170Gly， Tyr167Ala+Arg170Ala，

Tyr167Ala+Arg170Ser， Tyr167Ala+Arg170Thr，

Tyr167Ser+Arg170Gly， Tyr167Ser+Arg170Ala，

Tyr167Ser+Arg170Ser， Tyr167Ser+Arg170Thr，

Tyr167Thr+Arg170Gly， Tyr167Thr+Arg170Ala，

Tyr167Thr+Arg170Ser, and Tyr167Thr+Arg170Thr.

At relevant replacement, replacement, insertion or the deletion of expression, for example positively charged residue (K, R with amino-acid residue of particular common characteristics, H), electronegative residue (D, E) or conserved amino acid when modifying, this nomenclature is particularly useful, for example Tyr167{Gly, Ala, Ser, Thr}+Arg170{Gly, Ala, Ser, Thr} represent to replace another p1 amino acid with p1 amino acid.Detailed content is referring to " detailed Description Of The Invention " part.

Proteolytic enzyme

Can cut in the protein substrate that the enzyme of amido bond classifies as proteolytic enzyme or peptase (being used interchangeably) (is consulted Walsh, 1979, enzyme reaction mechanisms (Enzymatic ReactionMechanisms), W.H.Freeman and Company, San Francisco, chapter 3).

The numbering of amino acid sites/residue

If do not particularly point out, amino acid used herein numbering is corresponding to (BASBPN) numbering of sequence of subtilase enzymes BPN '.BPN ' sequence further describe people such as consulting Siezen, protein engineering (Protein Engng.) 4 (1991) 719-737 and Fig. 1.

Serine protease

Serine protease is meant energy catalysis peptide bond hydrolysis, and the enzyme (White that has essential serine residue at its avtive spot, Handler and Smith, 1973 " biochemical basis " (Principles of Biochemistry), the 5th edition, McGraw-Hill books company, New York, 271-272 page or leaf).

The molecular weight ranges of bacterial serine proteolytic enzyme is 20,000-45,000 dalton.They are suppressed by di-isopropyl phosphoric acid fluorine.The simple terminal ester of their hydrolysis, and active similar to Eukaryotic Chymotrypsin (also being serine protease).Narrower term-the Sumizyme MP (comprising a subgroup) of scope has reflected some serine protease optimal pH height, from pH9.0 to 11.0 (relevant summary is consulted Priest (1977) bacteriology and looked back (Bacteriological Rev.) 41711-753).

Subtilase enzymes

The subgroup of a temporary transient called after subtilase enzymes of serine protease is proposed by people such as Siezen [protein engineering 4 (1991) 719-737].They are determined by the amino acid sequence homologous analysis to more than 170 kinds of serine proteases (being known as subtilisin sample proteolytic enzyme (subtilisin-like protease) in the past).Subtilisin often was defined as in the past by gram positive bacterium or mycetogenetic serine protease, and now according to people's such as Siezen definition, it is a subgroup of subtilase enzymes.Identified multiple subtilase enzymes, and the aminoacid sequence of many subtilase enzymes after measured.Consult the document of people such as Siezen (1997) about these subtilase enzymes more detailed descriptions and their aminoacid sequence.

A subgroup of subtilase enzymes, I-S1 or " true subtilisin ", comprise " classical " subtilisin, as subtilisin 168, subtilisin BPN ', subtilisin Carlsberg (ALCALASE ^, Novo NordiskA/S) and subtilisin DY.

By another subgroup that people such as Siezen (seeing above) have divided subtilase enzymes, I-S2.I-S2 subgroup proteolytic enzyme is described as the highly subtilisin of alkalescence, comprises (the MAXACAL as subtilisin PB92 (BAALKP) ^, Gist-Brocades NV), subtilisin 309 (SAVINASE ^, Novo Nordisk A/S), subtilisin 147 (ESPERASE ^, Novo Nordisk A/S) and alkaline elastoser YaB.

The tabulation of subtilase enzymes acronym

I-S1

Subtilisin 168, BSS168 (BSSAS (Subtilisinamylosacchariticus), BSAPRJ (subtilisin J), BSAPRN (subtilisin NAT), BMSAMP (Mes-entericopeptidase),

Subtilisin BPN ', BASBPN,

Subtilisin DY, BSSDY,

Subtilisin Carlsberg, BLSCAR (BLKERA (M-Zyme), BLSCA1, BLSCA2, BLSCA3),

BSSPRC, serine protease C

BSSPRD, serine protease D

I-S2

Subtilisin Sendai, BSAPRS

Subtilisin ALP 1, BSAPRQ,

Subtilisin 147, Esperase ^BLS147 (BSAPRM (subtilisin AprM), BAH101),

Subtilisin 309, Savinase , BLS309/BLSAVI (BSKSMK (M-proteolytic enzyme), BAALKP (subtilisin PB92, Alkaliphilic bacillus (Bacillusalkalophilic) Sumizyme MP), BLSUBL (subtilisin BL)),

Alkalescence elastoser YaB, BYSYAB, " SAVINASE  "

SAVINASE ^Sell by Novo NordiskA/S.It is the subtilisin 309 that derives from bacillus lentus, and with BABP92 only in a site different (N87S consults this paper Fig. 1).SAVINASE ^Aminoacid sequence (referring to this paper Fig. 1) with called after BLSAVI.

Parent's subtilase enzymes

Term " parent's subtilase enzymes " refers to the subtilase enzymes according to people such as Siezen (protein engineering 4:719-737 (1991)) definition.The description that further details is consulted above " subtilase enzymes ".Parent's subtilase enzymes also can be to separate the subtilase enzymes obtain by natural origin, has carried out subsequently modifying and has kept the feature of subtilase enzymes simultaneously.Term " parent's subtilase enzymes " or can be called " wild-type subtilase enzymes ".

The modification of subtilase variant

Term used herein " modification " is defined as comprising the genetic manipulation of Bacillus subtilus Chemical Modification of Enzyme and coding subtilase enzymes.Modification can be in the replacement at interested amino acid sites place, deletion and/or insertion.

Subtilase variant

In this article, the subtilase enzymes of term subtilase variant or sudden change refers to the subtilase enzymes by the organism generation of expressing mutator gene, this mutator gene derives from the parent microorganism that has original or parental gene and produce corresponding parent enzyme, and wherein parental gene has been produced the mutator gene of sudden change subtilase enzymes when obtaining expressing in appropriate host by sudden change.

Homology Bacillus subtilus enzyme sequence

This paper has identified subtilase enzymes SAVINASE ^In ring district, specific activity site and described ring in the aminoacid insertion modified, to obtain subtilase variant of the present invention.

Yet the present invention is not limited to the modification of this special subtilase enzymes, and can expand to and SAVINASE ^Other parent (wild-type) subtilase enzymes that the homologous primary structure is arranged.

In order to identify the identity degree between other homology subtilase enzymes, can use identical parameter setting to utilize the GAP routine of GCG software package 9.1 editions to carry out.Output result by routine analysis arranges relatively side at amino acid, has calculated " identity per-cent " between two sequences

Based on description herein, identify that for those of ordinary skills suitable homology subtilase enzymes and corresponding homology avtive spot ring district are routine techniquess, it can be made amendment according to the present invention.

Scourability

Enzyme is the various natural ability that has the degraded of substrate on the catalysis object to be cleaned in for example cleaning process or hard-surface cleaning process, be commonly referred to as it washability (washingability), can wash power (washability), detergency (detergency) or scourability (washperformance).In this application, represent this specific character with the term scourability.

The separated DNA sequence

Term " isolating ", divide the period of the day from 11 p.m. to 1 a.m when being applied to dna sequence dna, represent that this dna sequence dna takes out from its natural genotypic environment, and thereby do not contain other irrelevant or unnecessary encoding sequence, and exist with the form that is suitable in the engineered protein production system, using.These isolating molecules are the molecules that separate with its natural surroundings, comprise cDNA and genomic clone.Isolated DNA molecule of the present invention does not contain their initial other associated genes, but may comprise naturally occurring 5 ' and 3 ' non-translational region such as promotor and terminator.The evaluation of these relevant ranges obviously be one of basic fundamental of this area (consult as Dynan and Tijan, From Right (Nature) 316: 774-78,1985).Term " DNA isolation sequence " also can be called " cloned dna sequence ".

Isolating protein

When being applied to protein, term " isolating " illustrates that this protein is present under the condition of the natural surroundings that is different from it.Isolating protein does not preferably contain other protein, especially other homologous protein (i.e. " homology impurity " (seeing below)) substantially.

Isolated protein is measured purity by SDS-PAGE and is higher than 10%, preferably is higher than 20%, more preferably is higher than 30%.The protein of highly purified form preferably is provided, promptly by SDS-PAGE identify that purity is higher than 40%, purity is higher than 60%, purity is higher than 80%, more preferably purity is higher than 95%, even more preferably purity is higher than 99%.

Term " isolating protein " also can be called " protein of purifying ".

Homology impurity

Term " homology impurity " refers to from any impurity (as the another kind of polypeptide except that polypeptide of the present invention) in the homologous cell in the initial source of polypeptide of the present invention.

Derive from

The term that uses with the peculiar microorganism source as this paper " derives from ", refers to that polynucleotide and/or polypeptide are by special source or by the cell generation of having inserted from the gene in this source.

Substrate

Term " substrate " use relevant with protease substrate is appreciated that the form that its scope is the wideest, as contains at least one easily by the compound of the peptide bond of protease hydrolysis.

Product

Term " product " use relevant with the product that derives from the proteolytic enzyme enzyme reaction is appreciated that the product that comprises the hydrolysis reaction that relates to the Bacillus subtilus protease enzyme in this article.Product may be the substrate of next hydrolysis reaction.

The concise and to the point description of figure

Fig. 1 shown utilize above-mentioned GAP ordinary method subtilisin BPN ' (a) and the arrangement between the Savinase (b) relatively.

Fig. 1 a has shown the subtilisin BPN ' and the comparison of the arrangement between the Savinase of taking from WO91/00345D.

Fig. 2 has shown subtilisin BPN ' and the comparison of the arrangement between the subtilisin Carlsberg that utilizes above-mentioned GAP ordinary method.。

Fig. 2 a has shown the subtilisin BPN ' and the comparison of the arrangement between the subtilase enzymes Carlsberg of taking from WO91/00345D.

Fig. 3 shows the three-dimensional structure of Savinase (Protein Data Bank (PDB) typing 1SVN).This figure has shown the interested avtive spot ring of this paper (b).

Detailed Description Of The Invention

The first aspect of subtilase enzymes of the present invention relates to the I-S1 of a kind of isolating (that is: purity＞10%) and the subtilase enzymes of I-S2 subgroup, it has at least one extra amino-acid residue at 100 from avtive spot ring (b) district of 95-103 position, and described thus additional amino acid residue is corresponding to the insertion fragment of at least one amino-acid residue between the 100-101 position.

In other words, subtilisin of the present invention is characterised in that, comprise an avtive spot ring (b) more than 9 amino-acid residues, compare with parent or known wild-type subtilisin, wherein extra amino-acid residue is maybe can be considered to 100 and 101 insertions.

The subtilase enzymes of first aspect present invention can be to identify and isolated parent or wild-type subtilase enzymes from nature.

Such parent's wild-type subtilase enzymes can screen specifically by standard technique well known in the art.

A kind of optimization approach of finishing this work is from various microorganism, in the preferred different Bacillus strains, uses the DNA district of the avtive spot ring of PCR specific amplification known coded subtilase enzymes.

Subtilase enzymes is one group of conservative enzyme, because their DNA and aminoacid sequence have homology.Therefore, might make up the relative special primer of avtive spot ring flank.

For example, contrast by analyzing different subtilase enzymes is (referring to people such as for example Siezen, protein science (Protein Science), 6:501-523,1997), those skilled in the art for example can make up the PCR primer corresponding to the avtive spot ring flank of the avtive spot ring (b) between the amino-acid residue 95-103 of any (as from BLSAVI) in I-S1 or the I-S2 group routinely.Utilize these PCR primers, from different microorganisms, preferred different Bacillus strain DNA amplification, subsequently the PCR fragment that amplifies is carried out dna sequencing, might identify and to produce the corresponding avtive spot district of containing 95-103 in the BLSAVI but longer avtive spot district, and wherein insert those bacterial strains that fragment can be considered to be present in the subtilase enzymes between position 100 and 101.After identifying the partial dna sequence of bacterial strain and interested subtilase enzymes, those skilled in the art can finish clone, expression and the purifying of purpose subtilase enzymes routinely.

Yet, estimate that subtilase enzymes of the present invention mainly is the variant of parent's subtilase enzymes.

Therefore, one embodiment of the invention relate to the isolating subtilase enzymes of first aspect present invention, wherein said subtilase enzymes is a kind ofly than its parent enzyme the structure variant of the ring of long-term job site more (b) to be arranged, and this variant contains at least one amino acid whose insertion between amino-acid residue 100 and 101.

Subtilase enzymes of the present invention shows outstanding scourability in washing composition, if this enzyme is the variant that makes up, compares with its maximally related subtilase enzymes (as subtilisin 309) and be presented at improved scourability in the washing composition.

Different Bacillus subtilus protease enzyme products will have different scourabilities in dissimilar detergent composition.Subtilase enzymes of the present invention has the scourability of improvement in most of dissimilar detergent composition.

Shown in this paper embodiment 3, preferred subtilase enzymes of the present invention has the scourability of improvement in detergent composition (seeing below).

For identifying that a kind of given Bacillus subtilus enzyme amino acid sequence (no matter this Bacillus subtilus enzyme sequence is parent's wild-type Bacillus subtilus enzyme sequence or the subtilase variant sequence that is produced by the method outside the rite-directed mutagenesis) whether within the scope of Bacillus subtilus enzyme sequence of the present invention, can carry out following step:

I) to described subtilase enzymes and subtilisin BPN ' (compare (seeing above) referring to this paper " definition " part;

Ii) according to step I) contrast carried out, identify in the described Bacillus subtilus enzyme sequence avtive spot ring corresponding to subtilisin BPN ' (comprising the zone between the 95-103 amino acids residue of two terminal amino acids) avtive spot ring district (b);

Iii) whether the one or more avtive spot rings in the described Bacillus subtilus enzyme sequence that identifies in ii) of determining step are longer than the corresponding avtive spot ring among the BLSAVI, and whether described prolongation is corresponding at least one amino-acid residue between the 100-101 position.

If satisfy such standard, then this subtilase enzymes is the Bacillus subtilus enzyme sequence in the scope of the invention.

Utilize the GAP ordinary method to carry out above-mentioned steps i) in arrangement relatively.

According to above describing, whether avtive spot ring (b) and described subtilase enzymes that those skilled in the art can identify subtilase enzymes routinely fall into scope of the present invention.If variant makes up by the rite-directed mutagenesis method, can know in advance certainly whether this subtilase variant falls into scope of the present invention.

Can be by standard technique known in the art, as the DNA rearrangement structure subtilase variant of fixed point/random mutagenesis or different Bacillus subtilus enzyme sequences.Further details is consulted the materials and methods (seeing below) of " production subtilase variant " part and this paper.

In further embodiment, the present invention relates to:

1. according to a kind of isolating subtilase enzymes of the present invention, at least one in the wherein said insertion amino-acid residue is selected from T, G, A and S;

2. according to a kind of isolating subtilase enzymes of the present invention, at least one in the wherein said insertion amino-acid residue is selected from charged amino-acid residue D, E, H, K and R, more preferably is selected from D, E, K and R;

3. according to a kind of isolating subtilase enzymes of the present invention, at least one in the wherein said insertion amino-acid residue is selected from hydrophilic amino-acid residue C, N, Q, S and T, more preferably is selected from N, Q, S and T;

4. according to a kind of isolating subtilase enzymes of the present invention, at least one in the wherein said insertion amino-acid residue is selected from little hydrophobic amino acid residues A, G and V; Or

5. according to a kind of isolating subtilase enzymes of the present invention, at least one in the wherein said insertion amino-acid residue is selected from big hydrophilic amino-acid residue F, I, L, M, P, W and Y, more preferably is selected from F, I, L, M and Y.

In further embodiment, the present invention relates to according to a kind of isolating subtilase enzymes of the present invention, wherein between the site 99 and 100 described insertion compare with the corresponding avtive spot ring in the BLSAVI and include at least two amino acid.

In further embodiment, the present invention relates to according to a kind of isolating subtilase enzymes of the present invention, wherein said subtilase enzymes comprises at least one insertion (BASBPN numerical system) that is selected from down group:

X100X{T，G，A，S}

X100X{D，E，K，R}

X100X{H，V，C，N，Q}

X100X{F，I，L，M，P，W，Y}

Or subtilisin 309 and the most relevant subtilase enzymes more specifically, as BAALKP, BLSUBL and BSKSMK.

G100GA

G100GT

G100GG

G100GS

G100GD

G100GE

G100GK

G100GR

G100GH

G100GV

G100GC

G100GN

G100GQ

G100GF

G100GI

G100GL

G100GM

G100GP

G100GW

G100GY

Or following arbitrary a kind of combination:

A98G+G100GA+S101A+S103T

S99G+G100GGT+S101T

Know an amino acid in this area and replaced the minor alteration that only causes the enzyme characteristic usually by so-called conserved amino acid.

Following Table III has been listed several groups of conserved amino acids.

The Table III conserved amino acid is replaced

Common property amino acid

Alkalescence: the R=arginine

K=Methionin

The H=Histidine

Tart: E=L-glutamic acid

The D=aspartic acid

Polar: Q=glutamine

The N=l-asparagine

Hydrophobic: the L=leucine

The I=Isoleucine

The V=Xie Ansuan

The M=methionine(Met)

Aromatic: the F=phenylalanine

The W=tryptophane

Y=tyrosine

Little: the G=glycine

The A=L-Ala

The S=Serine

The T=Threonine

Therefore, comprise subtilase variant such as G97A+A98AS+S99G that conservative property is replaced, the G97S+A98AT+S99A expection has does not have the very feature of difference to each other.

According to disclosed herein and/or be for example subtilase variant, those skilled in the art can identify the suitable conservative modification of these variants routinely according to all aspects of the invention and embodiment, with the subtilase variant of the scourability that obtains to have improvement.

In embodiments of the invention, interested is those subtilase enzymes that belong to I-S1 and I-S2 subgroup, and it is used for from natural or separate novel enzyme of the present invention from the different sorts of artificial generation, and is used for from the parent enzyme design and produces variant.

About variant from the I-S1 subgroup, preferred parent's subtilase enzymes is selected from BSS168 (BSSAS, BSAPRJ, BSAPRN, BMSAMP), BASBPN, BSSDY and BLSCAR (BLKERA, BLSCA1, BLSCA2, BLSCA3), BSSPRC, and BSSPRD, or their reservation the functional variant of I-S1 subgroup feature.

About the variant from the I-S2 subgroup, preferred parent's subtilase enzymes is selected from BSAPRQ, BLS147 (BSAPRM, BAH101), BLSAVI (BSKSMK, BAALKP, BLSUBL), BYSYAB and BSAPRS, or their reservation the functional variant of I-S2 subgroup feature.

More particularly, described parent's subtilase enzymes is BLSAVI (SAVINASE ^, NOVONORDISK A/S), and preferred subtilase variant of the present invention is SAVINASE ^Variant.

The present invention also comprises subtilase enzymes and any other combination to the modification of parent enzyme aminoacid sequence of the invention described above.Especially comprise combination with other modification in order to improvement enzyme characteristic known in the art.Many subtilase variant with different improved characteristics have been described in this area, and wherein a part is mentioned (seeing above) in this paper " background of invention " part.Those reference are open at this paper, can be advantageously and the reference of the combined subtilase variant of subtilase variant of the present invention as identifying.

These combinations comprise the site: the replacement in 222 (improving oxidative stability) and 218 (the improving thermostability), Ca binding site makes enzyme stable, as site 76 and known many other modifications of prior art.

In further embodiment, subtilase variant of the present invention can advantageously make up with one or more modifications of following any site:

27,36,57,76,87,97,101,104,120,123,167,170,206,218,222,224,235 and 274.

Especially, following BLSAYI, BLSUBL, BSKSMK and BAALKP variant are considered suitable for combination:

K27R, ^*36D, S57P, N76D, S87N, G97N, S101G, S103A, V104A, V104I, V104N, V104Y, H120D, N123S, Y167, R170, Q206E, N218S, M222S, M222A, T224S, K235L and T274A.

In addition, comprise that the variant that any variation and any one or a plurality of modification mentioned above of other combination of V104N+S101G, S87N+S101G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V104I or N76D+V104A variation or these sudden changes (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A) are made up has improved characteristics.

In addition, the subtilase variant of the main aspect of the present invention preferably with 129,131,133 and 194 in one or more modifications (more preferably 129K, 131H, 133P, 133D and 194P modify, and most preferably P129K, P131H, A133P, A133D and A194P modify) combination of any site.Any expression level of subtilase variant of the present invention that all makes during those are modified is higher.

Therefore, the present invention further embodiment relates to variant of the present invention, and wherein said modification is selected from down group:

Produce subtilase variant

Being used to clone subtilase enzymes of the present invention and being used for inserting many methods of introducing gene (as the subtilase enzymes gene) is well-known in the art, the document of quoting referring to " background of invention " part.

Usually, can use clone gene and in this gene, introduce to insert the standard method of (at random and/or fixed point) and obtain subtilase variant of the present invention.Consult this paper embodiment (seeing below) and (people (1989) molecular cloning such as Sambrook: laboratory manual, cold spring harbor laboratory, cold spring port, New York about further describing of appropriate technology; People such as F.M.Ausubel (chief editor) " molecular biological fresh approach ", John Wiley and Sons, 1995; C.R.Harwood and S.M.Cutting (chief editor) " molecular biology method of genus bacillus ", John Wiley andSons, (1990); With WO 96/34946).

In addition, the standard technique structure (WO95/22625 that is used for manually producing diversity that subtilase variant of the present invention can be reset by the DNA of for example different subtilase enzymes genes; Stemmer WPC, natural 370:389-91,1994).For example with Savinase ^Comprising of identifying compare Savinase with occurring in nature ^The DNA of one or more subtilase enzymes partial sequence of the respective area that avtive spot ring district is longer resets, and screens the variant that scourability makes moderate progress subsequently, and subtilase variant of the present invention can be provided.

Expression vector

The recombinant expression vector that contains the DNA construct of code book invention enzyme can be any carrier that can carry out the recombinant DNA operation easily.

The host cell that it will import is often depended in the selection of carrier.Thus, carrier can be the carrier of self-replicating, i.e. the carrier that exists with the outer entity form of karyomit(e), and it duplicates and is independent of THE REPLICATION OF CHROMOSOME, as plasmid.Perhaps, carrier can be such one type, when it is directed in the host cell, can partly or entirely be incorporated in the host cell gene group, and duplicate with the karyomit(e) of integrating.

Carrier is expression vector preferably, and wherein the dna sequence dna of code book invention enzyme is operably connected to DNA and transcribes required additional section.In general, expression vector derives from plasmid or viral DNA, perhaps can contain the element of the two." be operably connected " arrangement mode that refers to all sections of term makes them can exercise and the consistent function of desired use (as transcribing initial and run through the dna sequence dna of codase by promotor).

Promotor can be any dna sequence dna that shows transcriptional activity in selected host cell, can derive from coding and host cell homology or allogenic proteinic gene.

The example that is applicable to the promotor of bacterial host cell comprises the promotor of bacstearothermophilus maltogenic amylase gene, bacillus licheniformis alpha-amylase gene, bacillus amyloliquefaciens alpha-amylase gene, bacillus subtilis alkali proteinase gene or bacillus pumilus xylosidase gene, perhaps lambda particles phage P _ROr P _LPromotor, perhaps intestinal bacteria lac, trp or tac promotor.

If desired, the dna sequence dna of the code book invention enzyme suitable terminator that can also be operably connected.

Recombinant vectors of the present invention can also contain the dna sequence dna that carrier can be duplicated in selected host cell.

Carrier can also contain selective marker, as the gene of the complementary host cell defective of its product, perhaps encodes to microbiotic such as kantlex, paraxin, erythromycin, tsiklomitsin, spectinomycin or to the gene of the resistance of heavy metal or weedicide.

For enzyme of the present invention being imported the Secretory Pathway of host cell, can in recombinant vectors, provide secretory signal sequence (being also referred to as leader sequence, precursor sequence or presequence).Secretory signal sequence is connected with the dna sequence dna of codase with correct reading frame.Secretory signal sequence is usually located at 5 ' end of the dna sequence dna of codase.Secretory signal sequence can be usually the gene that the sequence relevant with enzyme maybe can be come other secretory protein of own coding.

Be used for connecting respectively the dna sequence dna, promotor of code book invention enzyme and randomly terminator and/or secretory signal sequence, or these sequences are assembled up with suitable substance P CR amplification scheme, with the method that their insertions is contained the suitable carrier that duplicates or integrate information needed, be those skilled in the art's well-known (consult as people such as Sambrook, ibid).

Host cell

The dna sequence dna that imports the code book invention enzyme of host cell can be and selected host's homology or allogenic sequence.If with the host cell homology, promptly by the natural generation of host cell, another promoter sequence that then usually it is operably connected, if perhaps feasible, thus another secretory signal sequence can be connected and/or the terminator sequence is not under its natural surroundings.Term " homology " means the dna sequence dna of the enzyme that comprises that the selected host living beings of coding is natural.Term " allos " means and comprises the natural dna sequence dna of not expressing of host cell.Therefore, dna sequence dna can perhaps can be a composition sequence from another kind of organism.

The host cell of DNA construct of the present invention to be imported or recombinant vectors can be any cell that can produce enzyme of the present invention, comprises bacterium, yeast, fungi and higher eucaryotic cells.

The example that can produce the bacterial host cell of enzyme of the present invention through cultivation is a gram positive bacterium, bacterial strain as bacillus, such as subtilis, Bacillus licheniformis, bacillus lentus, bacillus brevis, bacstearothermophilus, Alkaliphilic bacillus, bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, bacillus lautus, bacillus megaterium or bacillus thuringiensis bacterial strain, or the bacterial strain of streptomyces, such as shallow Streptomyces glaucoviolaceus or mouse ash streptomycete, perhaps gram negative bacterium is such as intestinal bacteria.The conversion of bacterium can be carried out (consult people such as Sambrook, see above) in known manner by protoplast transformation, electroporation, joint or by competent cell.

When bacterium is expressed in such as intestinal bacteria, enzyme can be retained in the tenuigenin, has (being called inclusion body) with insoluble particle usually, or can be directed to periplasmic space by the bacterium secretion sequence.In the previous case, lysing cell reclaims particle, and sex change is diluted denaturing agent then and made enzyme folding again.In the later case, enzyme can reclaim from periplasmic space, as with ultrasonic wave or osmotic shock smudge cells, with the composition of release periplasmic space, and reclaims enzyme.

When expressing enzyme in gram positive bacterium such as genus bacillus or streptomycete bacterial strain, enzyme can be retained in the tenuigenin, or can be directed in the substratum of extracellular by the bacterium secretion sequence.In the later case, enzyme can as mentioned belowly reclaim from substratum.

Produce the method for subtilase enzymes

The invention provides the method for production the present invention's isolating enzyme, the suitable host cell that wherein will transform the dna sequence dna of this enzyme of encoding is cultivated under the condition that allows enzyme to produce, and reclaims the enzyme that produces from culture.

When the expression vector of the dna sequence dna that contains codase is transformed in the heterologous host cell, might the allos reorganization produce enzyme of the present invention.

Might obtain highly purified Bacillus subtilus enzyme composition thus, it is characterized by and do not contain homology impurity.

In this article, homology impurity refers to any impurity that the homologous cell by the initial source of enzyme of the present invention produces (as be different from other polypeptide of the enzyme of the present invention).

The substratum that is used to cultivate transformed host cell can be any conventional substratum that is suitable for selected host cell growth.Can easily the subtilase enzymes of expressing be secreted in the substratum, and therefrom reclaim by well-known method, described method comprise through centrifugal or filter cell and substratum are separated, protein component in salt (as ammonium sulfate) precipitation substratum, pass through as chromatography method purifying such as ion exchange chromatography, affinity chromatographys subsequently.

The purposes of subtilase variant of the present invention

Subtilase variant of the present invention can be used for many industrial uses, in detergent industry.

The invention still further relates to the enzyme composition that contains subtilase variant of the present invention.

General introduction and corresponding preferred enzyme composition about preferred industrial application have hereinafter been described.

This general introduction is not all suitable application that are intended to list fully subtilase variant of the present invention.Subtilase variant of the present invention can be applicable in other industrial application of use proteolytic enzyme, especially subtilase enzymes known in the art.

The detergent composition that contains mutant enzyme

The present invention includes mutant enzyme of the present invention the cleaning and cleansing composition in purposes and this composition that contains mutant subtilisin.These cleanings and cleansing composition have good description in the art, consult WO 96/34946, WO 97/07202 and WO 95/30011 about further describing of suitable cleaning and detergent composition.

Can also show the scourability of many subtilase variant improvement of the present invention with reference to the following embodiment of this paper.

Detergent composition

Thereby enzyme of the present invention can be added into the composition that constitutes in the detergent composition.

Detergent composition of the present invention can for example be formulated into hand washing or machine washing detergent composition, add composition comprising being suitable for the pretreated laundry of polluted fabric, and the rinse composition that increases fabric softness, maybe can be formulated into the composition that is used for general family expenses hard-surface cleaning operation, or the quilt preparation is used for craft or machinery is washed the dish operation.

A special aspects, the invention provides a kind of detergent additive that contains enzyme of the present invention.Detergent additive and detergent composition can contain one or more other enzymes, as proteolytic enzyme, lipase, at, amylase, carbohydrase, cellulase, polygalacturonase, mannase, arabinase, Galactanase, zytase, oxydase, as laccase and/or peroxidase.

Usually, the character of selected enzyme should adapt with selected washing composition (that is: pH is the suitableeest, with other enzymes and non-enzyme component compatibility, etc.), and enzyme should exist with significant quantity.

Proteolytic enzyme: suitable proteolytic enzyme comprises animal, plant or microbe-derived.Microbe-derived is preferred.The mutant that comprises chemically modified or protein engineering.Proteolytic enzyme can be serine protease or metalloprotease, preferred alkaline microbial protease or trypsin-like proteolytic enzyme.The example of Sumizyme MP is a subtilisin, especially from the gemma deutero-those, for example subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (being described among the WO 89/06270).The trypsin-like examples of proteases is for example pig or Niu Laiyuan of trypsin) and be described in fusarium proteolytic enzyme among the WO84/25583.

Useful examples of proteases such as WO92/19729, WO98/20115, WO98/20116 and WO98/34946 are especially at the variant that has one or more replacements as upper/lower positions: 27,36,57,76,87,97,101,104,120,123,167,170,194,206,218,222,224,235 and 274.

Preferred commercially available proteolytic enzyme comprises: Novo Nordisk A/S (Denmark) is with trade(brand)name Alcalase ^TM, Savinase ^TM, Primase ^TM, Durazym ^TM, Esperase ^TMAnd Kannase ^TMThose that sell, by Geneneor International with trade(brand)name Maxatase ^TM, Maxacal ^TM, Maxapem ^TM, Properase ^TM, Purafect ^TM, and Purafect OXP ^TM, FN2 ^TMAnd FN3 ^TMThose that sell.

Lipase: suitable lipase comprises those of bacterium or originated from fungus.Comprise the mutant that chemistry or protein engineering are modified.The example of useful lipase comprises (synonym Thermomyces) lipase from Humicola, comprise for example being described among EP258068 and the EP305216 of Humicola lanuginosa, or from H.insolens described in WO96/13580, Rhodopseudomonas lipase for example is described in Pseudomonas alcaligenes or the pseudomonas pseudoalcaligenes lipase among the EP 218272, for example be described in the pseudomonas cepacia lipase among the EP 331376, for example be disclosed in Situ Ci Shi pseudomonas lipase among the GB1372034, Pseudomonas fluorescens lipase, pseudomonas strain SD705 (WO95/06720 and WO96/27002), P.wisconsinensis (WO96/12012), genus bacillus lipase is subtilis lipase (Dartois etc. for example, (1993), Biochemica et Biophsica acta 1131,253-260), bacstearothermophilus lipase (JP 64/744992) or bacillus pumilus lipase (WO91/16422).

The example of other Lipase variantses is as described in the following document:

WO 92/05249, and WO 94/01541, and EP 407 225, and EP 260 105, WO95/35381, and WO 96/00292, and WO 95/30744, and WO 94/25578, and WO 95/14783, WO95/22615, WO 97/04079 and WO 97/07202.

Preferred commercially available lipase comprises Lipolase ^TMWith Lipolase Ultra ^TM(Novo NordiskA/S).

Amylase:.Suitable amylase (α and/or β) comprises those of bacterium and originated from fungus.The mutant that comprises chemically modified or protein engineeringization.Amylase comprises: for example, and the α-Dian Fenmei that the specific bacterial strain of describing in detail in GB1296839 from Bacillus licheniformis obtains.

Useful diastatic example such as WO 94/02597, WO94/18314, described in WO 96/23837 and the WO 97/43424, especially at the variant that has one or more replacements as upper/lower positions: 15,23,105,106,124,128,133,154,156,181,188,190,197,202,208,209,243,264,304,305,391,408 and 444.

Commercially available amylase is Duramyl ^TM, Termamyl ^TM, Fungamyl ^TMAnd BAN ^TM(obtaining) and Rapidase from Novo Nordisk A/S ^TMWith Maxamyl P ^TM(obtaining) from Genencor.

Cellulase: suitable cellulase comprises those of bacterium and originated from fungus.The mutant that comprises chemically modified or protein engineeringization.Suitable cellulase comprises the cellulase from the kind of bacillus, Rhodopseudomonas, Humicola, Fusarium, Thielavia, little Acremonium (Acremonium), as be disclosed in US4435307, US 5648263, US 5691178, the fungal cellulase of producing from Humicola insolens, Myceliophthora thermophila and sharp sickle spore among US 5776757 and the WO 89/09259.

Especially the cellulase of Shi Heing is alkalescence or the neutral cellulase with colour-preserving function.The example EP 0495257 of the plain enzyme of this fibrid, EP0531372, WO96/11262, WO96/29397 is described in the WO98/08940.The example of other cellulase variants sees that as WO 94/07998, EP 0531315, and US 5457046, and US 5686593, and US 5763254, and WO 95/24471, described in WO 98/42307 and the PCT/DK98/00299.

Commercially available cellulase comprises Celluzyme ^TMAnd Carezyme ^TM(Novo NordiskA/S), Clazinase ^TMWith Puradax HA ^TM(Genencor International Inc.) and KAC-500 (B) ^TM(Kao Corporation).

Peroxidase/oxydase: suitable peroxidase/oxydase comprises those of plant, bacterium or originated from fungus.The mutant that comprises chemically modified or protein engineeringization.The example of useful peroxidase comprises from terrible Agaricus (Corpinus), for example from peroxidase and the variant thereof of C.cinereus, as WO 93/24618, described in WO 95/10602 and the WO 98/15257.Commercially available peroxidase comprises Guardzyme ^TM(Novo Nordisk A/S).

The independent additive that can contain one or more enzymes by adding, or contain the combined additive of all these enzymes by adding, these detergent enzymes are included in the detergent composition.Detergent additives of the present invention is independent additive or combined additive, can be mixed with for example particle, liquid or slurries or the like.Preferred detergent additives goods are particles, especially are non-dirt (non-dusting) particle, liquid, especially stable liquid, or slurries.

Non-dirt particle can be by disclosed method production among the US 4,106,991 and 4,661,452 for example, and can randomly wrap quilt by means known in the art.The example of wax sample capsulating material be molecular-weight average be poly-(oxyethane) product of 1000 to 20000 (polyoxyethylene glycol, PEG); Ethoxylized nonylphenol with 16 to 50 ethylene oxide units; Ethoxylized fatty alcohol, wherein alcohol contains 12 to 20 carbon atoms, and has 15 to 80 ethylene oxide units; Fatty Alcohol(C12-C14 and C12-C18); Lipid acid; And lipid acid single, two and Witepsol W-S 55.The film that is suitable for using by fluidization forms capsulating material to be seen described in the GB 1483591.The liquid enzymes goods can be stablized by adding polyvalent alcohol (as propylene glycol), sugar or sugar alcohol, lactic acid or boric acid according to prior art.Can prepare shielded enzyme according to disclosed method among the EP 238,216.

Detergent composition of the present invention can be any form easily, for example bar shaped, tablet, pulvis, particle, paste or liquid.Liquid washing agent can be a water-based, generally contains at the most 70% water and the organic solvent of 0-30%, or nonaqueous.

Detergent composition contains one or more tensio-active agents, described tensio-active agent can be non-ionic type comprise semi-polarity and/or anionic and/or cationic and/or amphoteric ionic surface active agent.These tensio-active agents generally exist with the level of 0.1% to 60% (weight).

When being included in wherein, washing composition contains about 1% to about 40% aniorfic surfactant usually, for example linear alkylbenzene sulfonate, α-Xi Jihuangsuanyan, alkyl-sulphate (aliphatic alcohol sulfate), alcohol ethoxy vitriol, secondary alkyl sulfonate, alpha-sulfo-fatty acid methyl ester, alkyl-or alkenyl succinic acid or soap.

When being included in wherein; washing composition contains about 0.2% to about 40% nonionogenic tenside usually; such as fatty alcohol ethoxylate, nonyl phenol ethoxylate, alkyl polyglucoside, alkyl dimethyl amine oxide, ethoxylated fatty acid single ethanol amide, fatty monoethanol amide, poly-hydroxyalkyl fatty acid amide, or the N-acyl group N-alkyl derivative of glucosamine (" glucamide ").

This washing composition can contain for example zeolite of the washing auxiliary detergent of 0-65% or coordination agent, diphosphate, triphosphate, phosphonic acid ester, carbonate, Citrate trianion, nitrilotriacetic acid(NTA), ethylenediamine tetraacetic acid (EDTA), diethylene triaminepentaacetic acid(DTPA), alkyl-or alkenyl succinic, soluble silicate or stratified silicate (for example from Hoechst SKS-6).

Washing composition can comprise one or more polymkeric substance.Example has carboxymethyl cellulose, poly-(vinyl pyrrolidone), poly-(ethylene glycol), poly-(vinyl alcohol), poly-(vinyl pyridine-N-oxide compound), poly-(vinyl imidazole), polycarboxylate be polyacrylic ester, toxilic acid/acrylic copolymer and 12-alkyl methyl vinylformic acid/acrylic copolymer for example.

Washing composition can contain bleach system, and it can comprise H ₂O ₂Source material, for example perborate or percarbonate, these materials again can with the bleach activator that forms peracid for example tetraacetyl ethylene diamine or nonanoyl hydroxy benzenesulfonic acid salt binding.Perhaps, bleach system can contain for example peroxy acid of acid amides, imide or sulfone class.

Enzyme in the detergent composition of the present invention can be stablized by using conventional stablizer; for example polyvalent alcohol (as propylene glycol or glycerol), sugar or sugar alcohol, lactic acid, boric acid or boric acid derivatives (as fragrant boric acid ester) or phenyl-boron dihydroxide derivative (as 4-formyl radical phenyl-boron dihydroxide), and said composition can be by for example WO 92/19709 and WO 92/19708 described preparation.

Washing composition can also contain other conventional decontamination composition, fabric conditioner for example, it comprises clay, foaming synergistic agent, froth suppressor, corrosion inhibitor, soil-suspending agent, anti-soil dirt deposition agent, dyestuff, sterilant, optical white, solubilizing agent, tarnish inhibitor or spices again.

Conceived at present and can in detergent composition, add any enzyme, especially enzyme of the present invention, add-on is to add the zymoprotein that is equivalent to 0.01-100mg in every liter of washings, the zymoprotein that adds 0.1-1mg in the zymoprotein that adds 0.05-5mg in preferred every liter of washings, the especially every liter of washings.

In addition, enzyme of the present invention can be mixed among the WO 97/07202 in the disclosed detergent body, be hereby incorporated by.

Application in leather industry

Subtilase enzymes of the present invention can be used for leather industry, in particular for the depilation of skin.

In this was used, subtilase variant of the present invention preferably used in the enzyme composition that also contains another kind of proteolytic enzyme.

Consult part (seeing above) about the more detailed description of other suitable proteolytic enzyme about being applicable to the enzyme in the detergent composition.

Application in woolen industry

Subtilase enzymes of the present invention can be used for woolen industry, in particular for containing the washing of sweater thing.

In this was used, subtilase variant of the present invention preferably used in the enzyme composition that also contains another kind of proteolytic enzyme.

Consult part (seeing above) about the more detailed description of other suitable proteolytic enzyme about being applicable to the enzyme in the detergent composition.

The present invention has more detailed description in the following example, these embodiment also are intended to the scope that restriction the present invention requires never in any form.

Material and method

Bacterial strain:

Subtilis DN1885 (people such as Diderichsen, 1990).

Bacillus lentus 309 and 147 is special bacterial strains of bacillus lentus, is preserved in NCIB, and numbering NCIB 10309 and 10147 and is described in U.S. Patent number 3,723, and in 250, this patent documentation this paper quotes as a reference.

Intestinal bacteria MC1000 (M.J.Casadaban and S.N.Cohen (1980); Molecular biology magazine 138 179-207), make r-with ordinary method, m+, it also is described in the Application No. 039,298.

Plasmid

PJS3: intestinal bacteria-subtilis shuttle vectors, contain the synthetic gene (being described in " protein and peptide communication " (Protein and Peptide letters) 3:39-44 (1996) by people such as Jacob Schiodt) of the subtilase enzymes 309 of encoding.

PSX222: subtilis expression vector (being described in WO 96/34946).

Molecular biology method commonly used

Unless otherwise indicated, DNA operation and transform with molecular biological standard method and carry out (people (1989) molecular cloning such as Sambrook: laboratory manual, cold spring harbor laboratory, cold spring port, New York; People such as E.M.Ausubel (chief editor) " molecular biology universal method " (CurrentProtocols in Molecular Biology), John Wiley and Sons, 1995; C.R.Harwood and S.M.Cutting (chief editor) " molecular biology method of genus bacillus " (Molecular Biological Methods for Bacillus), John Wiley and Sons, 1990).

Be used for of the specification sheets use of the enzyme of DNA operation according to supplier.

The enzyme that is used for the DNA operation

Unless otherwise indicated, the enzyme of the DNA that is useful on operation, as restriction enzyme, ligase enzyme or the like, all available from New England Biolabs, Inc..

Proteolytic activity

Proteolytic activity is with " thousand promise protein unit of enzyme " (Kilo NOVOProtease Units, KNPU) expression in the present invention.Activity is with respect to standard enzyme (SAVINASE ^) measure, and to measure be to be based upon on the basis of (promptly 50 ℃, pH8.3,9 minutes reaction times, Measuring Time 3 minutes) proteolytic enzyme digest dimethyl casein (DMC) solution under the standard conditions.Can ask for file AF 220/1 to Denmark Novo Nordisk A/S, this document is quoted as a reference herein.

GU is a glycine unit, is defined as with N-acetyl casein as substrate, under standard conditions, in the time of 15 minutes, produces the hydrolase of proteolysis of the amino amount that is equivalent to 1 mmole glycine in 40 ℃ of insulations.

Enzymic activity also can be tested with PNA, (be described in american petroleum chemistry association's magazine (Journal of American Oil Chemists Society) according to response measurement with soluble substrate succinyl--alanyl-alanyl-prolyl-phenylalanyl-p-NP, T.M.Rothgeb, B.D.Goodlander, P.H.Garrison and L.A.Smith, (1988)).

Fermentation

The fermentation of subtilase enzymes in 30 ℃ on rotary shaker (300r.p.m.) contain in the 500ml band baffle plate Erlenmeyer Erlenmeyer flask of 100ml BPX substratum and carried out 5 days.

Therefore, in order to obtain for example 2 liters of nutrient solutions, need 20 Erlenmeyer Erlenmeyer flasks to ferment simultaneously.

Substratum

BPX: form (every liter)

Potato starch 100g

The barley 50g that pulverizes

Soyflour 20g

Na ₂HPO ₄·12H ₂O 9g

Pluronic 0.1g

Sodium-caseinate 10g

Starch α-Dian Fenmeiyehua in the substratum, substratum by be heated to 120 ℃ and keep 45 minutes the sterilization.The sterilization back adds NaHCO ₃The pH of substratum is transferred to 9 to 0.1M.

Embodiment 1

The structure of enzyme variants and expression:

Site-directed mutagenesis:

Subtilase enzymes 309 site-directed mutagenesis mutant comprise the specific insertion between 100 and 101 on the avtive spot ring (b), it is (people (1989) molecular cloning such as Sambrook: laboratory manual that tradition clone by dna fragmentation obtains, cold spring harbor laboratory, the cold spring port), described dna fragmentation is produced by the PCR of the oligonucleotide (as follows) of the insertion fragment that contains expectation.

Template plasmid DNA is pJS3, or this plasmid contains the analogue of subtilase enzymes 309.

Instruct the segmental importing of insertion of sudden change to insert variant (arbitrary amino acid residue that X=inserts) between 100 and 101 with one section oligonucleotide, produce G100GX subtilase enzymes 309 variants to make up G100GX.

With subtilase enzymes 309 variant transformed into escherichia coli, use the DNA of purifying from the overnight culture of these transformants to transform subtilis, method is the conversion with purifying, connection and the subtilis of restriction enzyme digestion, dna fragmentation.People such as conversion of subtilis such as Dubnau, 1971, the molecular biology magazine is described in the 56:209-211.Localized random mutagenesis is determined in the zone will insert at random to import:

The complete strategy that carries out localized random mutagenesis is:

Synthetic corresponding to the mutagenic primer (oligonucleotide) in the dna sequence dna that inserts the site flank, this primer inserts segmental DNA base pair by definition and separates.

Then, the mutagenic primer that obtains and suitable subtend primer one are used from the PCR reaction.With the PCR fragment purification and the digestion that obtain and be cloned in intestinal bacteria-subtilis shuttle vectors (as follows).

Perhaps, if necessary, the PCR fragment that obtains is used from second PCR reaction as primer and second suitable subtend primer one, makes it possible to digest the mutagenesis district and it is cloned in the shuttle vectors.PCR is reflected under the normal condition and carries out.

Follow this strategy, make up the localization random library of SAVINASE, wherein import in the avtive spot ring district between the 100-101 position and insert.

Import sudden change by mutagenic primer (seeing below), represented all 20 seed amino acids (A of N=25%, T, C and G; And S=50%C and G.By the another PCR of wheel the PCR fragment that is produced is extended to the N of Savinase end, method is to utilize following primer, with have the PCR fragment that produces by pcr amplification and the combination of overlap: 5 ' CTA AATATT CGT GGT GGC GC 3 ' (justice arranged) and 5GAC TTT AAC AGC GTATAG CTC AGC 3 ' (antisense).The dna fragmentation that extends is cloned into HindIII and MluI site (on the seeing) of modified plasmid pJS3,10 intestinal bacteria bacterium colonies selecting is at random checked order, to confirm to contain designed sudden change.

Mutagenic primer (5 ' GTT AAA GTC CTA GGG GCG AGC GGT NNSTCA GGT TCG GTC AGC TCG ATT G3 ' (justice is arranged)) and the suitable subtend primer (as 5 '-CCC TTT AAC CGC ACA GCGTTT-3 ' (antisense)) that is arranged in downstream, pJS3 MluI site are used from the PCR reaction, and plasmid pJS3 is as template.With restriction enzyme HindIII and MluI with gained PCR product cloning in the pJS3 shuttle vectors.

With known method random library is transformed into intestinal bacteria then.

So about 100,000 mono-clonal/libraries are contained in the library of preparation.

10 sudden changes of bacterium colony order-checking to picked at random to confirm to design.

For purifying subtilase variant of the present invention, the subtilis pJS3 expression plasmid that will contain variant of the present invention is transformed in the competence bacillus subtilis strain, and as indicated abovely ferments in the substratum that contains 10 μ g/ml paraxin (CAM).

Embodiment 2

The purifying of enzyme variants:

This step relates to the purifying of the flat fermentation of 2 premium on currency, is used for producing subtilase enzymes at the subtilis host cell.

With about 1.6 liters of fermented liquids in 1 liter of Centrifuge Cup centrifugal 35 minutes in 5000rpm.With 10% acetic acid supernatant liquor is transferred to pH6.5, and on Seitz Supra S100 screen plate, filter.

The Amicon CH2A UF device that use has been equipped with Amicon S1Y10 UF cylinder is concentrated into about 400ml with filtrate.(room temperature, pH7) before the absorption, elder generation is centrifugal and filtration with the UF concentrated solution at last Bacitracin affinity column.Proteolytic enzyme is eluted from the Bacitracin post in room temperature with the 0.01M dimethylated pentanedioic acid that contains 25%2-propyl alcohol and 1M sodium-chlor, 0.1M boric acid and 0.002M calcium chloride damping fluid (pH7).

To have the fraction merging of protease activity from the Bacitracin purification step, and be added on the 750ml Sephadex G25 post (diameter 5cm, with containing 0.01M dimethylated pentanedioic acid, 0.2M boric acid and 0.002M calcium chloride, the damping fluid balance of pH6.5 is crossed).

To have the fraction merging of proteolytic activity from Sephadex G25 post, and be added to 150ml CM Sepharose CL 6B cationic exchange coloum (diameter 5cm, with containing 0.01M dimethylated pentanedioic acid, 0.2M boric acid and 0.002M calcium chloride, the damping fluid balance of pH6.5 is crossed) on.

With the linear gradient 0-0.1M sodium-chlor in 2 liters of same buffer (using 0-0.2M sodium-chlor under the situation of subtilisin 147) proteolytic enzyme is eluted.

In last purification step, will contain the fraction merging of proteolytic enzyme from CM Sepharose post, and in the Amicon ultrafiltration chamber that has been equipped with GR81PP film (available from Danish Sugar Factories Inc.), concentrate.

By technology and the above-mentioned separation method that uses embodiment 1 to be used to make up, production and separation obtain following subtilisin 309 variants:

G100GT

G100GA

G100GS

G100GD

G100GE

G100GP

G100GG

G100GH

G100GI

G100GT+Y167A

S99G+G100GT+S101T

A98G+G100GA+S101A+S103T

These variants show the scourability that is better than Savinase in initial analysis.

Embodiment 3

The scourability that contains the detergent composition of enzyme variants:

The following example provides the result of some washing tests that carry out under indicated condition.

Experiment condition

Table III: the experiment condition of estimating subtilisin 309 variants

Washing composition	Europe	Washing composition 95	US
				Detergent doses	4g/l	3.0g/l	1g/l
Wash temperature
		30℃	15℃	25℃
Washing time					30min	15min	10min
	The water hardness	18°dH(Ca 2+/Mg 2+ ＝5∶1)	6°dH	6°dH(Ca 2+/Mg 2+ ＝2∶1)
pH					Do not transfer	10.5	Do not transfer
	Enzyme concn	1，2，5，10， 30nM		1，2，5，10，30nM
Test macro					The 150ml glass beaker that has splash bar	10nm	The 150ml glass beaker that has splash bar
	Textiles/volume	5 textiless (φ 2.5cm) are in the 50ml washing composition	5 textiless (φ 2.5cm) are in the 50ml washing composition	5 textiless (φ 2.5cm) are in the 50ml washing composition
Test material					EMPA116	EMPAll7	EMPAll7

Washing composition

The washing composition or the pattern washing composition that use are called washing composition 95, perhaps respectively available from the supermarket (OMO, catalogue ED-9745105) and U.S. (Wisk, catalogue ED-9711893) of Denmark.Enzymic activity before using in all washing composition is all used the microwave treatment deactivation.

Washing composition 95 is simple pattern preparations.PH transfers to 10.5, and this is in the normal range of powder-type washing composition.95 type washing composition composed as follows:

STP(Na ₅P ₃O ₁₀) 25％

Na ₂SO ₄ 25％

Na ₂CO ₃ 10％

LAS(Nansa 80S) 20％

Nonionic surface active agent (Dobanol 25-7) 5.0%

Na ₂Si ₂O ₅ 5.0％

Carboxymethyl cellulose (CMC) 0.5%

Water 9.5%

Sample

The sample that uses is EMPA116 and EMPA117, and available from EMPA Testmareialen, Movenstrasse 12, CH-9015, St.Gall, Switzerland.

Reference

Use the reflectivity (R) of Macbeth ColorEye 7000 type photometer measurement test materials at 460nm.Measurement is carried out according to supplier's specification sheets.

Assessment

The improvement factor or the performance factor evaluation of the subtilase enzymes of the detergency the subject of knowledge and the object of knowledge research of subtilase enzymes:

Improvement factor IF _{Dosage/reaction}Be defined as the washing composition that contains the subtilase enzymes that remains to be studied, level off to the ratio of scourability slope of a curve of zero identical washing composition with reference to subtilase enzymes content with described.

IF _{Dosage/reaction}=a/a _Reference

Calculate scourability according to formula I:

$R=R_0+\frac{\mathrm{a\Δ}{R}_{\max }\·c}{\mathrm{\Δ}{R}_{\max }+a\·c}...\left(I\right);$

Wherein

R is the scourability of representing with reflectance unit; R ₀Be the intercept (blank) of matched curve to Y-axis; A levels off to the slope of matched curve 0 time as c, and c is an enzyme concn; And Δ R _MAXIt is the theoretical maximum detersive efficiency when c trends towards infinity.

Performance factor P calculates according to formula II:

P=(R _Variant-R _Blank)/(R _Savinase-R _Blank) (II)

Wherein

R _Variant: with the reflectivity of 10nM variant washing back test material; R _Savinase: use 10nMSavinase ^The reflectivity of washing back test material; R _Blank: without the reflectivity of enzyme washing back test material.

US (washing composition: US Wisk, Swatch:EMPA117)

Variant	P
		G100GA
	1，2
		S99G+G100GGT+S101T	1，6

● P calculates at [E]=5nM

This shows subtilase enzymes of the present invention and SAVINASE ^Compare aspect scourability and make moderate progress.

Sequence table

<110>Novo NordiskA/S

＜120〉in avtive spot ring district, have the I-S1 and the I-S2 of additional amino acid residue

The subtilase enzymes of subgroup

<130>5795.204

<140>

<141>

<160>2

<170>PatentIn Ver.2.1

<210>1

<211>275

<212>PRT

＜213〉Bacillus licheniformis

<400>1

Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val

1 5 10 15

Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Gln Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala

35 40 45

Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly

50 55 60

Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val

65 70 75 80

Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn

85 90 95

Ser Ser Gly Xaa Ser Gly Thr Tyr Ser Gly Ile Val Ser Gly Ile Glu

100 105 110

Trp Ala Thr Thr Asn Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly

115 120 125

Pro Ser Gly Ser Thr Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala

130 135 140

Arg Gly Val Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly

145 150 155 160

Asn Thr Asn Thr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala

165 170 175

Val Gly Ala Val Asp Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val

180 185 190

Gly Ala Glu Leu Glu Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr

195 200 205

Tyr Pro Thr Ser Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser

210 215 220

Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn

225 230 235 240

Leu Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr

245 250 255

Leu Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala

260 265 270

Ala Ala Gln

275

<210>2

<211>270

<212>PRT

＜213〉bacillus lentus

<400>2

Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala

1 5 10 15

His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

50 55 60

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

65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

85 90 95

Ser Gly Xaa Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp

100 105 110

Ala Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro

115 120 125

Ser Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg

130 135 140

Gly Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile

145 150 155 160

Ser Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp

165 170 175

Gln Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp

180 185 190

Ile Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr

195 200 205

Tyr Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly

210 215 220

Ala Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln

225 230 235 240

Ile Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn

245 250 255

Leu Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265 270

Claims (18)

1. the isolating subtilase enzymes of I-S1 and I-S2 subgroup, it has at least one extra amino-acid residue at 100 from avtive spot ring (b) district of 95-103 position, described thus extra amino-acid residue is corresponding to the insertion of at least one amino-acid residue between the 100-101 position, described insertion is selected from G100GT, G100GA, G100GS, G100GD, G100GE, G100GP, G100GG, G100GH and G100GI, and wherein said subtilase enzymes is selected from:

A) a kind of subtilase enzymes that belongs to the I-S1 subgroup, aminoacid sequence is composed as follows:

1 10 20 30

A-Q-T-V-P-Y-G-I-P-L-I-K-A-D-K-V-Q-A-Q-G-F-K-G-A-N-V-K-V-A-V

40 50 60

L-D-T-G-I-Q-A-S-H-P-D-L-N-V-V-G-G-A-S-F-V-A-G-E-A-*-Y-N-T-D

70 80 90

G-N-G-H-G-T-H-V-A-G-T-V-A-A-L-D-N-T-T-G-V-L-G-V-A-P-S-V-S-L

100a 110 120

Y-A-V-K-V-L-N-S-S-G-X-S-G-T-Y-S-G-I-V-S-G-I-E-W-A-T-T-N-G-M-D

130 140 150

V-I-N-M-S-L-G-G-P-S-G-S-T-A-M-K-Q-A-V-D-N-A-Y-A-R-G-V-V-V-V

160 170 180

A-A-A-G-N-S-G-S-S-G-N-T-N-T-I-G-Y-P-A-K-Y-D-S-V-I-A-V-G-A-V

190 200 210

D-S-N-S-N-R-A-S-F-S-S-V-G-A-E-L-E-V-M-A-P-G-A-G-V-Y-S-T-Y-P

220 230 240

T-S-T-Y-A-T-L-N-G-T-S-M-A-S-P-H-V-A-G-A-A-A-L-I-L-S-K-H-P-N

250 260 270

L-S-A-S-Q-V-R-N-R-L-S-S-T-A-T-Y-L-G-S-S-F-Y-Y-G-K-G-L-I-N-V

275

E-A-A-A-Q

Or

B) a kind of subtilase enzymes that belongs to the I-S2 subgroup, aminoacid sequence is composed as follows:

.1 10 20 30

.A-Q-S-V-P-W-G-I-S-R-V-Q-A-P-A-A-H-N-R-G-L-T-G-S-G-V-K-V-A-V-

. 40 50 60

.L-D-T-G-I-*-S-T-H-P-D-L-N-I-R-G-G-A-S-F-V-P-G-E-P-*-S-T-Q-D-

. 70 80 90

.G-N-G-H-G-T-H-V-A-G-T-I-A-A-L-N-N-S-I-G-V-L-G-V-A-P-S-A-E-L-

. 100a 110 120

.Y-A-V-K-V-L-G-A-S-G-X-S-G-S-V-S-S-I-A-Q-G-L-E-W-A-G-N-N-G-M-H-

. 130 140 150

.V-A-N-L-S-L-G-S-P-S-P-S-A-T-L-E-Q-A-V-N-S-A-T-S-R-G-V-L-V-V-

. 160 170 180

.A-A-S-G-N-S-G-A-*-G-S-I-S-*-*-*-Y-P-A-R-Y-A-N-A-M-A-V-G-A-T-

. 190 200 210

.D-Q-N-N-N-R-A-S-F-S-Q-Y-G-A-G-L-D-I-V-A-P-G-V-N-V-Q-S-T-Y-P-

. 220 230 240

.G-S-T-Y-A-S-L-N-G-T-S-M-A-T-P-H-V-A-G-A-A-A-L-V-K-Q-K-N-P-S-

. 250 260 270

.W-S-N-V-Q-I-R-N-H-L-K-N-T-A-T-S-L-G-S-T-N-L-Y-G-S-G-L-V-N-A-

. 275

.E-A-A-T-R。

2. according to the isolating subtilase enzymes of claim 1, wherein said 100 with 101 between insertion be selected from following one or more modifications and combine:

K27R, * 36D, S57P, N76D, S87N, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, Q206E, N218S, M222S, M222A, T224S, K235L, T274A, P219K, P131H, A133P, A133D and A194P.

3. according to the subtilase enzymes of claim 2, wherein said modification is selected from S101G+V104N, S87N+S101G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V101I or N76D+V104A.

4. according to the subtilase enzymes of claim 1, it comprises following modification: A98G+G100GA+S101A+S103T or S99G+G100GGT+S101T.

5. the separated DNA sequence of the subtilase enzymes of the claim 1 of encoding.

6. expression vector that contains the separated DNA sequence of claim 5.

7. microbial host cell that has transformed the expression vector of claim 6.

8. according to the microorganism host of claim 7, it is a bacterium.

9. microorganism host according to Claim 8, it is a bacillus.

10. according to the microorganism host of claim 9, it is such as bacillus lentus.

11. according to the microorganism host of claim 7, it is a fungi.

12. according to the microorganism host of claim 7, it is a yeast.

13. according to the microorganism host of claim 11, it is an Aspergillus.

14. the method for the subtilase enzymes of production claim 1 is wherein helping each host among the claim 7-13 to express and is secreting under the condition of described subtilase enzymes and cultivate, and reclaiming subtilase enzymes.

15. composition that contains the subtilase enzymes of claim 1.

16., wherein also contain cellulase, lipase, at, oxydo-reductase or amylase according to the composition of claim 15.

17. according to the composition of claim 15 or 16, wherein said composition is a detergent composition.

18. the enzyme composition of the subtilase enzymes of claim 1 or claim 15 or 16 is in laundry and/or wash purposes in the dish washing composition.

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