KR100660799B1 - Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region - Google Patents

Abstract

To subgroups of subgroups I-S1 and I-S2 having an additional amino acid residue at position 100 of the active site loop (b) region of positions 95-103. Variant subtilases show improved washing performance in detergents compared to parent enzymes.

Subtilases, I-S1, I-S2, variants, subtilisin

Description

SUBTILASE ENZYMES OF THE I-S1 AND I-S2 SUB-GROUPS HAVING AN ADDITIONAL AMINO ACID RESIDUE IN AN ACTIVE SITE LOOP REGION}

The present invention relates to novel subtilase enzymes of subgroups I-S1 and I-S2 having at least one additional amino acid residue in positions 100 of positions 95 to 103, the active site loop (b) region. This protease is a detergent; It is useful for exhibiting excellent or improved washing performance when used in laundry and detergent compositions. The present invention is a gene encoding the expression of the enzyme when inserted into a suitable host cell or living body; It is further related to methods of producing such host cells and novel enzymes which are transformed into them and are capable of expressing said enzyme variants.

Detergent industry enzymes have been used in washing formulations for over 30 years. Enzymes used in such preparations include proteases, lipases, amylases, cellulases, and other enzymes or mixtures thereof. The most important enzyme commercially is protease.

(Novo Nordisk A/S), RELASE

(Novo Nordisk A/S), MAXAPEM

(Gist-Brocades N.V.), PURAFECT

(Genencor International, Inc.)같은 변이체이다. Increasing numbers of commercially available proteases include DURAZYM, in which naturally occurring wild type proteases are protein engineered.

(Novo Nordisk A / S), RELASE

(Novo Nordisk A / S), MAXAPEM

(Gist-Brocades NV), PURAFECT

(Genencor International, Inc.).

In addition, many proteases include EP 130756 (corresponding to GENENTECH (US Reissue Patent 34,606 (GENENCOR))); EP 2154435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050 (GENEX); EP 260105 (GENENCOR); Thomas, Russell, and Fersht (1985) Nature 318 375-376; Thomas, Russell, and Fersht (1987) J. Mol. Biol. 193 803-813; Russel and Fersht Nature 328 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (Amgen); WO 95/27049 (SOLVAY S. A.); WO 95/30011 (PROCTER & GAMBLE COMPANY); WO 95/30010 (PROCTER & GAMBLE COMPANY); WO 95/29979 (PROCTER & GAMBLE COMPANY); US 5.543.302 (SOLVAY S. 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 WO 94/02618 (GIST-BROCADES N. V.).

However, while many useful protease variants have been described, there is still a need for new improved protease or protease variants for many industrial uses.

It is therefore an object of the present invention to provide for improved protease or protein engineered protease variants, particularly for use in the detergent industry.

Summary of the Invention

) 를 제조하는 중 이루어졌다. 이는 우리의 선행 출원인 DK1332/97에 기술되어 있다. The inventors have found that subtilisin, at least one active site loop longer than currently known, exhibits improved washability properties in the detergent composition. Its identification shows that subtilisin 309 (BLSAVI or Savinase) exhibits improved washability properties in detergent compositions compared to subtilisin variants, particularly parental wild type enzymes.

) During the manufacture. This is described in our prior application, DK1332 / 97.

Subgroups I-S1 (true "subtilisin") and I-S2 having at least one additional amino acid residue in positions 100 (ie between positions 100 and 101) of the active site loop (b) region 95-103; It is presently found that certain subtilases (or highly alkaline subtilisin) or variants thereof exhibit surprisingly improved washing performance compared to those currently known and those described in the above application.

An improved protease according to the invention is obtained by isolation of natural origin or by introducing at least one further amino acid residue in the active site loop (b) between positions 100 and 101 in wild-type subtilisin (active site definition and position See below for numbering).

Although this discovery has been made in subtilisin 309, it is expected that it will be possible to produce or isolate subtilase or subtilase variants with similar advantages.

Moreover, the subtilases contain longer active site loops than the active site loops in the corresponding known wild type subtilases, such as subtilisin 309 which can be considered to have amino acid residues inserted between positions 100 and 101. In particular, it would be possible to specifically screen natural isolates to identify novel wild-type subtilases that exhibit excellent washing performance in detergents as compared to its most closely related known subtilisin, such as subtilisin 309.

Related alignments and numbering criteria have been produced for FIGS. 1, 1a, 2, and 2a below to provide an alignment between subtilisin BPN '(BASBPN) (a) and subtilisin 309 (BLSAVI) (b). The alignment between subtilisin BPN '(a) (BASBPN) and subtilisin Carlsberg (g) is shown. 1A and 2A are the same as those shown in WO 91/00345, but the alignment in FIGS. 1 and 2 was completed using the GAP routine of the GCG package as described below. These alignments are used herein as a basis for numbering residues.

The seven active site loops (a) to (g) (including both terminal amino acids indicated) are fragments given below

(a) a region between amino acid residues 33 and 43;

(b) a region between amino acid residues 95 and 103;

(c) a region between amino acid residues 125 and 132;

(d) a region between amino acid residues 153 and 173;

(e) the region between amino acid residues 181 and 195;

(f) the region between amino acid residues 202 and 204;

(g) the region between amino acid residues 218 and 219

It is defined herein to encompass amino acid residues within.

Thus, in a first aspect the present invention has at least one additional amino acid residue at position 100 of positions 95 to 103 which is an isolated (i.e. more than 10% pure), active site loop (b) region, thereby The additional amino acid residues are associated with the subtilase enzymes of subgroups I-S1 and I-S2 corresponding to the insertion of at least one amino acid residue between positions 100 and 101.

In a second aspect the present invention relates to an isolated DNA sequence encoding a subtilase variant of the present invention.

In a third aspect the present invention relates to an expression vector comprising an isolated DNA sequence encoding a subtilase variant of the present invention.

In a fourth aspect the invention relates to a microbial host cell transformed with an expression vector according to the third aspect.

In a further aspect the present invention relates to the production of the subtilisin enzymes of the present invention.

The enzymes of the present invention can be used to culture microbial strains that will separate the enzymes therefrom and to recover the enzymes in significantly pure form; The expression vector according to the fourth aspect can be generally produced by inserting into an appropriate microbial host, culturing the host to express the desired subtilase enzyme, and recovering the enzyme product.

Furthermore, the present invention relates to compositions comprising the subtilase or subtilase variants of the present invention.

The invention further relates to the use of the enzymes of the invention in a number of industrially suitable uses, in particular in laundry compositions comprising laundry compositions and mutant enzymes, in particular in detergent compositions comprising mutant subtilisin enzymes.

Justice

Before describing the invention in more detail, the following terms and conventions will first be defined.

Amino acid naming

A = Ala = Alanine

V = Val = Valine

L = Leu = Leucine

I = Ile = Iroleucine

P = Pro = Proline

F = Phe = Phenylalanine

W = Trp = Tryptophan

M = Met = Methionine

G = Gly = Glycine

S = Ser = Serine

T = Thr = threonine

C = Cys = Cysteine

Y = Tyr = Tyrosine

N = Asn = Asparagine

Q = Gln = Glutamine

D = Asp = Aspartic Acid

E = Glu = glutamic acid

K = Lys = Lysine

R = Arg = Arginine

H = His = histidine

X = Xaa = any amino acid

Naming of Nucleic Acids

A = adenine

G = guanine

C = cytosine

T = Thymine (DNA only)

U = uracil (RNA only)

Naming and conventions for variant markings

In describing various enzyme variants produced or contemplated in accordance with the present invention, the following nomenclature and conventions apply for convenience of reference:

The backbone of the reference was initially defined by aligning the isolated parental wild type enzyme against subtilisin BPN '(BASBPN).

The alignment can be obtained by the GAP routines in version 9.1 of the GCG package and numbered variants using the following parameters: gap creation penalty = 8 and gap extension penalty = 8 and all other parameters change their default values. Keep it.

Another method is to use a known recognized alignment between subtilases, such as the alignment described in WO 91/00345. In most cases, the difference won't matter at all.

This alignment between subtilisin BPN '(BASBPN), subtilisin 309 (BLSAVI) and subtilisin Carlsberg (BLSCAR) is shown in Figures 1, 1A, 2 and 2A, respectively. This will define a number of deletions and insertions in relation to BASBPN. In FIG. 1, subtilisin 309 has six deletions at positions 36, 58, 158, 162, 163 and 164 compared to BASBPN, and in FIG. 1A subtilisin 309 has the same deletions compared to BASBPN at positions 36, 56, 159, 164, 165, and 166. In FIG. 2, subtilisin Carlsberg has one deletion at position 56 compared to BASBPN. These deletions in FIGS. 1, 1A, 2 and 2A are shown by an asterisk (*).

Various modifications performed on wild-type enzymes are generally represented using three factors:

Original amino acid position of substituted amino acid

The designation G195E thus means that glycine at position 195 is substituted with glutamic acid.

Where the original amino acid residue may be any amino acid residue, an abbreviation indicating only the position and the substituted amino acid may sometimes be used.

The position of the substituted amino acid

Such indications are particularly suitable in connection with the modification (s) in homologous subtilases (see below).

The identity of substituting amino acid residues is similar, even if not critical.

Original amino acid position

If both the original amino acid and the substituted amino acid include all amino acids, only the position is indicated, for example: 170.

If the original amino acid and / or substituted amino acid contains one or more amino acids but not all amino acids, the selected amino acid is indicated in parentheses {}.

Original amino acid position {substituted amino acid _One ,. . . Substituted amino acid _n }

For specific variants, specific three letter or single letter codes are used, including the codes Xaa and X to indicate any amino acid residue.

substitution:

If glycine is substituted for glutamic acid at position 195:

Is displayed as Glyl95Glu or G195E,

If glycine at position 195 is substituted with any amino acid residue:

Glyl95Xaa or G195X or Glyl95 or G195.

If any amino acid at position 170 is substituted with serine:

Displayed as Xaal70Ser or X170S or 170Ser or 170S.

Such indications are particularly suitable with regard to modifications in homologous subtilases (see below). 170Ser is thus meant to include both modified Lys170Ser in BASBPN and modified Arg170Ser in BLSAVI, for example (see FIG. 1).

For modifications in which the original amino acid and / or substituted amino acid is substituted for more than one but not all of the amino acids, the substitution of arginine for glycine, alanine serine or threonine at position 170

It will be designated Arg170 {Gly, Ala, Ser, Thr} or R170 {G, A, S, T} to refer to variants R170G, R170A, R170S, and R170T.

fruition:

The deletion of glycine at position 195 is indicated by Glyl95 * or G195 *.

Similarly, deletions of more than one amino acid residue, such as deletions of glycine and leucine at positions 195 and 196, would be represented by Glyl95 * + Leul96 * or G195 * + L196 *.

insertion:

Insertion of additional amino acids, for example G195 followed by lysine, is: Glyl95GlyLys or G195GK;

If more than one amino acid residue is inserted, for example, the insertion of Lys, Ala and Ser after G195 is: Glyl95GlyLysAlaSer or G195GKAS.

In such a case, the inserted amino acids are numbered by the addition of a lowercase alphabetic character to the amino acid residue position number preceding the inserted amino acid residue. In this example, SEQ ID NOs: 194-196 are thus:

194 195 196

BLSAVI A-G-L

194 195 195a 195b 195c 196

Variants A-G-K-A-S-L

Will be.

If amino acid residues identical to existing amino acid residues are inserted, it is evident that there is a lot of nomenclature. If, for example, glycine is inserted after glycine in the above example, it will be represented as G195GG.

194 195 196

BLSAVI A-G-L

from

194 195 195a 196

Variation A-G-G-L

194 194a 195 196

For changes in the furnace, the same actual change can only be indicated as A194AG. Such an example will be apparent to the skilled person, and the indication G195GG and its corresponding meaning for this type of insertion thus means including this equivalent multiplicity.

Filling the gap:

When there is an in-base deletion compared to the subtilisin BPN 'sequence used for numbering, it is expressed as * 36Asp or * 36D for aspartic acid insertion at position 36, which is the insertion at that position.

Multiple variants

Variants containing multiple variants are separated by a plus sign, for example:

Arg170Tyr + Gly195Glu or R170Y + G195E indicate that arginine and glycine were substituted with tyrosine and glutamine at positions 170 and 195, respectively.

Or for example Tyrl67 {Gly, Ala, Ser, Thr} + Argl70 {Gly, Ala, Ser, Thr} is a variant Tyr167Gly + Arg170Gly, Tyr167Gly + Arg170Ala, Tyr167Gly + Arg170Ser, Tyr167Gly + Arg170Thr, Tyr167Ala + Arg170Ala, Ayr170Ala Tyrl67Ala + Argl70Ser, Tyrl67Ala + Argl7OThr, Tyrl67Ser + Argl70Gly, Tyrl67Ser + Argl70Ala, Tyrl67Ser + Argl70Ser, Tyrl67Thr + Argl70Gly, Tyrl67hr + Arrl70Tla, Tyrl67Tla, Tyrl67Tla, Tyrl67 Arla

This nomenclature indicates the substitution of a positive charge (K, R, H), negative charge (D, E), or a small amino acid with another small amino acid, for example Tyrl67 {Gly, Ala, Ser, Thr} + Argl70 Conservative Amino Acid Modifications in {Gly, Ala, Ser, Thr} Particularly suitable in connection with modifications aimed at substitution, replacement, insertion or deletion of amino acid residues having certain general characteristics. See "Detailed Description of the Invention" for more details.

Protease

Enzymes that cleave amide bonds of protein substrates are classified as proteases, or (compatible) peptidase (Walsh, 1979, Enzymetic Reaction Mechanisms, W. H. Freeman and Company, San Francisco, Chapter 3).

Numbering amino acid positions / residues

If not stated otherwise, the amino acid numbering used herein corresponds to the numbering of the subtilase BPN '(BASBPN) sequence. Further description of the BPN 'sequence is shown in FIGS. 1 and 2 or Siezen et al., Protein Engng. 4 (1991) 719-737.

Serine Protease

Serine proteases are enzymes that catalyze the hydrolysis of peptide bonds and have essential serine residues at their active sites (White, Hadler and Smith, 1973 "Princeples of Biochemisty," Fifth Edition, McGraw-Hill Book Company, pp. 271- 272).

Bacterial serine proteases range in molecular weight from 20,000 to 45,000 Daltons. It is inhibited by diisopropyl fluorophosphate. It hydrolyzes simple terminal esters and is similar in activity to eukaryotic chymotrypsin, also a serine protease. In a narrower sense, alkaline proteases encompassing subgroups reflect a high optimal pH of pH 9.0 to 11.0 of some serine proteases (see Priest (1997) Bacteriological Rev. 41 711-753 for review).

Subtilases

A subgroup of serine proteases, temporarily called subtilases, are described in Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al., Protein Science 6 (1997) 501-523. It was defined by homology analysis of more than 170 amino acid sequences of the serine protease, previously named subtilisin-like protease. Subtilisin was previously defined as a serine protease, often produced by Gram-positive bacteria or fungi, and is now defined as a subgroup of subtilases according to Siezen et al. A wide variety of subtilases have been identified and the amino acid sequences of many subtilases have been determined. More detailed descriptions of such subtilases and their amino acid sequences are described in Siezen et al. (1997) is incorporated by reference.

Novo Nordisk A/S) 및 서브틸리신 DY (BSSDY)같은 "전통적" 서브틸리신을 포함한다.One subgroup of subtilases, I-S1 or "true subtilisin", is subtilisin 168 (BSS168), subtilisin BPN ', subtilisin Carlsberg (ALCALASE).

Novo Nordisk A / S) and "traditional" subtilisin, such as subtilisin DY (BSSDY).

, Gist-Brocades NV), 서브틸리신 309 (SAVINASE

, NOVO NORDISK A/S), 서브틸리신 147 (BLS147) (ESPERASE

, NOVO NORDISK A/S) 및 알칼리성 에라스타제 YaB (BSEYAB) 같은 효소를 포함한다. Further subgroups of subtilases, I-S2 or hyperalkaline subtilisin, are described in Siezen et al. Known by (above). Subgroup I-S2 protease is described as highly alkaline subtilisin and subtilisin PB92 (BAALKP) (MAXACAL

, Gist-Brocades NV), subtilisin 309 (SAVINASE)

, NOVO NORDISK A / S), subtilisin 147 (BLS147) (ESPERASE

, Enzymes such as NOVO NORDISK A / S) and alkaline Elastase YaB (BSEYAB).

List of abbreviations for subtilases

I-S1

Subtilisin 168, BSS168 (BSSAS (subtilisin amylosacaritis), BSAPRJ (subtilisin J), BSAPRN (subtilisin NAT), BMSAMP (mesenteric copeptidase),

Subtilisin BPN ', BASBPN

Subtilisin DY, BSSDY,

Subtilisin Carlsberg, BLSCAR (BLKERA (keratinase), BLSCA1, BLSCA2, BLSCA3),

BSSPRC, Serine Protease C

BSSPRD, Serine Protease D

I-S2

Subtilisin Sendai, BSAPRS

Subtilisin ALP 1, BSAPRQ,

BLS147 (BSAPRM (SubtilisinAprM), BAH101), Subtilisin 147, Esperase

BLS147 (BSAPRM (SubtilisinAprM), BAH101),

, BLS309/BLSAVI (BSKSMK (M-프로테아제), BAALKP (서브틸리신 PB92, Bacillus alkalophilic 알칼리성 프로테아제), BLSUBL (서브틸리신 BL)), Subtilisin 309, Savinase

, BLS309 / BLSAVI (BSKSMK (M-protease), BAALKP (subtilisin PB92, Bacillus alkalophilic alkaline protease), BLSUBL (subtilisin BL)),

Alkaline erythase YaB, BYSYAB,

""SAVINASE

"

는 NOVO NORDISK A/S에 의하여 판매된다. 이것은 B.leutus 유래 서브틸리신 309이고 BAALKP와 오직 한 위치 (N87S, 여기의 도 1 참조)만 상이하다. SAVINASE

는 도 1에서 b)로 표시된 아미노산 서열을 가진다. SAVINASE

Is sold by NOVO NORDISK A / S. This is B.leutus- derived subtilisin 309 and differs only from BAALKP in one position (N87S, see FIG. 1 here). SAVINASE

Has the amino acid sequence represented by b) in FIG.

Mosobtilase

The term "mostubylase" refers to Siezen et al. Subtilas as defined according to (1991 and 1997). See the description of "Subtillase" directly above for more details. Mosobtilase is also a subtilase isolated from its natural origin and subsequent modifications were carried out while retaining the properties of the subtilisin. Alternatively, the term "mosuttilase" is named "wild-type subtilase".

Modification of Subtilase Variants

The term "modification" as used herein is defined to include the genetic modification of the subtilase as well as the chemical modification of the subtilase. The modification can be replacement, substitution, deletion and / or insertion of the amino acid side chains in or within the amino acid of interest.

Subtilase variants

In the context of the present invention, the term subtylase variant or mutated subtilase refers to a subtilase produced by a living body expressing a mutant gene derived from a parent organism having the original parent gene and producing a corresponding parental enzyme. Meaning, the parent gene was mutated to produce a mutant gene in which said subtilase protease is produced when expressed in a suitable host.

Homologous subtilase sequences

서브틸라제의 루프 내 아미노산 삽입이 동정된다. For modification to obtain the subtilisin variant of the present invention, the specific active site loop region and the SAVINASE

Amino acid insertions in the loop of subtilases are identified.

의 1차구조에 상동인 1차구조를 가지는 다른 모 (야생형) 서브틸라제에도 미친다. 이 문맥에서 두 아미노산 서열 사이의 상동성은 "동일성" 기준에 의하여 기술된다. However, the present invention is not limited to this particular subtilase modification but is SAVINASE.

It also extends to other parent (wild type) subtilases that have a primary structure that is homologous to the primary structure. Homology between two amino acid sequences in this context is described by the "identity" criterion.

To determine the degree of identity between two subtilases, the GAP routines of GCG package version 9.1 can be applied using the same settings (below). The result from this routine is the calculation of "percent identity" between the two sequences in addition to the amino acid alignment.

It is easy for one skilled in the art to identify suitable homologous active site loop regions and corresponding homologous subtilases that can be modified according to the invention based on this technique.

Washing performance

For example, during washing or light surface washing, the ability of an enzyme to catalyze the decomposition of various naturally occurring substrates present on an object to be washed is often referred to as its washing power, washing power, washing power or washing performance. Throughout this application the term washing performance will be used to encompass this feature.

Isolated DNA sequence

The term "isolated", when used in a DNA sequence molecule, indicates that the DNA sequence is isolated in its natural genetic environment and thus is free of other exogenous or undesirable coding sequences and is suitable for use in the production of genetically engineered proteins. Form. Such isolated molecules are isolated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention may include native 5 'and 3' untranslated regions, such as promoters and terminators, although there are no other genes that are commonly linked. Identification of linked regions will be apparent to those of ordinary skill in the art (see, eg, Dynan and Tijan, Nature 316 : 774-78, 1985). The term “isolated DNA sequence” may alternatively be named “clone DNA sequence”.

Isolated protein

When applied to a protein, the term “isolated” refers to a protein that is isolated from its natural environment.

In a preferred form, the isolated protein is significantly free of other proteins, especially homologous proteins (ie "homologous impurities" (see below)).

When determined by SDS-PAGE, the isolated protein is more than 10% pure, preferably more than 20%, more preferably more than 30%. Furthermore, as determined by SDS-PAGE, in high-table form, ie more than 40%, more than 60%, more purely than 80%, more preferably more purely than 95% even more preferably more than 99% It is desirable to provide more pure protein. The term “isolated protein” is alternatively named “purified protein”.

Homologous impurities

The term “homologous impurity” refers to any impurity (eg, a polypeptide other than the polypeptide of the invention) derived from the homologous cell from which the polypeptide of the invention was first obtained.

Obtained from

The term “obtained from,” as used herein in connection with a particular microbial origin, refers to a polynucleotide and / or polypeptide produced by a cell into which a particular origin or gene of origin is derived.

temperament

The term "substrate" as used in connection with a substrate for a protease should be interpreted in its broadest form, including compounds containing at least one peptide bond that is susceptible to hydrolysis by the subtilisin protease.

product

The term "product" as used in connection with a protease enzymatic reaction should be interpreted in the context of the present invention to include the product of the hydrolysis to which the subtila protease is concerned. The product may be the substrate of the subsequent hydrolysis reaction.

(b) 사이의 정렬을 도시한다.1 is a subtilisin BPN '(a) and Savinase using the GAP routine

The alignment between (b) is shown.

사이의 정렬을 도시한다.1a shows subtilisin BPN 'and Savinase as taken from WO 91/00345

Shows the alignment between.

2 shows the alignment between subtilisin BPN 'and subtilisin Carlsberg using GAP routines.

2A shows the alignment between subtilisin BPN 'and subtilisin Carlsberg as taken from WO 91/00345.

3 shows a three-dimensional structure of Savinase (Protein data bank (PDB) entry 1SVN). In the figure the active site loop (b) is indicated.

Subtilas of the invention have at least one additional amino acid residue at position 100 of the active site loop region at positions 95 to 103 in the first aspect such that the additional amino acid residue is at least one amino acid residue between positions 100 and 101. Corresponds to the insertion of subgroups I-S1 and I-S2 into separate (ie purer than 10%) subtilase enzymes.

In other words, the subtilase of the present invention is characterized in that it comprises an active site loop (b) region of more than 9 amino acid residues, wherein the additional amino acid residues are in combination with position 100 as compared to the parent or known wild type subtylase. It may be inserted between 101, or considered to be inserted.

Subtilas of the first aspect of the invention are parental or wild-type subtilases that have been identified and isolated from nature.

Such parental wild type subtilases are specifically screened by standard techniques known in the art.

A preferred means of doing this is to specifically PCR amplify a DNA region known to encode an active site loop region of a number of different microorganisms, preferably of the Bacillus strain-derived subtilase .

Subtilases are a group of conserved enzymes in that their DNA and amino acid sequences are homologous. Thus, it is possible to construct relatively specific primers in contact with both sides of the active site loop.

One means of doing this is to perform alignment of different subtilases (eg Siezen et al. Protein Science 6 (1997) 501-523). From this, it is easy for a person skilled in the art to construct a PCR primer that contacts both sides of the active site loop corresponding to the active site loop (b) between the amino acid residues 95 to 103 in either group I-S1 or I-S2 such as BLSAVI. will be. Using such PCR primers amplify DNA from a number of different microorganisms, preferably different Bacillus strains and DNA sequencing of the amplified PCR fragments, for example, the active site region of positions 95 to 103 compared to BLSAVI Strains that produce subtilas from these groups that include longer active site regions corresponding to can be identified and insertions can be considered to exist between positions 100 and 101. Once subtilase strains of interest and partial DNA sequences have been identified, it is an easy task for those skilled in the art to complete, express and purify the cloning of such subtilases of the invention.

However, one can predict that the subtilase enzymes of the present invention are primarily variants of mosobtilase.

Thus, in an embodiment, the invention relates to a subtilase enzyme isolated according to a first aspect of the invention, said subtilase enzyme having at least one amino acid between amino acid residues 100 and 101 and its parent enzyme. Variants that have longer active site loops (b).

The subtilases of the present invention exhibit excellent wash performance in detergents and, if the enzyme is a prepared variant, it has improved wash performance compared to its closest related subtilas such as subtilisin 309.

Different subtilase products exhibit different washing performances in different types of detergent compositions. The subtilases of the present invention have improved washing performance compared to the closest cousin in many of these different types of detergent compositions.

Preferably, in the detergent composition shown in Example 3, the subtilase enzyme of the present invention has improved washing performance compared to its closest equivalent (see below).

Determining whether a given subtilase amino acid sequence is within the scope of the present invention (whether that subtilase sequence is a parental wild type subtilase sequence or a subtilase variant sequence produced by any method other than site specific mutagenesis) In order to do this:

i) aligning said subtilase sequence with the amino acid sequence of subtilisin BPN '(see "Definition" section above);

ii) in the subtilase sequence corresponding to the active site loop (b) region of subtilisin BPN 'comprising regions of amino acid residues 95-103 (including amino acids at both ends), based on the alignment of step i) Identifying the active site loop (b);

iii) whether the active site loop (b) in the subtilase sequence identified in step ii) is longer than the corresponding active site loop in the BLSAVI and inserting at least one amino acid between positions 100 and 101 Determining whether a correspondence can be used.

If so, the subtilase observed is a subtilase that is within the scope of the present invention.

The sorting performed in step i) is performed using a GAP routine as described above.

Based on this description, it is easy for a person skilled in the art to identify the active site loop (b) in subtilases and to determine if the subtilas in question are within the scope of the present invention. If the variant is prepared by site specific mutagenesis, it is natural to know in advance whether the subtilase is within the scope of the present invention.

Subtilase variants of the invention are prepared by standard techniques known in the art, such as site-specific mutagenesis / randomized mutagenesis, or by DNA shuffling of different subtilase sequences. For further details see the "Production of Subtylase Variants" and Materials and Methods section here (see above).

In a further embodiment the invention

1. An enzyme isolated according to the invention, wherein said at least one inserted amino acid residue is selected from the group comprising T, G, A and S;

2. A subtilase enzyme isolated according to the invention, wherein the at least one inserted amino acid residue is charged to comprise D, E, H, K, and R, more preferably D, E, K and R. A band is an enzyme selected from the group of amino acid residues;

3. A subtilase enzyme isolated according to the invention, wherein the at least one inserted amino acid residue is a hydrophilic amino acid residue comprising C, N, Q, S and T, more preferably N, Q, S and T Enzymes selected from the group;

4. A subtilase enzyme isolated according to the invention, wherein the at least one inserted amino acid residue is selected from the group of small hydrophobic amino acid residues comprising A, G and V;

5. A subtilase enzyme isolated according to the invention, wherein said at least one inserted amino acid residue is F, I, L, M, P, W and Y, more preferably F, I, L, M and Y Related to an enzyme selected from the group of large hydrophobic amino acid residues comprising:

In a further embodiment, the invention relates to an isolated subtilase enzyme according to the invention wherein the insertion between positions 100 and 101 comprises at least two amino acids as compared to the active site loop of the corresponding BLSAVI.

In a further embodiment, the present invention

(From BASBPN numbering)

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 more specifically for subtilisin 309 and near-relative subtilas such 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 the following combination

A98G + G100GA + S101A + S103T

S99G + G100GGT + S101T

It relates to an isolated subtilase enzyme comprising at least one insertion, selected from the group comprising any one of the following.

It is well known in the art that the so-called conservative substitution of one amino acid residue for a similar amino acid residue will only slightly change the properties of the enzyme.

Table III below lists the groups of conservative amino acid substitutions.

TABLE 3

Conservative Amino Acid Substitutions

General Character Amino Acids

Basic (positive charge) K = lysine

H = histidine

Acid (negative charge) E = glutamic acid

D = aspartic acid

Polarity Q = glutamine

N = asparagine

Hydrophobic L = leucine

I = isoleucine

V = valine

M = methionine

Aromatic F = phenylalanine

W = tryptophan

Y = tyrosine

Small G = glycine

A = alanine

S = serine

T = threonine

According to this principle, subtilase variants comprising conservative substitutions such as G97A + A98S + S99G, G97S + A98AT + S99A are expected to exhibit significantly different properties from each other.

It is an easy task for a person skilled in the art to identify suitable conservative modifications for such variants to obtain other subtilase variants that exhibit similar improved washability based on the subtilase variants disclosed and / or described herein. to be.

According to the present invention, the subtilases of the present invention may be used in combination with the subgroups I-S1 both in the separation of the novel enzymes of the present invention from natural or various artificial productions, and in the design and production of variants from mosobtilase. I-S2, in particular subgroup I-S2.

Regarding variants derived from subgroup I-S1, retain the characteristics of BSS168 (BSSAS, BSAPRJ, BSAPRN, BMSAMP), BASBPN, BSSDY, BLSCAR (BLKERA, BLSCA1, BLSCA2, BLSCA3), BSSPRC and BSSPRD or subgroup I-S1 It is preferred to select mosobtilase from the group comprising one of its functional variants.

With respect to subgroup I-S2 derived variants, BSAPRQ, BLS147 (BSAPRM, BAH101), BLSAVI (BSKSMK, BAALKP, BLSUBL), BYSYAB and its functional variants retaining the characteristics of BSAPRS or subgroup I-S2 It is preferable to select mosobtilase from the group.

NOVO NORDISK A/S)이고, 본 발명의 바람직한 서브틸라제 변이체는 따라서 SAVINASE

의 변이체이다.In particular, the mosobtilase is BLSAVI (SAVINASE

NOVO NORDISK A / S), and preferred subtilase variants of the present invention are therefore SAVINASE

Is a variant of.

The invention also includes any one of the above subtilases of the invention in combination with any of the other modifications to its amino acid sequence. In particular, combinations with other modifications known in the art that provide improved properties to enzymes are envisaged. The art describes a number of subtilase variants with different improved properties and a number of variants are described herein in the "Background" section (see above). The references are disclosed herein as references for the identification of subtilase variants that can advantageously be combined with the subtilase variants of the present invention.

Such combinations include positions: 222 (improve oxidative stability), 218 (improve thermal stability), substitutions in Ca-binding sites that stabilize enzymes, eg, position 76 and many others apparent from the prior art.

In a further embodiment, the subtilase variant of the invention is:

Advantageously combined with one or more variations in any one of 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 206, 218, 222, 224, 235 and 274.

Specifically, the following BLSAVI, BLSUBL, BSKSMK, and BAALKP variants:

K27R, * 36D, S57P, N76D, S87N, G97N, S101G, S103A, V104A, V104I, V104N, V104Y, H120D, N123S, Y167, R170, Q206E, N218S, M222S, M222A, T224S, K235L and T274A are suitable for combination Is thought to be

S101G + V104N, S87N + S101G + V104N, K27R + V104Y + N123S + T274A, N76D + S103A + V104I or N76D + V104A or any combination of these mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A) Further variants, including those in which any one is combined with one or more of the above modifications, exhibit improved properties.

Further variants of the main aspect of the invention are in any of positions 129, 131, 133 and 194, preferably as 129K, 131H, 133P, 133D and 194P modifications, and most preferably P129K, P131H, A133P And one or more variations as A133D and A194P variants. Any of these modifications are expected to provide higher expression levels of the subtilase variants of the invention in their production.

Accordingly, further embodiments of the invention relate to variants according to the invention wherein the modification is selected from the group comprising.

Production of Subtilase Variants

Numerous methods are known in the art for cloning subtilases of the present invention and for introducing insertions into genes (eg subtilase genes) (see references cited in the "Background" section).

In general, standard methods of cloning a gene and introducing (randomly and / or site-specifically) insertion into the gene are used to obtain the subtilase variants of the invention. For other techniques of suitable techniques, see examples herein (see below) and Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM et al. (eds.) "Current protocols in Mollecular Biology", John Wiley and Sons, 1995; Harwood, CR, and Cutting, SM (eds.) "Molecular Biological Methods for Bacillus"; John Wiley and Sons, 1990; and WO 96 / 34946 is a reference.

의 활성부위 (b) 루프보다 긴 활성부위 (b) 루프 영역을 포함하는 하나 이상의 천연에서 동정된 서브틸라제 부분서열을 가지는 Savinase

를 코딩하는 유전자의 DNA 셔플링은 이어지는 개선된 세척수행능 변이체에 대한 스크리닝 후 본 발명에 따르는 서브틸라제 변이체를 제공할 것이다.In addition, the subtilase variants of the present invention are constructed by standard techniques for the artificial production of a variety of DNA shuffling of different subtilase genes (WO 95/22625; Stemmer WPC, Nature 370: 389-91 (1994)). do. For example Savinase

An active site of (b) an active site that is longer than the loop (b) a savinase having one or more naturally-identified subtilase sequences comprising a loop region

DNA shuffling of the gene encoding A will provide the subtilase variants according to the invention following screening for subsequent improved washability variants.

Expression vector

The recombinant expression vector comprising the DNA construct encoding the enzyme of the invention may be any vector that will conveniently carry out a recombinant DNA process.

The choice of vector will often depend on the host cell into which it is introduced. Therefore, a vector is a self-replicating vector whose replication is independent of the chromosome, ie, a vector, for example a plasmid, present as an extrachromosomal gene. Alternatively, the vector may be introduced into a host cell and all or part of it may be inserted into the host cell genome and replicate with the inserted chromosome.

The vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is operably linked to additional fragments essential for DNA transcription. In general, expression vectors contain a factor derived from both plasmids or viral DNA, or both. The term "operably linked" refers to a fragment that is arranged to function for its intended purpose, for example, to initiate transcription in a promoter and continue in a DNA sequence encoding an enzyme.

The promoter may be any DNA sequence that exhibits transcriptional activity in the host cell of choice and may be derived from a gene encoding a homologous or nonhomologous protein to the host cell.

Examples of promoters suitable for use in bacterial hosts include Bacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis α-amylase gene, Bacillus amyloliquefaciens α-amylase gene, Bacillus subtilis alkaline protease gene, or Lamda phage of Bacillus pumilus xylosidase gene. P _R or P _L promoters or E. coli lac , trp or tac promoters.

The DNA sequences encoding the enzymes of the invention are also operably linked to appropriate terminators, if desired.

The recombinant vector of the present invention further comprises a DNA sequence which allows the vector to replicate in the host in question.

The vector also encodes resistance to heavy metal resistance or resistance to antibiotics, such as kanamycin, chloramphenicol, erythromycin, tetramycin, spectinomycin, etc. It includes genes to make.

Secretion signal sequences (also known as leader sequences, prepro sequences, and free sequences) are provided in recombinant vectors to guide the enzyme of the invention into the secretory pathway of the host cell. The secretory signal sequence is linked to the frame of the enzyme coding sequence. The secretory signal sequence is generally located 5 'of the DNA sequence encoding the enzyme. Secretory signal sequences are normally associated with enzymes or derived from genes encoding other secretory proteins.

Used to nucleotide DNA sequences encoding each of the present enzymes, promoters and optionally terminator and / or secretory signal sequences, or to assemble these sequences into suitable vectors containing the information necessary for replication or insertion by means of suitable PCR amplification strategies. Methods are well known to those skilled in the art (see, eg, Sambrook et al., Cited above).

Host cell

The DNA sequence encoding the enzyme introduced into the host cell may be homologous or nonhomologous to the host cell in question. If homologous to a host cell, ie produced naturally by the host cell, it will typically be operably linked to a different promoter sequence than to its natural environment or to other secretory signal sequences and / or terminator sequences if applicable. . The term “homology” is intended to include DNA sequences encoding enzymes that are native to the host organism in question. The term “nonhomologous” is intended to include DNA sequences that are not naturally expressed by the host cell. Thus, DNA sequences are derived from other organisms or are synthetic sequences.

The host cell into which the DNA construct or recombinant vector of the present invention is introduced may be any cell capable of producing the enzyme and includes higher eukaryotic cells including bacteria, yeasts, fungi and plants.

Examples of bacterial host cells capable of producing the enzyme of the present invention in culture include B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. Gram-positive bacteria such as Bacillus strains, such as circulans, B. lautus, B. megatherium , or B. thuringiensis , or Streptomyces strains such as S. lividans or S. murinus, or Gram-negative bacteria such as Escherichia coli .

Bacterial transformation is accomplished using competent cells by protoplast transformation, electroporation, conjugation, or by means known in the art (see Sambrook et al., Supra).

When expressing enzymes in bacteria such as E. coli, enzymes are typically retained in the cytoplasm as insoluble granules (known as inclusion bodies) or transported to the periphery space by bacterial secretion sequences. In the former case, the cells are lysed, the granules are recovered and denatured, and the enzyme is then refolded by dilution of the denaturing agent. In the latter case, the enzyme destroys the cells, and for example, causes the contents of the cell surrounding space to be ejected by sonication or osmotic shock, and the enzyme is recovered to recover from the cell surrounding space.

When the enzyme is expressed in Gram-positive bacteria, such as Bacillus or Streptmyces strains, the enzyme is maintained in the cytoplasm or transferred to extracellular medium by bacterial secretion sequences. In the latter case, the enzyme is recovered from the culture as described below.

Production method of subtilase

The present invention provides a method for producing an enzyme isolated according to the present invention, wherein a suitable host cell transformed with a DNA sequence encoding the enzyme is cultured under conditions allowing the production of the enzyme and the resulting enzyme is recovered from the culture. .

Expression vectors comprising DNA sequences encoding enzymes are transformed into non-homologous host cells to allow for heterologous recombination production of the enzymes of the invention.

Thus, it is possible to produce a fixed subtilase composition characterized by the absence of homologous impurities.

Homologous impurity in this context means any impurity (eg, a polypeptide other than the enzyme of the invention) derived from a homologous cell from which the enzyme of the invention was first obtained.

The medium used to culture the transformed host cell may be any conventional medium suitable for growing the host cell in question. The expressed subtilase is conveniently secreted into the culture medium, the cells are separated from the medium by centrifugation or filtration, the protein composition is precipitated by salts such as ammonium sulfate, and then chromatographed with ion exchange resins, affinity chromatography, and the like. It may be recovered from it by a known method including performing a.

Use of Subtilase Variants of the Invention

The subtilase variants of the invention are used in a number of industrial applications, especially in the detergent industry.

In addition, the present invention relates to enzyme compositions comprising the subtilase variants of the present invention.

An overview of preferred enzyme compositions corresponding to preferred industrial applications is given below.

This Summary is not intended in any way to complete an enumeration of suitable applications of the subtilase variants of the invention. Subtilase variants of the invention can be used in other industrial applications known in the art, including the use of proteases, in particular subtilases.

Detergent composition comprising a mutant enzyme

The present invention includes such compositions comprising mutant enzymes of the invention for use in washing and detergent compositions and mutant subtilase enzymes. Such cleaning and detergent compositions are well described in the art and described in WO 96/34946; WO 97/07202; WO 95/30011 is a reference for further description of suitable cleaning and detergent compositions.

Moreover, the following example (s) describe improvements in washability for the subtilase variants of the invention.

Detergent composition

The enzyme of the present invention is added to the detergent composition and thus becomes a component of the detergent composition.

The detergent composition of the present invention is a detergent composition for use as formulated, for example, as a hand wash or machine wash detergent composition comprising a pretreated laundry additive composition of stained fabric and a rinse agent added fabric softener composition or commonly used in home hard surface washing operations. Or for dishwashing or mechanical dishwashing operations.

In a specific aspect, the present invention provides a detergent additive comprising the enzyme of the present invention. In addition to detergent compositions, detergent additives may include proteases, lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, metnanases, arabinases, galactanases, xylanases, oxidases, for example One or more other enzymes such as laccases and / or peroxidases.

In general, the properties of the enzyme chosen should be compatible with the detergent chosen (ie, optimal-pH, compatibility with other enzymes and non-enzymatic components, etc.) and the enzyme should be present in an effective amount.

Proteases : Suitable proteases include those of animal, plant or bacterial origin. Bacterial origin is preferred. Chemically modified or protein engineered mutations are included. Proteases may be serine proteases or metallo proteases, preferably alkaline microbial proteases or trypsin-like proteases. Examples of alkaline proteases are subtilisin, in particular subtilisin from Bacillus , for example subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (WO 89/06279). (Described in). Examples of trypsin-like proteases are trypsin (eg of swine or bovine origin) and Fusarium proteases described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116 and WO 98/34946, in particular the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120 , 123, 167, 170, 194, 206, 218, 222, 224, 235, 274.

Preferred commercially available protease enzymes include Alcalase ^TM , Savinase ^TM , Primase ^TM , Duralase ^TM , Esperase ^TM , Kannase ^TM (Novo Nordisk A / S), Maxatase ^TM , Maxacal ^TM , Maxapem ^TM , Properase ^TM , Purafect ^TM , Purafect OxP ^TM , FN2 ^™ and FN3 ^™ (Genencor International Inc.).

Lipase : Suitable lipases include bacterial or fungal origin. Chemically modified or protein engineered mutations are included. Examples of useful lipases are Humicola ( syn. Thermomyces ), for example H. lanuginosa ( T. lanuginosus ) described in EP 258 068 and EP 305 216 or H. insolens derived lipases described in WO 96/13580, Pseudomonas lipases, eg For example, P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonous species strains SD 705 (WO 95/06720 and WO 96 / 27002), lipases derived from P. wisconsinesis , Bacillus lipases, for example B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B . the pumilus (WO 91/16422) containing the lipase derived.

Other examples are WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615 , Lipase variants as described in WO 97/04079 and WO 97/07202.

Preferred commercially available lipase enzymes include Lipolase ^™ and Lipolase Ultra ^™ (Novo Nordisk A / S).

Amylase : Suitable amylases (α and / or β) include bacterial or fungal origin. Chemically modified or protein engineered mutations are included. Amylases include, for example, α-amylases obtained from certain strains of B. licheniformis described in more detail in Bacillus , for example GB 1,296, 839.

Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, in particular the following 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 are variants having one or more substitutions.

Commercially available amylases are Duramyl ^™ , Termamyl ^™ , Fungamyl ^™ and BAN ^™ (Novo Nordisk A / S), Rapidase ^™ , and Purastar ^™ (from Genencor International Inc.).

Cellulase : Suitable cellulase includes those of bacterial or fungal origin. Chemically modified or protein engineered mutations are included. Suitable cellulases are celluloses derived from the genus Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium , for example from US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259 from Humicola insolens, Myceliophthora thermophilapor Fusarium Cellulase produced.

Particularly suitable cellulase are alkaline or neutral cellulase with color protection advantages. Examples of such cellulase are the cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples of cellulase variants are those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT / DK98 / 00299.

Commercially available cellulases include Celluzyme ^™ , Carezyme ^™ (Novo Nordisk A / S), Clazinase ^™ , Puradax HA ^™ (Genencor International Inc.) and KAC-500 (B) ^™ (Kao Corporation).

Peroxidase / Oxidase : Suitable peroxidase / oxidases include plant, bacterial or fungal origin. Chemically modified or protein engineered mutations. Examples of useful peroxidases include peroxidases derived from C. cinereus and variants thereof, such as those described in Coprinus , for example WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme ^™ (Novo Nordisk A / S).

Detergent enzymes are included in the detergent composition by the addition of individual additives containing one or more enzymes or by the addition of a combination additive comprising all of these enzymes. Detergent additives of the present invention, ie individual additives or combination additives, can be prepared, for example, as granules, liquids, slurries and the like. Preferred detergent additive preparations are granules, in particular dust-free granules, liquids, especially stabilizing liquids, or slurries.

Dust-free granules are produced, for example, as described in US Pat. Nos. 4,106,991 and 4,661,452 and are optionally coated by methods known in the art. Examples of waxy coating materials include poly (ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1000 to 20000; Ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; Alcohols include ethoxylated fatty alcohols containing 12 to 20 carbons and having 15 to 80 ethylene oxide units; Fatty alcohol; fatty acid; And mono-, di- and triglycerides of fatty acids. Examples of film-forming coatings suitable for use in liquid phase technology are given in GB 1483591. Liquid enzyme preparations are stabilized according to established methods, for example by adding polyols such as propylene glycol, sugars or sugar alcohols, lactic acid or boric acid. Enzymes to be protected can be prepared according to the methods disclosed in EP 238,216.

The detergent composition of the present invention may be in any convenient form, for example rods, tablets, powders, granules, dough or liquid. Liquid detergents are typically aqueous or non-aqueous, containing up to 70% water and 0-30% organic solvent.

The detergent composition may be non-ionic and includes one or more surfactants that include semi-polar and / or anionic and / or cationic and / or zwitterionic systems. Surfactants are typically present at levels of 0.1% to 60% by weight.

When included therein, detergents typically comprise from about 1% to about 40% linear alkylbenzenesulfonates, α-sulfo fatty acid methyl esters, anionic surfactants such as alkyl- or alkenylsuccinic acids, or soaps.

When included therein, detergents typically contain from about 0.2% to about 40% of alcohol ethoxylates, nonylphenol ethoxylates, alkylpolyglycosides, alkyldimethylamine oxides, ethoxylated fatty acid amides, fatty acid monoethanolamides, Nonionic surfactants such as polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (“glucamide”).

Detergents include 0-65% zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid. Admixtures or complex agents such as alkyl- or alkenylsuccinic acids, soluble silicates or layered silicates (eg, SKS-6 from Hoechst).

Detergents may contain one or more polymers. Examples include polycarboxes such as carboxymethylcellulose, poly (vinylpyrrolidone), poly (ethyleneglycol), poly (vinylalcohol), poly (vinylpyridine-N-oxide), poly (vinylimidazole), polyacrylates Carboxylates, malic acid / acrylic acid copolymers and loryl methacrylate / acrylic acid copolymers.

The detergent may contain a bleach system comprising a H ₂ O ₂ source such as perborate or percarbonate that can be combined with a peracid-forming bleach activator such as tetraacetylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleach system may comprise peroxy acid, for example in the form of amide, imide, or sulfone.

Enzymes of the detergent composition of the present invention are conventional stabilizing agents, for example polyols such as propylene glycol or glycol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives, for example aromatic borate esters, 4-formylphenyl boric acid phenylboric acid derivatives Can be stabilized and the composition can be prepared, for example, as described in WO 92/19709 and WO 92/19708.

Detergents also contain other conventional detergent ingredients such as, for example, fabric conditioners, foam accelerators, suds inhibitors, anti-corrosive agents, dyes, antibacterial agents, optical gloss agents, fragrances, discoloration inhibitors, or perfumes, including muds. can do.

Certain enzymes, particularly enzymes of the present invention, are added to the detergent composition in amounts corresponding to 0.01 to 100 mg enzyme protein per liter of wash liquor, preferably 0.05 to 5 mg enzyme protein per liter of wash liquor, in particular 0.1 to 1 mg enzyme protein per liter of wash liquor. Currently intended.

The enzymes of the present invention may additionally be inserted into the detergent formulations disclosed in WO 97/07202 which are incorporated herein by reference.

Application to the leather industry

The subtilases of the invention are used in the leather industry, in particular for hair loss.

In this application, the subtilase variants of the present invention are preferably used in enzyme compositions which further comprise other proteases.

For a more detailed description of suitable other proteases, see the section on enzymes suitable for use in detergent compositions (see above).

Application to the wool industry

The subtilases of the present invention are used in the wool industry, in particular for washing wool containing fabrics.

In this application, the subtilase variants of the present invention are preferably used in enzyme compositions which further comprise other proteases.

For a more detailed description of suitable other proteases, see the section on enzymes suitable for use in detergent compositions (see above).

The invention is described in more detail in the following examples which are not intended to limit in any way the scope of the claimed invention.

Materials and methods

Strains :

B. subtilis DN1885 (Diderichsen et al., 1990).

B. lentus 309 and 147 are specific strains of Bacillus lentus and are described in US Pat. No. 3,723,250, deposited with NCIB, assigned accession numbers NCIB 10309 and 10147, and incorporated herein by reference.

E. coli MC 1000 (MJ Casadaban and SN Cohen (1980); J. Mol. Biol. 138 179-207) became r ⁻ , m ⁺ by conventional methods and is also described in US Pat. No. 039,298.

Plasmids :

pJS3: E. coli-B. subtilis shuttle vector containing a synthetic gene encoding subtilase 309 (Jacob SchiØdt et al. Protein and Peptide letters 3: 39-44 (1996)).

pSX222: B. subtilis expression vector (described in WO 96/34946).

General Molecular Biology Methods :

Unless otherwise noted, DNA manipulation and transformation were performed using molecular biology standard methods (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM et (eds.) "Current protocols in Molecular Biology", John Wiley and Sons, 1995; Harwood, CR and Cutting, SM (eds.) "Molecular Biological Methods for Bacillus", John Wiley and Sons, 1990).

DNA manipulation enzymes were used according to the supplier's specifications.

DNA manipulation enzymes

Unless stated otherwise, all DNA manipulation enzymes such as, for example, restriction endonucleases, ligases, etc., are purchased from New England Biolabs, Inc.

Proteolytic activity

)에 대하여 상대적으로 측정되고, 측정은 표준 조건, 즉 50℃, pH 8.3, 9분의 반응시간, 3분의 측정시간에서 단백질분해효소에 의한 디메틸 카제인 (DMC) 용액의 가수분해에 기초한다. 폴더 AF 220/1이 요구 에 따라 Novo Nordisk A/S, Denmark로부터 이용가능하고 이 폴더는 여기 참고문헌으로 수록되어 있다.Proteolytic activity in the context of the present invention is expressed in kilo NOVO protease units (KNPU). Activity is enzyme standard (SAVINASE

Relative to, and the measurement is based on the hydrolysis of dimethyl casein (DMC) solution by protease at standard conditions, ie 50 ° C., pH 8.3, reaction time of 9 minutes, measurement time of 3 minutes. Folder AF 220/1 is available on request from Novo Nordisk A / S, Denmark, which is hereby incorporated by reference.

GU is a glycine unit defined as protease activity that produces an amount of NH ₂ -group equivalent to 1 mmole glycine with N-acetyl casein as a substrate at 40 ° C. for 15 minutes under standard conditions.

Enzyme activity also depends on the reaction with the soluble succinyl-alanine-alanine-proline-phenyl-alanine-para-nitro-phenol, and the Journal of American Oil Chemists Society, Rothgeb, TM, Goodlander, BD, Garrison, PH, And PNA analysis described in Smith, LA, (1988).

Fermentation :

Fermentation for the production of subtilase enzymes is carried out on a shake table (300 r.p.m.) in a 500 ml Erlenmeyer flask with a baffle containing BPX medium at 30 ° C. for 5 days.

As a result, 20 Erlenmeyer flasks were fermented simultaneously, for example to produce 2 liters of gravy.

Badge :

BPX medium composition (per liter)

Potato Starch 100g

Barley Powder 50g

20g soybean powder

Na ₂ HPO ₄ x 12H ₂ O 9g

Pluron 0.1g

10 g of sodium caseate

Starch in the medium is liquefied with α-amylase and the medium is sterilized by heating at 120 ° C. for 45 minutes. After sterilization, the pH of the medium is adjusted to 9 by the addition of 0.1M NaHCO ₃ .

Example 1

Preparation and Expression of Enzyme Variants

Site-specific mutagenesis :

The subtilase 309 site-specific mutations of the invention comprising a specific insertion between positions 100 and 101 in the active site loop (b) were produced by DNA fragments produced by PCR of oligos containing the desired insertion (Sambrook et al., Molecular Cloning: produced by traditional cloning of A Laboratory Manual, 2nd Ed. Cold Spring Harbor, 1989 (see below).

The template plasmid DNA was a derivative thereof containing a variant of pJS3 or subtilase 309.

Insertion was introduced into the preparation of the G100GX (X = any amino acid residues inserted between positions 100 and 101) by oligo-induced mutagenesis, resulting in the G100GX subtylase 309 variant.

Subtilase 309 variant was transformed into E. coli . It is transformed with DNA purified from the overnight culture of the transformants were transformed into B. subtilis by transformation of the purified restriction endonuclease hydrolysis, DNA fragments, ligation, B. subtilis.

Transformation of B. subtilis is described by Dubnau et al., 1971, J. Mol. Biol. 56, pp. It was performed as described by 209-221.

Occasional random mutations to insert random insertions into ubiquitous regions :

The overall strategy used to perform ubiquitous random mutations was as follows:

Mutagenesis primers (oligonucleotides) corresponding to DNA sequences flanking both sides of the insertion site and separated by DNA base pairs defining insertion were synthesized.

The resulting mutagenesis primers were then used in a PCR reaction with appropriate relative primers. The resulting PCR fragment was hydrolyzed by endonucleases and E. coli-B. Prior to being cloned into the subtilis shuttle vector, it was purified and extended by secondary PCR-reaction (see below).

Alternatively, if necessary, the resulting PCR fragment can be used as a primer of the secondary PCR reaction with a second suitable relative primer to allow cloning of the hydrolysis and mutagenesis regions into the shuttle vector. PCR reactions are performed under normal conditions.

Following this strategy, a ubiquitous random mutation library was constructed in which insertion was introduced in the active site loop region between positions 100 and 101 in SAVINASE.

Mutations were introduced by mutagenesis primers (see below) so that all 20 amino acids were present (N = 25% A, T, C and G, S = 50% C and G). PCR fragments produced include primers; Once again by combination of overlapping sequences with PCR fragments produced by PCR-amplification by 5 'CTA AAT ATT CGT GGT GGC GC 3' (sense) and 5 'GAC TTT AAC AGC GTA TAG CTC AGC 3' (antisense). PCR cycles were extended in the N-terminal direction of Savinase. The extended DNA-fragment was cloned into the Hind III- and Mlu I- sites (see above) of the modified plasmid pJS3 and sequenced ten randomly selected E. coli colonies to identify the designed mutations.

Mutagenesis primers (5 ′ GTT AAA GTC CTA GGG GCG AGC GGT NNS TCA GGT TCG GTC AGC TCG ATT G3 ′ (sense)) are suitable antisense relative primers (eg, 5 ′) downstream of the Mlu I site in plasmid pJS3. CCC TTT AAC CGC ACA GCG TTT-3 '(antisense)) was used in the PCR reaction with the plasmid pJS3 as a template. This resulting PCR product was cloned into the pJS3 shuttle vector by the use of restriction enzymes HindIII and MluI.

Random libraries were transformed into E. coli by known techniques.

The prepared library contained approximately 100,000 individual clones per library.

Randomly selected colonies were sequenced for identification of the designed mutations.                 

In order to purify the subtilase variants of the invention, B. subtilis pJS3 expressing plasmids comprising the variants of the invention were transformed into competent B. subtilis strains and in a medium containing 10 μg / ml chloramphenicol (CAM). Fermented as described above.

Example 2

Purification of Enzyme Variants :

This process involves the purification of a 2-liter scale fermentation for the production of the subtilase of the present invention in Bacillus host cells.

Approximately 1.6 liters of fermentation broth was centrifuged in a 1 liter beaker for 35 minutes at 5000 rpm. The supernatant was adjusted to pH6.5 with 10% acetic acid and filtered on a Seitz Supra S100 filtration plate.

The filtrate was concentrated to approximately 400 ml using Amicon CH2A UF units equipped with Amicon S1Y10 UF cartridges. The UF concentrate was centrifuged and filtered before absorption on a Bacitracin affinity column at room temperature, pH7. The protease was eluted from the Bacitracin column at room temperature using 25% 2-propanol and 1M sodium chloride in a buffer containing 0.01M dimethylglutalic acid, 0.1M boric acid and 0.002M calcium chloride adjusted to pH 7.

Fractions with protease activity from the Bacitracin purification step were combined and applied to a 750 ml Sephadex G25 column (5 cm diameter) equilibrated with buffer containing 0.01 M dimethylglutalic acid, 0.2 M boric acid and 0.002 M calcium chloride adjusted to pH 6.5. It was.                 

Fractions with proteolytic activity from Sephadex G25 columns were combined to equilibrate 150 ml CM Sepharose CL 6B cation exchange resin with buffer containing 0.01 M dimethylglutalic acid, 0.2 M boric acid and 0.002 M calcium chloride adjusted to pH 6.5. Applied to the column (5 cm diameter).

Proteases were eluted using 0-0.1 M sodium chloride linear gradient (0-0.2 M sodium chloride for subtilisin 147) in 2 liters of the same buffer.

In the final purification step, the protease containing fractions from the CM Sepharose column were combined and concentrated in Amicon ultracentrifuge units equipped with GR81PP membranes (from Danish Sugar Factories Inc.).

Using the preparation and fermentation techniques of Example 1 and the above separation step, the following subtilisin 309 variants were produced and isolated:

G100GT

G100GA

G100GS

G100GD

G100GE

G100GP

G100GG

G100GH

G100GI                 

G100GT + Y167A

S99G + G100GT + S101T

A98G + G100GA + S101A + S103T

These variants showed better washing performance than Savinase in preliminary analysis.

Example 3

Washing Performance of Detergent Compositions Containing Enzyme Variants

The examples below provide results from multiple wash tests performed under the indicated conditions.

Small scale washing

Washing conditions:

Europe Detergent 95 United States of America Detergent dosage 4g / l 3g / l 1 g / l Washing temperature 30 ℃ 15 ℃ 25 ℃ Cleaning time 30 minutes 15 minutes 10 minutes Hardness of water 18 ° dH (Ca ²⁺ / Mg ²⁺ = 5: 1) 6 ° dH 6 ° dH (Ca ²⁺ / Mg ²⁺ = 2: 1) pH Do not adjust 10.5 Do not adjust Enzyme concentration 1, 2, 5, 10, 30, nM 1, 2, 5, 10, 30, nM Test system 150ml glass beaker with stirring rod 10 nm 150ml glass beaker with stirring rod Fabric / bulk 5 pieces of fabric (Ø2.5cm) in 50ml detergent 5 pieces of fabric (Ø2.5cm) in 50ml detergent 5 pieces of fabric (Ø2.5cm) in 50ml detergent Test substance EMPA116 EMPA117 EMPA117

Detergent:

The detergents used were model detergents named detergent 95 or were purchased from supermarkets in Denmark (OMO, Datasheet ED-9745105) and USA (Wisk, Datasheet ED-9711893), respectively. All enzymatic activities in the detergent were inactivated by microwave treatment before use.

Detergent 95 is a simple model preparation. The pH is adjusted to 10.5, which is within the normal range for powdered detergents. The composition of model detergent 95 is as follows:

25% STP (Na ₂ P ₃ O ₁₀ )

25% Na ₂ SO ₄

10% Na ₂ CO ₃

20% LAS (Nansa 80S)

5.0% nonionic tenside (Dobanol 25-7)

5.0% Na ₂ Si ₂ O ₅

0.5% Carboxymethylcellulose (CMC)

9.5% water

Sample:

Samples used were EMPA Testmaterialen, Movenstrasse 12, CH-9015 EMPA116 and EMPA117 purchased from Gall, Switzerland.

reflectivity

Measurement of the reflectance (R) on the test material was performed using a Macbeth ColorEye 7000 photometer at 460 nm. The measurement was performed according to the manufacturer's protocol.

evaluation

The evaluation of the wash performance of the subtilase is determined by the coefficient of improvement or the coefficient of performance for the observed subtilase.

Improvement factor, IF _{dose / response,} is defined as the ratio of the slope of the wash performance to the detergent containing subtilase and the detergent containing reference subtilase observed where the subtilase concentration is asymptotically zero.

IF _{dose / response} = a / a _ref

Detergency is formula I:

_{R = R 0 + a · ΔR} max · c / (ΔR max + a · c) (I)

Is calculated according to

R is the washing performance in the reflection unit; R ₀ is the Y-intercept of the curve; a is the slope of the curve at c → 0; c is the enzyme concentration; ΔR _max is the theoretical maximum washing capacity at c → ∞.

Coefficient of performance, P is Equation II:

P = (R _variant -R _blank ) / (R _Savinase -R _blank )

Is calculated according to

R _variant is the reflectance of the test material washed with 10 nM variant; R _Savinase is the reflectance of the test material washed with 10 nM Savinase; R _blank is the reflectance of the test material washed without enzyme.

US (detergent: US Wisk, Swatch: EMPA117)

Variant P G100GA 1.2 S99G + G100GGT + S101T 1.6

* P is the value measured at [E] = 5 nM

에 비하여 개선된 세척능을 나타내는 것으로 보인다.














The subtilase of the present invention is thus Savinase

It appears to show improved washability as compared to.

<110> NOVOZYMES A / S <120> Subtilase enzymes of the I-S1 and I-S2 sub-groups having an          ad-ditional amino acid residue in an active site loop region <130> 5795.204 <150> PA 1998 01673 <151> 1998-12-18 <160> 2 <170> KopatentIn 1.71 <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 (34)

By having one or two additional amino acid residues at position 100 of the active site loop (b) region from positions 95 to 103, the additional amino acid residues correspond to the insertion of one or two amino acid residues between positions 100 and 101. The amino acid residue is selected from the group consisting of T, A, S, D, E, P, G, H, and I; Subtilase enzymes (a) amino acid sequence: 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 Subtilas belonging to subgroup I-S1 having (b) amino acid sequence: 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 Subtilas belonging to subgroup I-S2 having       (c) a subtilase enzyme, characterized in that it is a homologous subtilase having at least 95% identity with the subtilase of (a) or (b). The method of claim 1, G100GA, G100GT, G100GG, G100GS, G100GD, G100GE, G100GH, G100GI, and G100GP Isolated subtilase enzyme, characterized in that selected from the group comprising. The method of claim 1, wherein K27R, * 36D, S57P, N76D, S87N, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, Y167X, R170X, Q206E, N218S, M222S, M222A, T224S, K235L, T274A, P129K And a modification selected from the group comprising P131H, A133P, A133D, and A194P. The method of claim 3, wherein the modification is S101G + V104N, S87N + S101G + V104N, K27R + V104Y + N123S + T274A, N76D + S103A + V104I or N76D + V104A, or these mutations (V104N, S101G, K27R, V104Y, N123S). , T274A, N76D, V104A). 3. The subtilase enzyme of claim 2, comprising a modification selected from the group comprising A98G + G100GA + S101A + S103T and S99G + G100GGT + S101T. An isolated DNA sequence encoding the subtilase enzyme of any one of claims 1 to 5. An expression vector comprising the isolated DNA sequence of claim 6. A microbial host cell transformed with the expression vector of claim 7. 9. The microbial host cell of claim 8, wherein the microbial host cell is Bacillus lentus . 9. The microbial host cell of claim 8, wherein the microbial host cell is Aspergillus . A method for producing the subtilase enzyme of any one of claims 1 to 5, wherein the method comprises transducing with an expression vector comprising an isolated DNA sequence encoding the subtilase enzyme of any one of claims 1 to 5. Culturing the converted microbial host cell under conditions conducive to the expression and secretion of the enzyme and recovering the enzyme. A composition comprising the subtilase enzyme according to any one of claims 1 to 5. 13. The composition of claim 12, further comprising cellulase, lipase, cutinase, oxidoreductase, other protease or amylase. 13. The composition of claim 12, wherein the composition is a detergent composition. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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