PL184399B1 - Varieties of subtilisin - Google Patents

Abstract

Detergent compositions are provided comprising variants of subtilase, which have been produced by mutating the genes for a number of subtilases and expressing the mutated genes in suitable hosts. The enzymes exhibit improved stability and/or wash performance in detergents, especially liquid detergents and soap bars, in comparison to their wild type parent enzymes. The enzymes are well-suited for use in liquid detergent compositions and soap bars.

Description

The present invention relates to novel liquid detergent compositions containing enzyme mutants or enzyme variants which exhibit improved storage stability while maintaining or enhancing their detergency.

Background of the invention

In the detergent industry, enzymes have been introduced into washing formulations for over 30 years. The enzymes used in such formulations are proteases, lipases, amylases, cellulases as well as other enzymes or mixtures thereof. From a commercial point of view, proteases are the most important.

Although proteases have been used in the detergent industry for over 30 years, it has not been explained in detail how enzymes interact with substrates and / other substances present in e.g. detergent compositions. Some factors related to specific residues and influencing certain properties, such as oxygen and thermal stability, have generally been elucidated, but many have remained undiscovered. Also, it is not yet known exactly what physical or chemical properties correspond to good detergency or protease capacity in specific detergent compositions.

The proteases currently used have been largely discovered by isolating proteases from nature and testing them in detergent formulations.

Proteases

Amide-cleaving enzymes in protein substrates are classified as proteases, or (alternatively) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms.

W.H. Freeman and Company, San Francisco, Chapter 3). Bacillus bacteria secrete two extracellular protease species, a neutral or metalloprotease and an alkaline protease which is functionally serine endopeptidase and is usually referred to as subtilisin. The secretion of these proteases has been linked to the bacterial growth cycle with increased protease secretion in the stationary phase, when sporulation also occurs. Joliffe et al (1980) J. Bacteriol 1411199-1208, suggested that the functions of Bacillus proteases are altered in the cell wall.

Subtilases

Serine protease is an enzyme that catalyzes the hydrolysis of peptide bonds in which the serine residue at the active center is essential (White, Handler, and Smith, 1973 "Principles of Biochemistty", fifth edition, McGraw-Hill Book Company, NY, pp. 271-272) .

Bacterial serine proteases have a molecular weight in the range of 20,000 to 45 °. They are inhibited by diisopropyl fluorophosphate. Hydrolysis is simple terminal esters and is similar in activity to eukaryotic chymotrypsin as well as serine protease. In a narrower sense, an alkaline protease, including the subgroup, reflects a high pH optimum, pH in the range 9.0 to 11.0, from other serine proteases (see Priest (1977) Bacteriological Rev 41 711-753 for comparison).

A subgroup of serine proteases similarly called subtilases was proposed by Siezen et al., Protein Engng. 4 (1991) 719-737. They were determined by sequence homologous analysis of more than 40 amino acids of serine proteases previously described as subtilisin-like proteases. Previously, subtilisin was referred to as a serine protease produced by gram-positive bacteria or fungi and, according to Siezen et al., Now forms a subset of subtilases. A wide variety of subtilisins have been identified, and the amino acid sequences of many subtilisins have been determined. These include more than six Bacillus chain subtilisins named subtilisin 168, BPN 'subtilisin, Carlsberg subtilisin, subtilisin Y, amylosaccharyticus subtilisin, and mesentericopeptidase (Kurihara et al (1972) J. Biol. Chem. 247 5629-5631; Welle and et al. (1983) Nucleic Acids Res. 117911-7925; Stahl and Ferrari (1984) J. Bacterial. 159 811-819; Jacobs et al. (1985) Nucl. Acids Res. 13 8913-8926; Nedkov et al ( 1985) Biol. Chem. Hoppe-Seyler 366 421-430; Svenseni et al. (1986) FEBSLett. 196 228-232), one actinomycetes subtilisin, thermitase from Thermoactinomyces vulgaris (Meloun et al. (1985) FEBS Lett. 198 195 -200), and one fungal subtilisin, Proteinase K from Tritirachium album (Jany and Mayer (1985) Biol. Chem. Hoppe-Seyler 366 584-492). Further literature references are provided below in Table I of Siezen et al.

The subtilisins are physically and chemically well characterized. In addition to the knowledge of the primary structure (amino acid sequence) of these enzymes, more than 50 subtilisin structures were determined by high resolution X-ray method for which substrate junctions, transitions, products, at least three different protease inhibitors were plotted, and the structural consequences of natural variability ( Kraut (1977) Ann. Rev. Biochem. 46 331-358).

In the context of the present application, the substrates should be interpreted in their broadest form as consisting of a compound containing at least one peptide bond susceptible to hydrolysis by a subtilisin protease.

Also, the term "product" should be interpreted in the context of the present invention as including the products of the subtilisin protease induced hydrolysis reaction. The product may be a substrate for the next hydrolysis reaction.

One subgroup of subtilases, I-S1, includes "classic" subtilisins such as subtilisin 168, subtilisin BPN ', Carlsberg subtilisin (ALCALASE®, NOVO NORdIsK A / S) and DY subtilisin.

A further subgroup of subtilases I-S2 was recognized by Siezen et al. (supra). The I-S2 protease subgroup has been described as highly alkaline subtilisins containing enzymes such as subtilisin PB92 (MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, NOVO NORDISK A / S), subtilisin 147 (ESPERASE®, NOVO NORDISK A / S) and YaB alkaline elastase.

In the context of the present invention, variants of subtilases or mutated subtilases means a subtilase that has been produced by an organism that expresses a mutant of the gene po184 399 derived from a parent microorganism having the original or parent gene and that produces the corresponding parent enzyme, that parent gene has been mutated to produce the mutant of the gene from which a mutant subtilisin protease is produced when expressed in a suitable host.

Statistical and targeted mutations of the subtilase gene arise from knowledge of the physical and chemical properties of the enzyme and provide information regarding the catalytic activity of subtilases, substrate specificity, tertiary structure, etc. (Wells et al. (1987) Proc. Natl. Acad. Sci. USA 84; 1219-1223; Wells et al (1986) Phil. Trans. R. Soc. Lond A. 317 415-423; Hwang and Warshel (1987) Biochem. 262669-2673; Rao et al. (1987) Nature 328 551 -554.

Later publications on this subject include Carter et al. (1989) Proteins 6 240-248 relating to the design of variants that cleave specific sequence sites in a substrate (positions 24 and 64); Graycar et al. (1992) Annals of the New York Academy of Science 672 72-79 discuss previously published results; and Takagi (1993) Int. J. Biochem. 25 307-312 also gives an overview of past results.

Targeted mutations of the subtilisin genes in particular have attracted a lot of attention and a variety of mutations have been described in the following patent applications and patents:

EP-A-130 756 (GENENTeCh) (corresponds to US Reissue Patent No. 34 606 (GENENCOR)) refers to targeted or statistically generated mutations in "carbonyl hydrolases" and the subsequent study of various properties of mutant enzymes such as the _kcat / K ratio _m , the activity profile of pH and aerobic stability. This publication reveals that a certain mutation is feasible and that a BPN subtilisin mutation 'at certain specific positions i.e.' TYR, ³ Asp, ¹⁵⁵ ASN, ¹⁰⁴ Tyr, ²²² Met, ¹⁶⁶ Gly, 6 * His, ¹⁶⁹ Gly, ¹⁸⁹ Phe, ³³ Cheese, ²²¹ Ser, ²¹⁷ Tyr, ¹⁵ 6Glu, or ^ 2 Ala, provides enzymes exhibiting altered properties. Since all of these positions except for the -1 position were known to be related to enzyme function prior to filing, and therefore are evident to be selected, this application does not add much to the problem of deciding where to mutate in order to obtain enzymes with the desired properties.

EP-A-214 435 (HENKEL) relates to the cloning and expression of Carlsberg subtilisin and its two mutants. This application does not disclose a reason to mutate ¹⁵⁸ Asp to ^ 8 Ser and * 61 Ser to ^ Asp.

WO-A-87/04461 (AMGEN) proposes to reduce the number of Asn-Gly sequences present in the parent enzyme in order to obtain mutant enzymes exhibiting improved pH stability and heating, the application notes the removal, mutation or modification of 1 ⁰⁹ Asn residues and 2 ¹⁸ Asn in BPN subtilisin '. There are no examples of deletions or modifications to Gly residues.

WO-A-87/05050 (GENEX) discloses statistical mutations and studies of a large number of BPN 'subtilisin mutants to improve properties. Mutations of the following positions 21 ⁸ Asn ^than Gly, 254Thr, ^{1 < '6} Gly' → Ala, ¹⁸⁸ Ser, ¹² 6Leu and 53Ser.

EP-A-251446 (GENENCOR) describes how homologous considerations at the primary and tertiary structure level can be used to identify equivalent amino acid residues, whether or not to keep. This information, together with the inventors 'knowledge of the tertiary structure of BPN' subtilisin, led the inventors to select positions suitable for mutations in anticipation of obtaining mutants with altered properties. The items thus identified are: ¹²⁴ Met, ²²² Met, ^ Tyr, * 52Ala, ^ Gly, ^ Gly, ^ Gly, ^ Phe, ^ Tyr. And also * 55 Asn, 21Tyr, 22Tyr, 24Ser, 32 Asp, 33Ser, 36Asp, 4 ⁶ Gly, 48 Ala, 49Ser, 5 ° Met, 77Asn, 8 ^? Ser, 94Lys, 94Val, 96Leu, ^L07 Ile, Gly ^ll0, 17 ° Lys ^ Tyr, ¹⁷² Pro, Asp '97, 99Met ^{1, £} 20 Ser, 21 Lys i221Ser ^3, the items have been identified as having influence on various properties enzyme. To support these suggestions, many mutations are illustrated by examples. In addition to single mutations at these positions, the inventors have carried out multiple multiple mutations. Further, the inventors have identified 21 ⁵ Gly, 6 ^? His, ¹²⁶ Leu, ^B 5 Leu, and amino acid residues within segments 97-103, 126-129, 213-215, and 152-172 are interesting but mutations at any of these positions are not exemplified.

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It is of particular interest for the purposes of the present invention that the inventors of EP-A-251446 suggest a substitution of 1 ⁷⁰ Lys (in BPN 'subtilisin type I-S1), especially suggest the introduction of Glu or Arg instead of the original Lys. The Glu variant appeared to have been generated and found to be highly suitable for autolytic degradation (cf. pages 48.121,123 (Table XXI gives obvious error but shows a reduction in autolysis half time from 86 to 13 minutes) and Figure 32).

EP-A-260 105 (GENENCOR) describes modification of certain properties of enzymes comprising a catalytic triad by selecting amino acid residues within about 15a of the catalytic triad, and replacing selected amino acid residues with other amino acids. The subtilase enzymes described in this application are specifically mentioned as belonging to the class of enzymes containing the catalytic triad. Subtilisin positions 222 and 217 are indicated as favorable for the conversion.

Thomas, Russell, and Fersht (1985) Nature 318 375-376 disclosed that Asp to Ser exchange in BPN * subtilisin * alters the pH dependency of the enzyme.

In the next article (1987) J. Mol. Biol. 193 803-813, the same authors of the discussion) changed ¹⁵⁶ Serwmiejsce ¹⁵⁶ Glu. Both of these mutations are within approximately 15A of an active ⁶⁴ His.

In Nature 328 496-500 (1987), Russel and Fersht discuss the results of their experiments and present principles for altering the pH activity profile due to enzyme mutation to obtain surface charge changes.

WO-A-88/08028 (Genex) and WO-A-88/08033 (Amgen) relate to modification of amino acid residues in the calcium binding sites of BPN 'subtilisin. It is said to stabilize the enzyme by substituting a more negatively charged residue for the original one.

wO-A-89/06279 (NOVO NORDISK A / S) identified position 170 as interesting and suggested for Tyr replacement of an existing residue. However, no data are available for this variety. In WO-A-91/00345 (NOVO NoRDISK A / S) the same suggestion was made, and it was shown that variant Tyr at position 170 of subtilisin 309 (type I-S2) exhibits improved washing performance in detergents at about pH 8 (variant S003 in tables III, IV, V, VI, VIII, X). The same substitution in combination with other substitutions in other positions also shows an improved washing performance (S004, S011-S014, S022-S024, S019, S020, S203, S225, S227 in the same table and table VII) all according to the general concept of this applications.

EP-A-525 610 (SOLVAY) suggests an improvement in the stabilization of the enzyme (subtilase type I-S2 closely related to subtilisin PB92) towards ionic surfactants by reducing the hydrophobicity in a particular surface region. Consequently, the substitution of Gln for Arg at position 164 (170 if BPN 'numbering is used) is suggested. The application does not disclose variants containing such a substitution.

WO-A-94/02618 (GIST-BROCADES N.V.) describes many variants of entry 164 (170 if BPN numbering is used) of the PB92 type I-S2 subtilisin. The examples disclose the substitutions of Met, Val, Tyr, Ile for the original Arg. Detergent powder testing of these varieties shows a slight improvement. Especially it has been shown in washing performance tests on cocoa, for a variety Ile an improvement of about 20-30%. No stability data available.

Industrial applications of subtilases

Proteases such as subtilisins have found great industrial use, especially in detergent compositions, because of their utility in removing protein stains.

At present, at least the following proteases are known to be commercially available, and many are sold in large quantities in many countries around the world:

Subtilisin BPN 'or Novo available from e.g. SIGMA, St. Louis, USA.

Carlsberg subtilisin, marketed by NOVO NORDISK A / S (Denmark) as ALCALASE® and by IBIS (Netherlands) as MAXATASE®; Both of them belong to the subgroup I-S1 of the subtilase.

Among the subtilases of the I-S2 subgroup, the following are known and sold:

Bacillus lentus subtilisin, subtilisin 309, marketed by NOVO NORDISK A / S (Denmark) as SAVINASE®. A protein engineered variant of this enzyme is marketed as DURAZYM®.

Enzymes very similar to SAVINASE® such as Subtilisin PB92, MAXACAL® marketed by Gist-Brocades N.V. (a protein engineered variant of this enzyme is marketed as MAXAPEM®), OPTICLEAN® marketed by SOLVAY et al. and PURAFECT® marketed by GEnEnCOR International.

Bacillus lentus subtilisin, subtilisin 147, marketed by NOVO NORDISK A / S (Denmark) as ESPERASE®.

However, to be effective, such enzymes not only need to be active under the washing conditions, but must also be compatible with the other ingredients during detergent preparation and storage.

For example, the subtilisins may be used in combination with other enzymes active against other substrates, and the selected subtilisin should be stable against such enzymes, and the selected subtilisin preferably should not catalyze the degradation of other enzymes. The selected subtilisin should be resistant to the action of other components of the detergent composition, such as bleaching agents, oxidizing agents, etc. in particular, enzymes for use in detergent compositions should be stable with respect to the oxidizing force, calcium binding properties, and pH conditions caused by non-enzymatic detergent ingredients, during storage and in the washing liquid during washing.

The ability of an enzyme to catalyze the degradation of varieties of naturally occurring substrates present on items to be cleaned during e.g. washing, often relates to their washing ability, washability, detergency or washing performance. Throughout this application the term washing performance will be used to encompass these properties.

The ability of an enzyme to remain active in the presence of the other ingredients of a detergent composition before use (usually by adding water in the wash process) is typically related to storage stability or shelf life. It is often measured as half-life, t _1/2 . The term storage stability will be used in this application to represent this property.

Naturally occurring subtilisins have been found to have properties that vary widely with respect to the strength or detergency of the varieties in various parameters such as pH. Several of the aforementioned protease detergents indeed perform better than those marketed around 20 years ago, but for optimal performance each enzyme has its own specific conditions with respect to formulation and washing conditions, e.g. pH, temperature, ionic strength (= Γ), system active (surfactants, surfactants, bleach, etc.), fillers, etc.

Consequently, it has been found that an enzyme having suitable properties at low pH and low I may be less attractive under more basic conditions and higher I, or an enzyme showing good properties at higher pH and higher I may be less attractive under low pH conditions and Low I. It has also been found that the storage stability varies between enzymes, but then specific enzymes have been found to show large differences in storage stability depending on different detergent formulations, depending on many parameters such as pH, pi, bleach system, tensides etc. , and depending on the physical form of the detergent composition, which may be a powder, dust or liquid. Moreover, it can be in concentrated or diluted form.

The use and development of recombinant DNA techniques has had a profound effect on protein chemistry.

While it is now possible to use these technologies to construct enzymes having the desired amino acid sequences, but as previously shown, little research has been devoted to designing subtilisins with altered properties.

Many techniques for producing and testing a large number of mutant enzymes are described in EP-A-130 756 (GENENTECH) (US Reisue Patent No. 34,606 (GENENCOR), and WO-A-87/05050 (GENEX) refers to much extended classical methods of isolating simple enzymes, subjecting them to classical mutagenic programs (using radiation or chemical mutagens) and studying their properties The differences in these methods are more effective in knowing about the presence of a large number of enzyme variants substituted at specific positions.

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The subtilisin enzyme typically has about 275 amino acid residues. Each residue may have from 1 to 20 possible naturally occurring amino acids. Therefore, one of the very serious difficulties occurring in this procedure is the very large number of generated mutations, which must be taken into account in the preliminary tests determining their properties.

The procedure described in this application will be only slightly better than the traditional statistical mutation methods known for years.

Other known techniques relate to changing specific properties such as aerobic stability, thermal stability, Ca stability, transesterification rate and hydrolysis rate (EP-A-260 105 (GENENCOR)), pH activity profile (Thomas, Russell, and Fersht, supra ) and substrate specificity (WO-A-88/07578 GENENTECH)). None of these publications deal with changes in enzyme washing performance or storage stability.

PCT / DK / 88/00002 (NOVO NORDISK A / S) proposes to use the concept of comparing homologues to determine which amino acid positions should be selected for mutations and which amino acids should be substituted at these positions in order to obtain the desired changes in hot action .

Using such a procedure, the test procedure can be drastically shortened because the number of mutants generated is much smaller, but with this procedure it can only be predicted that enzymes will be obtained exhibiting the combined useful characteristics of the parent enzyme and the enzyme used in the comparison.

Thus, as shown above, no relationship has yet been identified between well-defined enzyme properties as defined above and the washing performance and storage stability of the enzyme in a variety of detergent compositions.

The problem is that despite many studies aimed at determining the mechanism of enzyme activity, still little is known about the factors in the structure and combination of amino acid residues that determine properties such as storage stability in detergents, enzymes depending on most of their properties, especially when enzymes are present in mixed complex.

As a consequence, there is still a need for further improvement and adaptation of enzymes to detergent systems as well as for a better understanding of protease mechanism of action and degradation in practical use in cleaning or washing compositions. Such understanding can result in principles used to select the mutations that, to a reasonable degree of certainty, will result in the enzyme showing improved storage stability and / or improved performance under certain conditions in detergent compositions.

It has been surprisingly found that a subtilase variant with improved storage stability and / or performance in detergent compositions can be obtained by substituting one or more amino acid residues located in or adjacent to the hydrophobic parent domain of the subtilase with an amino acid residue more hydrophobic than the original residue.

The present invention relates to a liquid detergent composition containing a subtilase variant which, in addition to the usual components, includes a subtilase in which one or more amino acid residues located in or adjacent to the hydrophobic parent domain of the subtilase has been substituted by an amino acid residue more hydrophobic than the original residue, the subtilase variant being substituted by the subtilase variant. is R170L or R170I.

Preferably the composition comprises a subtilase whose parent subtilase is selected from the group consisting of ABSS168, BASBPN, BSsDy and BLSCAR.

In a preferred embodiment the composition comprises a subtilase whose parent subtilase is selected from the subgroup I-S2, in particular from the group consisting of BLS147, BLS309, BAPB92 and BYSYAB, and particularly preferably comprises a subtilase whose parent subtilase is TVTHER.

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In another preferred embodiment it preferably comprises a subtilase according to the invention, which is a variant combined with further substitutions, deletions and / or insertions in any one or more of the positions: 36, 57, 76, 218, 222 and 224.

In this embodiment, it preferably comprises a subtilase belonging to the subgroup I-S2 with a further variation selected from the group consisting of * 36D, S57P, N76D, N218S, M222S, M222A and T224S.

Preferred compositions of the invention contain a subtilase variant selected from the group of variants including:

a) S57P + R170L a ') S57P + R170I

b) R170L + N218S b ') R170I + N218S

c) S57P + R170L + N218S c ') S57P + R170I + N218S

c) S57P + V104Y + R170L + N218S c) S57P + V104Y + R170I + N218S

d) R170L + N218S + M222A d ') R170I + N218S + M222S

e) S57P + R170L + S188P + A194P

e) S57P + R170I + S188P + A194P

f) Y167L + R170L

f) Y167L + R170I

g) Y167I + R170L g ') Y167I + R170I

h) N76D + R170L + N218S h ') N76D + R170I + N218S

i) S57P + N76D + R170L + N218S i ') S57P + N76D + R170I + N218S

j) N66D + R170L + N21S S + M222A j ') ^ 6DD +: ^ 7001 + ^ HS S + M22SS j) N76D + NUDL + N ^ LS + MUSA j ^w ) N76D + RfZDL + N208S + M12SS

k) S57P + + S188P + A144P + NUSS

k) S57P + R170L + S188P + A194-P + N218S

l) * 36D + N76D + H120D + R170L + G195E + KK33L l ') * 36D + N76D + H120D + + G195E + K235L

m) N76D + H120D + R ^ L + G195E + K235L m ') N76D + H ^ D + R170I + G195E + K235L

n) * 36D D- G97N + V104Y + H120D D RHOL + A114P + G195E + K235L n ') * 36D + NNH + + + RHW + A194P + G195E + K235L

o) S57P + RUdL + o ') S57P + R170I + Q306E

p) R170L + Q206E p ') R1701 + Q206E

q) Y1167I + R170L + Q206E q ') Y667I + RHOI + Q206E

r) Y ^ F + RHOL r ') Y667F + RUOI

t) Y ^ I + RnOL + A ^ P t ') Y667 I + R170I + A994P

u) Y167 I + R170L + N288S u ') Y167I + R170I + R318S

v) Y167 I + RnOL + AWP + roUS

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V) Y167I + R170I + A194P + N218S

x) R170L + P131V x ') R170I + P131V

y) * 36D + Y167I + R170L y ') * 36D + Y167I + R170I aa) Y167V + R170L aa') Y167V + R170I

In a preferred embodiment, the composition of the invention further comprises a deflocating polymer.

A particularly preferred composition as a subtilase variant comprises S57P + V104Y + R170L + N218S or S57P + V104Y + R170I + N218S.

The following abbreviations are used in the above symbols and in the rest of the description. Shortcuts

Amino acids

AND Ala Alanine V Val Valina L. Leu Leucine AND How much Isoleucine P. Pro Prolma F. Phe Phenylalarntine IN Trp Tryptophan M. Underworld Methionine G. Gly Glycine S. Cheese Serine T. Thr Threonin C. Cys Cysteine Y Tyr Tyrosine N Asn Asparagine Q Gin Glutamine D Asp Aspartic acid E. Glu Glutamic acid K. Lys Lysine R Arg Arginine H. His Histidine

Nucleic acid bases

A = Adenine

G = Guanina

C = Cytosine

T = Thymine (only in DNA)

U = Uracil (only in RNA)

Variations

The following nomenclature is used to describe the different enzyme variants made or contemplated in the present invention: original amino acid (s) - position (s) - substituted amino acid (s).

Accordingly, the glycine at position 195 of glutamic acid is defined as:

Gly 195 Glu or G195E deletion of glycine at the same position is defined as:

Gly 195 * or G195 * and the insertion of an additional amino acid residue such as lysine is defined as:

Gly 195 GlyLys or G195GK

Whereby the deletions indicated in Table I, or the presence in subtilisin not indicated in Table I, insertions at such positions are defined as:

18-4399 * 36 Asp or * 36D for insertion of aspartic acid at position 36.

Multiple mutations are separated by pluses, e.g .:

Arg 170 Tyr + Gly 195 Glu or R170Y + G195E are mutations at position 170 and 195 substituted with tyrosine and glutamic acid instead of arginine and glycosine, respectively.

Items

For describing the variants in this application and in the appended claims, an alignment of the subtilase variants as in Siezen et al., Supra is used. In other publications relating to subtilase, a different ordering or numbering for specific enzymes has been used. It is a routine matter for a skilled person to determine a position with a specific number in the numbering used. Reference is made to Fig. 1 for an alignment of residues suitable for the present invention from a large number of subtilases. In Table I, reference is made to WO-A-91/00345 showing the ordering of residues suitable for the present invention from a large number of subtilases.

Table I.

Currently known subtilases (from Siezen et al., Supra).

Organism cDNA, gene enzyme acronym 1 2 3 4 PRO-CARIOTIC Bacteria: gram positive Bacillus subtilis 168 apr A subtilisin 1168, apr ABSS168 Bacillus amyloliquefaciens apr subtilisin BPN '(NOVO) BASPPN Bacillus subtilis DY - subtilisin DY BSSDY Bacillus licheniforms + Carlberg subtilisin BLSCAR Bacillus lentus + subtilisin 147 BLS147 Bacillus alcalophilus PB92 + subtilisin PB BAPB92 Bacillus sp. DSM 4828 - alkaline protease BDSM48 Bacillus YaB but rules elastase. YaB BYSYAB Bacillus subtilis 168 epr min. extracell.prot. BSEPR Bacillus subtilis bpf bacillopeptidase F BSBPF Bacillus subtilis IFO3013 ispl intracell.ser. BSISP1 Bacillus subtilis A50 - intracell.ser.prot. BSIA50 Bacillus thuringiensis - extracell.ser.prot. ETFINI Bacillus cereus - extracell.ser.prot. BCESPR Nocardiopsis dassonvillei - basic cheese prot. NDAPII Thermactinomyces vulgaris - termitase TVTHER Enterococcus faecalis cylA cytolysin component A EFCYLA Staphylococcus epidermidis epiP epidermin lead.prot. SEEPIP Streptococcus pyrogenes scpA C5a peptidase SPSCPA Lactococcus lactis SK11 prtP SK11 cell wall prot. LLSK11 Gram-negative bacteria Dichelobacter nodosus + alkaline protease DNEBPR Xanthomonas campestris + extracellular prot. XCEXPR Serratia marcescens + Exrtacell. ser.prot. SMEXSP Thermus aquaticus YT-1 pstI aqualysin I TAAQUA Thermus rT41A + T41A protease TRT41A Vibrio alginolyticus proA protease A VAPROA Streptomyces rutgersensis - proteinase D SRESPD Archaea halophilic strain 172P1 - halophil extra.prot. ARB172 Cyanobacteria E. Anabaena variabilis work Ca-dependent protease AVPRCA

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Table 1 - continued

1 2 3 4 Lower eukaryotic Mushrooms Tritirachium album Limber + proteinase K TAPROK Tritirachium album + proteinase R. TAPROR TritreacCium album proT proteinase T. TAPROT Aspergillus oryzae + alkaline protease AOALPR Malbranchea pulchella - thermomycoline MPTHMY Acremonium chrysogenum alp alkaline protease ACALPR Yeast Klnyweromyces lactis kex1 kex1 (tr. proteinase KLKEX ^ 1 Sacharomyces ahehvrsrae kex2 kex2 ser. proteinase SCKEX2 Sacharomyces ahrhvrsiae prb1 protease B SCPRB1 Yarrowia hpolytica xpr2 as. extrαctll.prot. YLXPR2 Higher karyotic Worms Cahnorhabditis elegans closer4 protease cuticle CEBLI4 Insects Drosophila (fruit fly) furl furin1 DMFUR1 Drosophila (fruit fly) fur2 furin2 DMFUR2 Plants. Cucumis melo (melon) « cncnmi (in CMCUCU Mammals Man (also rat, mouse) fur furin HSFURI Human (also mouse) + insulinoma PC2 prot. HSIPC2 Mouse + pituitary PC3 prot. MMPPC3 Man + tripeptidil peptid. II HSTPP

References used in Table I.

Amino acid sequence references (links to GenBank® / EMBL Data Bank data are given in parentheses):

ARB172 Kimiekura and Seno. (1990) Biochem. CellBioL 68 352-359 (sequenced amino acids of mature protease residues 1-35; residue 14 not specified).

BSS168 Stahl) and Feraari. 11984) J. Bacteriol. 158411-418 (K01988). Yoshimoto. Oyamai et al. (1488) J Biochem. 103 1060-1065 (mature subtilisin from B. subtilis var. Amylosacchariticus distinguished by having T130S and T162S). Svendsen et al. (1986) FEBS Lett. 196 228-232 (PIR A23624; amino acid sequencing; B. meseμμ mature alkaline mesentericopeptidase distinguished by having S85A, A88S, S89A, S183A and N259S).

BASBPN WeHsinm) 11983) Nuci Acids Res 11 7911-7925 XC00165. Vasantha and irmi (1984) J. Bacteriol. 159 811-814 (K02496).

BSSDY Nedkov et al. (1983) Hoppe-Seylers Z. PhysioL Chem. 364 1537-1540 (PIR A00969; amino acid sequencing).

BLSCAR Jacobs and ΐηηΐ (1985) NucL AcidsRes 13 8913-9926. Smith and ϊηηΐ (1968) J. Biol Chem. 243 2184-2191 (PIR A00968; amino acid sequencing; mature protease sequence that differs from T103S, P129A, S158N, N161S and S212N).

BLS147 Hastnip and hmi (1989) PCT patent WO ') 89/06279. publ. July 13

1989 (Esperase® from B. lentus). Takami et al. (1990) Appl. Microbiol. Biotechnol. 33 519-523 ((Equivalent amino acid residues of the alkaline protease residues 1-20 from Bacillus sp. No. AH-101; this sequence differs from BLS147 by having N11S).

184 399

ΒΑΒΡ92

BDSM48

BYSYAB

BSEPR

BSBPF

BSISP1

BSIA50

BTFINI

BCESPR

NDAPII

TVTHER

EFCYLA

SEEPIP

SPSCPA

DNEBPR

LLSK11

XCEXPR

SMEXSP

TAAQUA

TRT41A

VAPROA

SRESPD

AYPRCA van der Laan et al. (1991) Appl. Erwiron. Microbiol. 57 901-909. (Maxacal®). Hastrp et al. (1989) PCT patent application WO 89/06279, published July 13, 1989 (subtilisin 309, Savinase® from B. lentus is only distinguished by the possession of N873). Godette et al. (1991) Abstracts 5th Protein Society Symposium, June 6, Baltimore: abbreviation M8 (highly alkaline protease from B. lentus is distinguished by having N87S, S99D, S101R, S103 A, V1041, and G159S). Rettenmaier et al. (1990) PCT patent application WO 90/04022, published April 19, 1990.

Kaneko et al. (1989) J. Bacteriol. 171 5232-5236 (M28537).

Slomai others (1988) 7. Bacteriol. 1705557-5563 (M22407). Bruckner (1990) Mol. Gen. Genat. 221 486-490 (Χ53307).

Słoma et al. (1990) J. Bacteriol. 172 1470-1477 (M29035; revised). Wu et al. (1990) J. Biol. Chem. 265 6845-6850 (J05400); this sequence differs in having A169V and 586 less C-terminal residues according to structure).

Koide et al. (1986) J. Bacteriol. 167 110-116 (M13760).

Strongin et al. (1978) 7. Bacteriol. 133 1401-1411 (mature protease sequenced amino acids 1-54; residues 3; 39; 40; 45; 46; 49 and 50 not specified).

Chestukhina et al (1985) Biokhimiya 50 1724-1730 (amino acid sequenced mature protease of residues 1-14 from B. thurigiensis variety israeliensis, and residues 1-16 and 223-243 from cultivar initimus). Kunitate et al. (1989) Agric. Biol. Chem. 53 3251-3256 (amino acid sequenced mature protease of residues 6-20 from cultivar kurstaki. BTKURS).

Chestukhina et al (1985) Biokhimiya 50 1724-1730 (mature amino acid sequences 1-16 and 223-243).

Tsujibo et al (1990) Agric. Biol. Chem. 54 2177-2179 (mature amino acid sequencing residues 1-26).

Meloun et al. (1985) FEBS Lett. 183 195-200 (PIR A00973; (mature protease amino acid sequencing of residues 1-274).

Segarra et al. (1991) Infect. Immun. 59 1239-1246.

Schnell et al. (1991) personal report (Siezen et al. (Supray).

Chen et al. (1990) 7. Biol. Chem. 265 3161-3157 (J05224).

Kortt et al. (1991) Abstract% th Protein Society Symposium, June 22-26, Baltimore, acronym S76.

Vos et al. (1989) 7. Biol. Chem. 264 13579-13585 (J04962). Kok et al. (\ 9% $) Appl. Environ. Microbiol. 54231-238 (M24767); the sequence from the Wg2 chain differing at position 44, includes 18 protease domains differences, and deletion of residues 1617-1676). Kiwaiki et al. (1989) Mol. Microbiol. 3 359-369 (X1413 0; CD0763 N sequence differing in position46, includes protease domain 22 and deletions of residues 1617-1676).

Liu et al. (1990) Mol. Gen. Genet. 220 433-440.

Yanagida et al. (1986) 7. Bacteriol. 166 937-994 (M13469).

Terada et al. (1990) 7 Biol. Chem. 265 6576-6581 (J054I4).

McHale et al. (1990) Abstracts 5th Eur. Congr. Biotechn. Christiansen, Munck and Villadsen (eds), Munksgaard Int. Publishers, Copenhagen.

Deane et al. (1989) Gene 76 281-288 (M25499).

Lavrenova et al. (1984) Biochemistry USSR. 49 447-454 (amino acids sequenced from residues 1-23; residues 13, 18, and 19 not specified).

Maldener et al. (1991) Mol. Gen. Genet. 225 113-120 (the published sequence has 28 undefined residues near positions 2000-210 according to the error in reading the structure).

184 399

TAPROK

TAPROR

TAPROT

AOALPR

MPTHMY

ACALPR

KLEX1

SCKEX2

SCPRB1

YLXYPR2

CEBL14

DMFUR1

DMFUR2

CMCUCU

HSFURI

HSIPC2

MMPPC3

HSTPP

Gunkel and Gassen (1989) Eur. J Biochem. 179 185-194 (X14688 / XI4689). Jany et al. (1986) J. Biol. Chem. Hoppe-Seyler 367 87 (PIR A24541; amino acid sequencing; mature protease differs in having S745G, SILST204-208DSL and VNLL264-267FNL).

Samai et al. 11990) Mol. Mirrobiol. 4 17894792 ¢ 556116).

Samai and mni 0989) Gnni 85 329-333.

Tatsumi et al. (1989) Mol. Gen. Genet. 219 33-38. Cheevadhanarah et al. (1991) EMBL Data Library (X54726).

Gaucher and Stevenson 91976) Methods Enzymol. 45,415-433 (sequenced amino acids residues 1-28, and LSGTSM hexapeptide with serine active center).

Isogal 1 ńmi 11991) Agbic. ΒίοΙ. Chem. 55 471477. Stepimov et al. (1986) Int. J. Biochem. 18 369-375 (amino acids of residue L27 were phoned; mature protease differing in having H13 [1] Q, R13 [2] N and S13 [6] A).

Wescdowkiii-Louel and Fukthaaaa 11988i FEBS lett. 234 464-470 (X07038).

Mizuno et al. 0988i Bucchem. Bóopyys. Rss. Commnn. 156 246-254 (M24201).

Moehle and ίηηί 0987i Mol. Clll. Βίοι 7 4390-4399 (Ml8097).

davidow and union 11987IJ. ΒαΡ ^ Μ. 169 4627 4629 (M1774)). Maooba et al. (1988) Mol. Cell. Biol. 8 4904-4916 (M23353).

Peter and Roee 099) and Des Noem BeeedrEs Gazette 1 and 2 ..

Roebooek and Mni 199) and FEBS Lett. 289 133437 0559384).

Roebooek and mni Ο992) 267 ^ 12 ^^ 1 ^ 255.

Kaneda and ΐηη i 19,984) J. Βϊαοίκτη. 95 825-829 IequencSonated ammo acids of the NIISGTSM octapeptide with an active serine center). vrni den ΟποιΙ ^ Ι ibrni 09901 NuoI. AiiSs Rss. The murine furin seelection, which differs at position 51, comprises five catalytic domains: A15E, Y21F, S223F, A232V and N258 [2] D0. MISUMII OTHERS (1990) Nucl. Acids Res. 18 6719 (X55660: Rat furin sequencing differing at position 49, includes 3 catalytic domains: A15E, Y21f, H24R). Dragon and Slemrr0990) 7. ΒΐοΙ. Chem. 26i 29974ΟΟΙ (J05252). Seddah et al. (1990) DNA Cell Biol. 9 415-854 (mouse pituitary PC2 protease sequence different at position 23, including seven protease domains: I4F, S42 [2] Y, E45D, N76S, D133E, V134L and G239 [1] D).

Smeekens imni 0991) PPooc. Natl.Aadd Uci. USAi 88, 340-344 (58572). Stidah et al. (1990) DNA Cell Biol. 9 415-424 (M55668 / M55669; partial sequence)

Tonkemson and ennISon099) and Bóohhemietry 30 168 474 (0 ^ 299 ^).

Brief description of the figures

In the drawings, figure 1 shows the alignment listed in table I;

Figure 2 is a three-dimensional representation of subtilisin 309 showing the position of the hydrophobic domains and some substituted amino acid residues in their vicinity.

It has surprisingly been found that the storage stability and / or performance in detergent compositions is improved when the amino acid residues adjacent to the hydrophobic domains contain residues P129, P131,1165, Y167, Y171 of subtilisin 309 substituted with more hydrophobic residues. Especially the rest of R170.

Figure 2 shows the hydrophobic domains in subtilisin 309 and the adjacent residue numbers substituted to increase the hydrophobicity of the domain. This can be achieved by substituting hydrophobic residues for non-hydrophobic residues and / or by substituting residues to make them more hydrophobic than in the parent enzyme.

The same principles have been applied to the corresponding hydrophytic domains of other subtilases, the identification of which is apparent to one of ordinary skill in the art. Graphical representations, as in Figure 2, may be made for other subtilases to identify targeted residues for substitution in accordance with the invention.

Their number is presented below in Table II:

Table II

Rests of hydrophobic domains and their neighborhoods

Pos. \ Enzyme BASBPN BLSCAR BLS309 BLS147 TVTHER Domain 129 P. AND P. T. T. 131 G. G. P. G. G. 165 V AND AND V P. 167 Y Y Y Y Y 171 Y Y Y Y Y Neighborhood 136 K. K. E. E. Q 159 S. S. G. G. T. 164 T. T. S. G. AND 170 K. K. R R Y 194 P. AND AND P. S. 195 E. E. G. E. V

Table II is constructed according to the alignment shown in Figure 2. It will be appreciated that similar or larger tables covering other subtilases can be readily produced by those skilled in the art.

The invention uses variants of subtilase in which the amino acid sequences have been changed by mutations in the gene of the subtilisin enzyme that is designed to be modified (parent enzyme or gene) in the codon responsible for expressing the amino acid residues at positions 129, 131, 165, 167, 171, 136, 159, 164, 170, 194 and 195, the residues of which are more hydrophobic than the residue (s) in the parent enzyme, especially those hydrophobic residues that contain a relatively long hydrophobic chain, such as Ile, Leu, and Val, where, if the mutant gene is expressed, the amino acid residue is substituted with a more hydrophobic residue which increases the hydrophobicity of the domain as such.

Generally the hydrophobic residues are as follows: Val (V), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), and Trp (W). Among them, Val, Ile and Leu are preferred. Looking at Table II and applying the principles of the invention, a number of suitable candidates for substitution can be identified.

For both BASBPN and BLSCAR seem suitable positions 136, 159, 164, 167, 170 and 195 for substitutions. In BLS309, positions 136, 164, 167 and 170 would be selectable first, and positions 159 and 195 would be second. In BLS147, positions 136, 167, 170, and 195 would be first and positions 159 and 164 second. Finally, on TVTHER, positions 136, 167 and 194 are first and 164 are second.

This will have the benefit of substituting the Gly residue in the hydrophobic domain for a more bulky and hydrophobic residue.

Such considerations apply to hydrophobic or hydrophobic residues that may occupy any of the listed positions, meaning that any increase in hydrophobicity appears to be beneficial. This means that e.g. highly hydrophytic residues such as charged residues Arg (A), Asp (D), Glu (E), or Lys (K) can be substituted with any residue that is

184,399 less hydrophilic. Such less hydrophilic residues include Gly (G), Cys (C), Ser (S) Ala (A), Thr (T), Tyr (Y), Gln (Q), His (H), or Asn (N) residues.

Similar considerations apply to other subtilases having hydrophobic domains at this portion of the enzyme surface.

In the context of the present invention, subtilase is defined according to Siezen et al. above. In a narrower sense, suitable subtilases are those which belong to the subgroups I-S 1 and I-S2, applicable to many embodiments of the invention. In a more specific sense, many embodiments of the invention pertain to the serine protease of gram-positive bacteria, which may be subsumed with substantially unambiguous homology in their primary structure, to the subtilases listed in Table I above.

One or more substitutions at the above-defined positions are possible in combination with another substitution, deletion or addition to an amino acid sequence in the parent enzyme. In particular, combinations with other substitutions known to improve enzyme properties are contemplated.

Such combinations include items: 222 (improved aerobic stability), 218 (improved thermal stability), substitutions at Ca binding sites stabilizing the enzyme, e.g., item 76, and many others distinguishing from the prior art. Moreover, specific combinations are also contemplated with the variations indicated in EP-A-405 901.

Variations

A: single varieties:

Subtilisin BPN ', Subtilisin Carlsberg, Subtilisin 168 and Subtilisin DY varieties:

K136V, K13 ^ I, K136L, K136M, K136F, S159V, S1591, S159L, S159M, S159F, T164V, T1641, SKUL, SI UHM, S164F, K170V, K1701, KI70L, K170M, K170F, E195V, E1951, Varieties of termitase: E195L, E195M, E195F, Q136V, Q136L, ęiUM, Q136F, T159V, Tl 591, T159L T159M, T159F, A164V, Al ^^ I, A164L, A164M, A164F, Y167V, YH7I, Y167L, Y167M, Y167F Y170V, Y110I, Y170L, Y170M, Y170F, S194V, H44L, SH4M, S194F, Variants of Subtilisin 309, Subtilisin 147 and Bacillus PB92 protease: E136V, E136I, E134L, E136F, G159V, G1 ^^ 9, G194L, G159M, G164V, G1 ^^^ 4, dUL, GlUtM, <^ 1 ^^ «,, (BLS147) S164V, S ^ L ^ L, SHHM S164F, (BLS309 and BAPPB92) Y167V, Y ^ H, YHLL, Y167M, R170V, RUM, (both off for PAPB92) R170L, R170M, (disabled for PAPB92), RI00F, R170GIRI00C A194V, Aim, A194L, A194F, (BLS309 ΐ BAPB99) P194V, P19U, P194L, PWM, P194F, (BLS147) E195V, E ^, E195L, EmM, E195F, (BLS447) G195V, G195I, B: combination variations: Gl95L, G195M, G195F, ^^ LS399 and BAPB92)

Each of the above-mentioned varieties is contemplated to provide benefit when combined with another variation in any of the following items: 27, 36, 57, 76, 101, 104, 123, 218, 222, 224, and 274.

In particular, the following BLS309 variants are considered suitable for the combination:

K27R, * 36D, S57P, N76D, S101G, V104A, V104N, V104Y, N123S, A194P, Q206E, N218S, M222S, M222A, T224S and T274A.

184 399

Such variations also contain any one or two of X167L, X167I, X170L, and / or X170I in combination with any one or more of the other substitutions, deletions, and / or insertions indicated above as being preferred.

Moreover, the variants include any of the variants V104N + S101G, K27R + V104Y + + N123S + T27A or N76D + V104A, in combination with any one or more substitutions, deletions and / or insertions indicated above as appearing to exhibit improved properties.

Specific combinations are listed previously.

The cleaning and detergent compositions of the invention are generally in the form of liquid detergent compositions, but subtilase forms can be incorporated into detergent compositions in the form of bars, tablets, sticks and the like for direct use which exhibit improved enzyme stability.

Preferably the compositions according to the invention are especially aqueous liquid detergents having, for example, homogeneous physical properties, e.g. they may contain a micellar solution of surfactants in a continuous aqueous phase, so-called isotropic liquids.

Alternatively and advantageously, they may be physically heterogeneous and structured, e.g. containing a dispersion of lamellar droplets in a continuous aqueous phase, e.g. containing an anti-flocculation polymer having a hydrophilic backbone and at least one hydrophobic side chain as described in EP-A -346,995 (Unilever) (incorporated herein by reference). The latter liquids are heterogeneous and may contain a suspension of solids, such as particles of filler material, e.g. of the types described below.

Preferably, the inventive liquid cleaning and detergent compositions should have a high concentration of electrolytes, as described in EP-A-328177, EP-A-359308, EP-A-328176, EP-A-346995 (all from Unilevera).

Such compositions contain one or more subtilisin enzyme variants in combination with any of the ingredients usually included in such compositions, well known to those skilled in the art.

Such ingredients include fillers such as phosphate or zeolite fillers, surfactants such as anionic, cationic, nonionic or zwitterionic surfactants, polymers such as acrylate polymers or their equivalents, bleaching systems such as perborate or amine-containing bleach precursors or activators, structuring agents such as silicone structurants, a base or acid to adjust the pH, humectants and / or neutral inorganic salts.

In addition, many optional ingredients typically found in compositions of the invention, such as:

A. Possible co-surfactants

B. Tartrate succinate filler

C. Neutralizing system

D. suds eliminating agent

E. Other enzymes

F. Any other ingredients

The weight ratio of synthetic anionic surfactant to ethoxylated nonionic surfactant is from 1: 1 to 5: 1. The compositions have a 10% by weight solution in solution at 20 ° C, a pH range of 7.0 to 9.0; a Critical Micelle Concentration less than or equal to 200 ppm and an air / water interfacial tension at critical micelle concentration less than or equal to 32 dyne / cm at 35 ° C in distilled water. Preferably the compositions are clear, homogeneous and phase stable and have good cleaning performance and good enzyme stabilization.

184 399

Various ingredients

1. Synthetic anionic surfactants

The liquid detergent compositions of the invention contain from about 10% to about 50%, preferably from about 15% to about 50%, more preferably from about 20% to 40%, and most preferably from 20% to about 30% by weight of natural or synthetic surfactants. . Suitable natural or synthetic surfactants are e.g. soaps and those disclosed in US-A-4,285,841, and US-A-3,929,678.

Suitable anionic surfactants include water-soluble salts, especially alkali metal, ammonium and alkylammonium salts (e.g. monoethanolamine or triethanolamine), sulfonation reaction products of organic compounds having an alkyl group in their molecular structure having from about 10 to about 20 carbon atoms, and sulfonic acid or sulfuric acid ester. (The term "alkyl" also includes the alkyl portion of an aryl group). Examples of this group of synthetic surfactants are alkyl sulfates, especially those obtained by sulfonating higher alcohols (C ₈ -C] 8 carbon atoms) such as those prepared by reducing tall oil or coconut oil glycerides; Alkylbenzene sulfates in which the alkyl groups contain from about 9 to about 15 carbon atoms in a straight or branched chain, e.g. as described in US Pat. Nos. 2,220,099 and US 2,477,383. Especially valuable are straight chain alkyl benzene sulfates with medium a number of carbon atoms in the alkyl group from about 11 to 14.

Other anionic surfactants are the water-soluble salts: paraffin sulfates containing from 8 to about 24 (preferably about 12 to 18) carbon atoms; alkylglyceryl ether sulfates, especially C8-C6 alcohol ethers (e.g., made from tall oil and coconut); alkylphenol ethoxylated ether sulfates containing from 1 to about 4 ethylene oxide units per molecule and from 8 to 12 carbon atoms in the alkyl group; and ethoxylated alkyl ether sulfates having 1 to 4 ethylene oxide units per molecule and from 10 to 20 carbon atoms in the alkyl group.

Other suitable anionic surfactants include water-soluble salts of α-sulfonated fatty acid esters containing from 6 to 20 carbon atoms in the fatty acid group and from 1 to 10 carbon atoms in the ester group; water-soluble salts of 2-acyloxyalkene-1-sulfonic acids containing from 2 to 9 carbon atoms in the acyl group and from 9 to 23 carbon atoms in the alkane moiety; water-soluble olefin sulfate salts containing from 12 to 24 carbon atoms; and β-alkyloxy alkane sulfates containing from 1 to 3 carbon atoms in the alkyl group and from 8 to 20 carbon atoms in the alkane moiety.

Preferred anionic surfactants are sulfates, C? -C? Alkyl and ethoxylated alkyl sulfates containing an average of 4 units of ethylene oxide per mole of alkyl sulfate, linear C _n -C | 3 alkyl benzene sulfates, and mixtures thereof.

2. Ethoxylated nonionic surfactants

The second optional ingredient is an ethoxylated nonionic surfactant in an amount of 2% to 14%, preferably 2% to 8%, more preferably 3% to 5% by weight. The weight ratio of synthetic anionic surfactant (acid base) to non-ionic surfactant is from 1: 1 to 5: 1, preferably from 2: 1 to 5: 1, more preferably from 3: 1 to 4: 1. Such ratios ensure the formation and adsorption of sufficiently hard surfactants at the air / water interface to ensure good removal of greasy / oily stains.

Ethoxylated nonionic surfactants are of the formula R (OC2H4) _n OH, wherein R1 represents -C _I0 -C ₁₆ group, or a Cs-C ^ alkylphenyl, n has a value of from 3 to 9, the nonionic surfactant has an HLB (hydrophilic-lipophilic balance) of from 6 to 14, preferably from 10 to 13. These surfactants are further described in US-a-4 285 841 and US-a-4 284 532. particularly preferred sąprodukty condensation of alcohols C ₁₂ - C 'with 3 to 8 moles of ethylene oxide per mole of alcohol, e.g., a C1-C6 alcohol condensed with about 6.5 moles of ethylene oxide per mole of alcohol. Other non-ionic agents

The surfactants that should be mentioned are APG, EGE and glucamide surfactants.

3. Detergent fillers

Among the typical detergent ingredients that may be present in conventional amounts in the detergent compositions of the present invention are the following: the compositions may or may not be builder-supported, and may be of the zero-P type (e.g., may not contain any phosphorus-containing builders). The compositions may contain, in the aggregates, for example from 1-50%, e.g. at least about 5% and often up to 35-40% by weight of one or more organic and / or inorganic fillers. Typical examples of fillers include the above-mentioned and, more broadly, alkali metal ortho-, pyro- and tripolyphosphates, alkali metal carbonates, both alone and in admixture with calcite, alkali metal citrates, alkali metal nitrilotriacetates, carboxymethyloxysuccinates, zeolites, polyacetalcarboxylates, etc.

More specifically, the compositions of the invention contain from 5% to 20%, more preferably from 10% to 15% by weight of a detergency builder which may be a fatty acid containing 10 to 18 carbon atoms and / or a polycarboxylate, zeolite, polyphosphate and / or polyphosphonate builder. Preferably, the compositions contain from 0 to 10% (more preferably from 3% to 10%) by weight of saturated fatty acids containing from 12 to 14 carbon atoms together with from 0 to 10%, more preferably from 2% to 8%, most preferably from 2% to 8%. % to 5% by weight of a polycarboxylated builder, most preferably citric acid, in a weight ratio of from 1: 1 to 3: 1.

Since the proteolytic enzymes of the invention have the advantageous optimal storage stability unlike other enzymes when the ratio of filler to water hardness is close to one, the compositions preferably contain a sequestration-effective filler of from 2 to 10, preferably from 3 to 8, grains per gallon of hardness.

Suitable saturated fatty acids can be obtained from natural sources such as vegetable or animal esters (e.g. palm kernel oil, palm and coconut oil) or synthetically produced (e.g. by oxidation of crude oil or hydration of carbon monoxide by the Fisher-Tropsch process). Examples of saturated fatty acids suitable for use in the compositions of the invention are capric, larinic, myristic, coconut and palm kernel fatty acids. The saturated coconut fatty acids are preferred; in a 5: 1 to 1: 1 (preferably about 3: 1) weight ratio of a mixture of lauric and myristic acids; mixtures of the above-mentioned with minor amounts (e.g. 1% -30% of total fatty acids) of oleic acid and palm kernel fatty acid.

The compositions of the invention preferably also contain the polycarboxylate, polyphosphate and polyphosphonate builders described in US-A-4,284,532; water-soluble polycarboxylate builders, especially citrates, are preferred. Suitable polycarboxylate builders include various amino polycarboxylates, cycloalkane polycarboxylates, alkyl polycarboxylates, polycarboxylates, tetrahydropolycarboxylates, benzene polycarboxylates, and polyacetyl polycarboxylates.

Examples of such polycarboxylate builders are sodium and potassium ethylenediaminetetraacetate; sodium and potassium nitrilotriacetate; water-soluble salts of phytic acid, e.g. sodium and potassium phytate as disclosed in US-A-1,739,942, the polycarboxylate materials described in US-A-3,364,103; and the water-soluble salts of the polycarboxylate polymers and copolymers described in US-A-3,308,067.

Other suitable detergency builders include water-soluble salts of polymeric aliphatic polycarboxylic acids having the following structural and physical properties: (a) a minimum molecular weight of about 350 calculated as the acid form; (b) an equivalent weight of 50 to 80 calculated as the acid form; (3) at least 45 mole% of the monomeric fragments have at least two carboxyl radicals separated from each other by no more than two carbon atoms; (d) the attachment point for the polymer chain 20

The chain of any carboxyl-containing radical is located not more than three polymer chain atoms from the point of attachment of the next carboxyl-containing radical. Specific examples of such fillers are polymers and comonomers of itaconic, aconitic, maleic, mesoinic, fumaric, methylene malonic and citraconic acid.

Other suitable polycarboxylate builders include the water-soluble salts, especially the sodium and potassium salts of mellitic acid, citric acid, pyromellitic acid, benzene pentacarboxylic acid, oxodiacetic acid, carboxymethyloxy succinic acid, carboxymethyloximalonic acid, cis-cyclohexanhexuric acid and cetisarboxylic acid, and cis-cyclohexanhexuric acid and cetisarboxylic acid.

Other polycarboxylates are the polyacetyl carboxylates described in US-A-4 144 226 and US-A-4 146 495.

Other detergency builders include zeolites such as the aluminosilicate ion exchange materials described in US-A-4,405,483.

Other preferred fillers have the general formula R-CH (COOH) _CH2 (COOH) ie. Derivatives of succinic acid, wherein R is a C ₁ 7 alkyl or C37-alkene, preferably C ^ -CN, or wherein R may be substituted by by a substituent which is hydroxy, sulfo, sulfoxy or sulfone. Such succinate builders are preferably used in the form of their water-soluble salts, including the sodium, potassium or alkanolammonium salts. Specific examples of succinate fillers include: lauryl succinate, myristyl succinate, palmitic succinate, dodecenyl succinate and the like.

4. Proteolytic enzymes

The enzymes of the invention can be used in detergent compositions in well-known standard amounts. These amounts can vary widely, for example about 0.0002-0.1, e.g. about 0.005-5, Anson units per gram of the detergent composition. Expressed in alternative units, proteases may be included in the compositions in an amount, depending on the formulation of the composition, from about 0.1 to 100 GU / mg (e.g. 1-50, especially 5-20 GU / mg), or any broadly varying amount. centering at about 0.01-4, e.g., 0.1-0.4 KNPU per gram of the detergent formulation.

For example, it may be convenient to use the enzymes in an amount of about 0.25 mg protein enzyme per liter of washing liquid, corresponding to an enzyme activity of 0.08 KNPU per liter *. Suitable detergent formulations may contain the enzymes, for example, in an amount to provide 0.1-0.4 KNPU / g.

In other words, the inventive composition comprises from about 0.01% to about 5%, preferably from about 0.1% to about 2%, by weight of the proteolytic enzymes of the invention.

The described proteolytic enzyme is preferably included in an amount effective to provide an activity of from 0.05 to about 1.0; more preferably from about 0.1 to 0.75; most preferably from about 0.125 to about 0.5 mg of active enzyme per gram of composition.

5. Enzyme stabilizing system

The liquid detergents according to the invention may contain an enzyme stabilizing An system containing calcium ion, boom acid, propylene glycol and / or short chain carboxylic acids. The enzyme stabilizing system comprises from about 0.5% to about 15% by weight of the composition.

The composition preferably contains from about 0.01 to about 50, preferably from about 0.1 to about 30, more preferably from about 1 to 20 millimoles of calcium ion per liter. The calcium level should be selected so that there is always a certain minimum level available for the enzyme after complexation with the filler in the composition. Any water-soluble salt can be used as the calcium ion source, including calcium chloride, calcium formate, and calcium acetate. Small amounts of calcium ion, typically from about 0.05 to 0.4 millimoles per liter, are often present in the composition due to the presence of calcium in the enzyme slurry and water. From about 0.03% to about 0.6% of calcium formate is preferred.

184 399

The second preferred enzyme stabilizer are polyols containing only carbon, hydrogen and oxygen atoms. Preferably they contain from 2 to 6 carbon atoms and from 2 to 6 hydroxyl groups. Examples include propylene glycol (especially 1,2-propanediol, which is preferred), ethylene glycol, glycerol, sorbitol, mannitol, and glucose. The polyols are typically present in an amount from about 1.5% to about 8% by weight of the composition. Preferably, the weight ratio of polyols to added boric acid is at least 1, more preferably at least 1.3.

In addition, the compositions preferably contain the water-soluble short chain carboxylates described in US-A-4,318,818. Formates are preferred and may be used in an amount of from about 0.05% to about 5%, preferably from about 0.2% to about 2%. %, most preferably from 0.4% to 1.5% by weight of the composition. Sodium formate is preferred.

The compositions of the invention preferably contain 3% to 3% boric acid in an amount from about 0.25% to about 5%, more preferably from about 0.5% to about 3% by weight. The boma acid may be, but is preferably not, formed by compounds capable of generating boric acid in the composition. Boma acid is preferred, however other compounds such as boric anhydride, borax, and other alkali metal borates (e.g. sodium ortho-, meta- and pyroborate, sodium pentaborane) are suitable. Substituted boric acids (e.g., phenylboronic acid, butanoboric acid, and p-bromophenylboronic acid) may also be used in place of boric acid.

6. Water

The liquid detergent compositions of the present invention may be aqueous or non-aqueous liquids. If they are aqueous liquids, they contain from about 5% to about 60%, preferably from about 25% to about 45%, by weight of water.

Further optional ingredients

A. Possible co-surfactants

Optional co-surfactants for use with these toxic nonionic surfactants include amides of the formula:

OR ²

X! ' 1 3 wherein R is an alkyl, hydroxyalkyl or alkenyl radical containing from 8 to 20 carbon atoms, and R2 and ^R3 are selected from hydrogen, methyl, ethyl, propyl, isopropyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, and said radicals additionally contain up to 5 ethylene oxide units with the proviso that at least one of R2 and R3 contains a hydroxyl group.

Preferred amides are alkilooli _C8-C20 fatty acids, in which each alkiloolu group contains from 1 to 3 carbon atoms, and additionally can contain up to 2 ethylene oxide units. Particularly preferred are the mono- and diethanols of the C, 1 -C, 6 fatty acid amides.

If used, the amides are present in the compositions in a weight ratio of ethoxylated nonionic surfactant to amide surfactant of from 4: 1 to 1: 4, preferably from 3: 1 to 1: 3.

Preferred optional co-surfactants used in an amount of 0.15% to 1% are the quaternary ammonium, amine and amine oxide surfactants described in US-A-4,507,219.

Of these, the salts of _{C) 0} C _M alkyl sulfates, for example. Methyl, decyl, lauryl trimethylammonium chloride, bromide mirystylotnmetyloamoniowy chloride and methylsulfate and cocotrimethylammonium. 0.2% to 0.8% of monoalkyl trimethyl ammonium chloride is preferred.

B. Tartrate succinate carrier

Preferred compositions of the invention may contain from 0 to about 10%, preferably from 0 to about 6% by weight in acid, of a tartrate succinate carrier material selected from the groups consisting of

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i) HOCH - CH - O - CH - CH ₂ and I and I coox coox coox coox wherein X is a salt-forming cation;

ii) CH ₂ - CH - O - CH lilii coox coox coox coox coox

CH - O - CH - CH ₂

And coox wherein X is a salt-forming cation; and iii) mixtures thereof.

Tartrate succinate compounds for use in the present invention are described in US-A-4,663,071.

C. Neutralizing system

The compositions of the invention may also optionally contain from about 0 to about 0.04 moles, preferably from about 0.01 to 0.035 moles, most preferably from about 0.015 to about 0.03 moles, per 100 grams of the composition, alkanolamines selected from: monoethanolamine, diethanolamine, triethanolamine and mixtures thereof. The low content of alkanolamines, especially monoethanolamine, is beneficial in enhancing product stability, detergency and fragrance. However, the amount of alkanolamine should be minimized for best chlorine bleaching compatibility.

Additionally, the compositions contain sodium ions, and preferably potassium ions, in an amount effective to neutralize the ammonium fragments and to provide an appropriate pH.

D. suds eliminating agent

Another optional ingredient for use in the liquid detergent compositions of the invention in an amount of from 0 to about 1.5%, preferably from about 0.5% to about 1.0% by weight of the silicone based suds eliminator.

Silicones are widely known and used agents with high suds elimination efficiency. For example, US-A-3,455,839 discloses compositions and methods for defoaming aqueous solutions by incorporating therein small amounts of liquid polydimethylsiloxane.

Suitable suds regulating silicones are a mixture of silicone and silanated silica as described in the publication of German patent application DE-A-2 124 526.

Silicone defoamers and suds regulating agents have been successfully incorporated into granular detergent compositions by protecting them from detergent surfactants as described in US Patents 3,933,672 and US 4,652,392.

Preferably the silicone suds eliminating agent is used in the suds eliminating amount in the present invention, the suds eliminating agent consists essentially of:

(i) a polydimethylsiloxane fluid having a viscosity from about 20 cs. to about 1500 cs. at 25 ° C;

(ii) from about 5 to about 50 parts per 100 parts by weight of (i) a siloxane resin consisting of (CH ₃ ) ₃ SiO _{I / 2} units and SiO ₂ units in the ratio of (CH ₃ ) ₃ SiO _1/2 units to units S1O2 from about 0.6: 1 to about 1.2: 1; and (iii) from about 1 to about 20 parts per 100 parts by weight of (i) solid silica gel.

The term "suds-eliminating amount" means that the formulator can select the suds controlling amount to control suds to the desired level. The amount of suds depends on the selected surfactant. For example, high suds control surfactants use relatively more suds control agent to achieve the desired suds control than low suds surfactants.

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E. Other enzymes

The detergent compositions according to the invention may contain further enzymes.

For example, the lipase may be added in the form of a granular composition (alternatively a solution of the enzyme lipsIityczeogs slurry with a carrier material (e.g. as wEP-A-258 068 (Novo Nordisk A / S).

The amount of lipase added can vary widely, e.g. 50 to 30,000 LU / g per gram of surfactant system or detergent composition, e.g. often at least 100 LU / g, preferably at least 500 LU / g, sometimes preferably above 1,000, greater than 2,000 LU / g or greater than 4,000 LU / g or greater, very often in the range from 50 - 4,000 LU / g, and acceptable in the range of 200 - 1,000 LU / g. In this specification, the lipase units are defined as in EP-A-258 068.

The lipolytic enzyme may be selected from a wide range of lipases, in particular, the lipases described in, for example, the following patents: EP-A-214 761 (Novo Nordisk A / S), EP-A-258 068, and in particular lipases having immunological cross-activity. with antiserum against lipase from Thermomyces lanuginosus ATCC 22070, EP-A-505 208 and EP-A-206 390, in particular lipases showing immunological cross-activity with antisera against lipase from ChromoOacter viscosum var Lipolyticum NRRL B-3673, or against lipase from Alcaligenes PL.-679, ATCC 31371 and FERM-P 3783, also lipases described in WO 87/00859 (Gist-BrscedoI) and EP-A-504 284 (Sapporo Breweries). In particular, the following commercially available lipase formulations, for example, are suitable: LipsleIo® Novo Nordisk A / S, Amano lipases CE, P, B, AP, M-AP, AML and CES, and Meito lipases MY-30, OF and PL. , also Esterne® MM, Lipozym, SP225, SP285 (all Novo Nordisk A / S), Saiken lipase, Enzeco lipase, Toyo Jozo lipase and Dio ^ are lipid (trademarks), Lumafast® (Genecor Inc.), Lipomax® ( GiIt-Brocedo I.

N. V.), and lipases described in WOiA-98/03578 (Uni-yer).

Amylases may be used when needed, in an amount ranging from about 1 to about 100 MU (units of maltose) per gram of detergent composition (or 0.014-1.

0.17-0.7 KNU / g (Noyo units)). Examples of suitable amylases are e.g. Termamyl® and BAN (Novo Nordisk A / S). For example, when desired, cellulases may be used in an amount ranging from about 0.3 to about 35 CEVU units per gram of detergent composition. Suitable coIuIazemi are e.g. CeH ^ yme® and Canz ^ e® (Novo Nordisk A / S).

Other enzymes suitable for use in the present invention are oxidases and porsxidases.

F. Other Optional Ingredients

Other optional ingredients for use in the liquid detergents of the invention are stain removers, stain-disrupting polymers, anti-deposition agents such as tetraethyleopentoxide (from about 0.5% to 3%, preferably from about 1% to about 3% by weight), suds regulators, hydrotropes such as sodium coumensulphoniums, fluffs, entericides, bactericides, dyes, perfumes and bleaches known in the art. Such optional ingredients generally constitute less than 15%, preferably from about 0.5% to about 10%, more preferably from about 1% to about 10% by weight of the composition.

The composition may contain from about 0% to about 8%, preferably from about 0% to about 5%, by weight of Cu-Cm alkee succinic acid or its soybean. These materials have the general formula R-CH (COOX) CH ₂ (COOX), wherein R is a Cu-Cn group and each X is hydrogen or a suitable cation such as sodium, potassium, ammonium or eikeno-ion (e.g. mono-, di- or trieteeoIoemiea). Specific examples are 5-dsdeceeyIsbursztyeian (preferred) and 2-tetredeceeyIsbursztyeiee.

Compositions of the invention optionally contain from about 0.1% to about 1%, preferably from about 0.2% to about 0.6% by weight of the water-soluble salts of ethyleotriemieotetramethylphosphate, diethylene triemin pentemethyleophosphonic acid, ethylensdiemiestetraoctswegs (preferred) or diethyleneglycol ester (most preferred) ) to increase the cleaning effect of the fabric treatment.

In addition, the detergent compositions may contain from 1-35% of a bleach or bleach precursor or system containing a bleach and / or its precursor.

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Further optional ingredients are foam extinguishing agents, anti-corrosion agents, soil suspending agents, sequestering agents, anti-redeposition agents, etc.

The compositions of the invention contain up to about 10% ethanol.

G. Other Properties

Direct use compositions typically at a dilution of 10% by weight in water at 20 ° C have a pH of from about 7.0 to 9.0; preferably from about 8.0 to about 8.5.

Compositions for immediate use have a Critical Micelle Concentration (CMC) of less than or equal to 200 parts per million (ppm) and an air / water surface tension above the CMC of less than or equal to 32, preferably less than or equal to 30 dynes per centimeter at 35 ° C. C in distilled water. These measurements are described in "Measurement of Interfacial Tension and Surface Tension - General Review for Practical Man" C. Weser, GIT Fachzeitschrift fur das Laboratorium, 24 (1980) 642-648 and 734-742, FIT Verlag Ernst Giebeler, Darmstadt and " Interfacial Phenomena - Equilibrium and Dynamic Effects ”, CA Miller and P. Neogi, Chapter 1, pp. 29-36 (1985), Maecel Dekker, Inc. New York.

The compositions according to the invention may be used in the washing of textiles, especially, but not limited to, cotton and polyester-based textiles, and mixtures thereof. For example, a washing process conducted at a temperature of about 60-65 ° C or less, e.g., about 30-35 ° C or less, is particularly suitable. It may be very suitable for applying the composition at a rate effective to provide about, e.g., 0.4-0.8 g / L surfactant in the wash liquor, however, it is of course possible to use lower or higher concentrations if desired. For example, without limitation it can be maintained so that a use rate of from about 1 to 10 g / L, e.g. from about 3-6 g / L, of the detergent formulation is suitable for use where the formulations are essentially of the composition as in the examples.

In this aspect, the invention particularly relates to:

a) a detergent composition in the form of an aqueous detergent liquid containing an anionic surfactant, nonionic surfactant, humectant, organic acid, caustic soda, with a pH adjusted between 9 and 10.

b) a detergent composition in the form of a non-aqueous detergent liquid containing a liquid non-ionic surfactant consisting essentially of a linear alkoxylated primary alcohol, triacetin, sodium triphosphate, caustic soda, perborate monohydrate bleach precursor and a quaternary amine bleach activator, with a pH adjusted to between 9 a 10.

c) an enzymatic liquid detergent composition providing a washing liquid having a pH of 9 or less when used at a rate corresponding to 0.4-0.8 g / L of surfactant.

d) an enzymatic liquid detergent composition providing a washing liquid having a pH of 8.5 or more when used at a rate corresponding to 0.4-0.8 g / L of surfactant.

e) an enzymatic liquid detergent composition providing a wash liquor with an ionic strength of 0.03 or less, e.g. 0.02 or less, using at a rate corresponding to 0.4-0.8 g / l of surfactant.

f) an enzymatic liquid detergent composition providing a wash liquor with an ionic strength of 0.01 or more, e.g. 0.02 or more, using at a rate corresponding to 0.4-0.8 g / l of surfactant.

It has been found that the above-defined subtilase variants can be advantageously incorporated into a detergent composition in the form of bars, tablets, sticks and the like for direct application to fabrics, hard surfaces or other surfaces. In particular, they can be incorporated into soap or soap / synthetic bar compositions which exhibit exceptional enzyme stability.

Detergent compositions in the form of bars, tablets, sticks and the like for direct use are e.g. described in South African patent 93/7274, incorporated herein by reference. Preferred such bar compositions contain, in addition to the subtilase variant:

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i) 25 to 80%, most preferably 25 to 70% by weight of an active detergent which is a soap or a mixture of soap and synthetic detergent active, calculated as anhydrous;

ii) 0 to 50% and most preferably 10 to 30% by weight of water;

iii) 0 to 35% and most preferably 0.1 to 30% by weight of filler.

In general, the amount of subtilase variant to be included in such compositions is such that the proteolytic activity corresponds to 0.1 to 100 GU / mg of the composition, preferably 0.5 to 20 GU / mg, most preferably 1.0 to 10 GU / mg. where GU / mg is glycine units per milligram.

Methods of producing mutations in subtilase genes

There are many ways to introduce mutations in genes. After briefly discussing the cloning of subtilase genes, methods for generating both statistical and specific mutations within the subtilase gene will be discussed.

Cloning of the subtilase gene

The subtilase-encoding genes can be cloned from any of the organisms indicated in Table I, especially gram-positive bacteria or fungi, by various methods well known in the art. First, genomes and / or a DNA cDNA library must be constructed using chromosomal DNA or template RNA from the subtilase producing organism. Then, if the subtilase amino acid sequence is known, homology tagged sample oligonucleotides can be synthesized and used to identify the encoding subtilisin clones from a bacterial DNA genome library or from a cDNA library. Alternatively, samples of the tagged oligonucleotide containing subtilase homolog sequences from a different bacterial chain or organism can be used as samples to identify subtilase encoding clones using hybridization and washing under lower strength conditions.

Yet another method of identifying subtilase producing clones consists of inserting DNA genome fragments into an expression vector such as a plasmid, transforming protease negative bacteria with the resulting DNA library genome, and then placing the transformed bacteria on agar containing a subtilase substrate such as skim milk. These bacteria containing the subtilase bearing plasmid will produce colonies around the halo of pure agar according to the consumption of skim milk by the secreted subtilase.

Generation of statistical mutations in the subtilase gene

After the subtilase gene has been cloned with an appropriate vector, such as a plasmid, a variety of methods can be used to introduce statistical mutations in the gene.

One method will be to incorporate the cloned subtilase gene as part of the retrieval vector into an Eschericia coli mutant chain.

Another method involves generating a single chain from a subtilase gene and then annealing a DNA fragment containing the subtilase gene with another DNA fragment such that part of the subtilase gene remains a single chain. This separated single chain region can be exposed by any number of mutagenizing agents, including, but not limited to, sodium bisulfite, hydroxyamine, nitrous acid, formic acid, or hydralazine. Specific examples of this method of generating statistical mutations are described by Shortle and Nathans (1978, Proc. Natl. Acad. Sci. U.S.A. 75 2170-2174). According to the method of Shortle and Nathans, the plasmid carrying the subtilase gene would be threaded by restriction of the cleavage enzyme of the gene. This strand will be inserted into the gap using the action of polymerase I DNA exonuclease. The resulting single chain gap can be mutagenized using any of the above-mentioned mutagenizing agents.

Alternatively, the subtilisin gene from pieces of the Bacillus strain containing the natural promoter and other control sequence can be cloned into a plasmid vector containing replication of both E. coli and B. subtilis, a selective phenotypic marker and M 3 replication parent to generate a single DNA plasmid chain during superinfection with aiding with the IR1 phage. A single-chain DNA plasmid containing the cloned subtilisin gene is isolated and annealed to a DNA fragment containing the sequence vector but not the subtilisin coding region, producing a dashed double section.

184,399 sticks. Mutations are introduced into the subtilisin gene either by sodium bisulfite, nitrous acid or formic acid, or by replication of the mutating E. coli chain as described above. Since sodium bisulfite only reacts with cytosine in a single DNA strand, the mutation produced with this mutagen is limited to the coding regions only. The reaction time and concentration of sodium bisulfite vary from experiment to experiment, so that on average, one to five mutations in the subtilisin gene are formed. Incubation of 10 µg of the broken DNA double molecule in 4M sodium bisulfite, pH 6.0 for 9 minutes at 37 ° C in a reaction volume of 400 µl, deaminates approximately 1% of the cytosine in the single chain region. The coding region of the parent subtilisin contains approximately 200 cytosines, depending on the twist of the DNA. Preferably, the reaction time ranges from about 4 minutes (until a mutation frequency is one in 200) to about 20 minutes (about 5 in 200).

After mutagenesis, the disrupted molecules are treated in vitro with DNA polymerase I (Klevow fragment) to generate a complete double chain and to protect the mutations. The appropriate E. coli is then transformed with the mutagenized DNA to generate an extended library of subtilisin mutants. Extended mutant libraries can also be generated by growing plasmid DNA in the Mut D of the E. coli chain, which enhances the mutation range according to the pron error of the DNA polymerase.

The mutagenic nitrous acid and formic acid can also be used to generate a mutant library. Since these chemicals are not as specific to a single DNA chain as sodium bisulfite, the mutagenesis reactions are performed according to the following procedure. The coding portion of the subtilisin gene is cloned into the M13 fab by standard methods and a single DNA phage chain is generated. Single chain DNA is then reacted with 1M nitrous acid pH 4.3 for 15-60 minutes at 23 ° C, or with 2.4M formic acid for 1-5 minutes at 23 ° C. These reaction time ranges form a mutation frequency of 1 in 1,000 to 5 in 1,770. After mutagenesis, the universal primer is fused with M 3 DNA, duplex DNA is synthesized using the mutated single-chain DNA as template such that the coding portion of the subtilisin gene becomes completely double chain. At this point, the M13 vector can be cleaved from the coding region with a restriction enzyme and ligated into the non-mutagenized expression vector such that mutations occur only in the restriction fragment. (Myers et al., Science 229 242-257 ^ 1 ^^ 5) ^^.

Generation of targeted mutations in subtilase genes

If the cloned subtilase gene and the center and residues for the original substitutions selected for the identified mutation have been determined, then such mutations can be introduced using a synthetic oligonucleotide. These oligonucleotides contain side nucleotide sequences of a particular mutation site; the nucleoside mutant is inserted during oligonucleotide synthesis. In a preferred method, a single broken DNA chain carrying the subtilase gene is created in a vector carrying the subtilase gene. Then, a synthetic nucleotide bearing the desired mutation is fused to the homologous part of a single DNA strand. The remaining gap is then filled with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described by Morinaga et al. (1984, Biotechnology 2 646-639). According to Morinag et al., An intra-gene fragment is removed using a restriction endonuclease. The vector / gene now containing the gap is then denatured and hybridized to the vector / gene that, instead of containing the gap, has been cleaved with another restriction endonuclease at a site outside the region associated with the gap. The single-chain gene region is then hybridized with the mutant oligonucleotide, the remaining gap is filled with the Klenow fragment of DNA polymerase I, the insertions are ligated with T4 DNA ligase, and after the replication cycle, a double-chain plasmid carrying the specified mutation is generated. The Morinaga method requires additional manipulations to construct a new restriction site, and thus facilitate the generation of multiple mutations. U.S. Reissue Patent No. 34,606 (Estell et al.) Published May 10, 1994, allows the introduction of oligonucleotides carrying multiple mutations by introducing smaller changes

184,399 in the cassette, however, an even greater variety of mutations can be introduced at the same time by the Morinag method, as many nucleotides of different lengths can be introduced.

Expression of subtilase mutants

Mutant subtilase genes produced by the methods described above, or alternatively known in the art, can be secreted as an enzyme using an expression vector. An expression vector can generally be referred to as a cloning vector, since the expression vector typically comprises the components of a conventional cloning vector, i.e. an element that allows the vector to replicate autonomously in a microorganism independently of the microorganism's genome, and one or more phenotype markers for the selected target. The expression vector contains the control sequence coding for a promoter, operator, ribosome binding site, translation initiation signal, and optionally a repressor gene or various activating genes. To allow the expressed protein to be secreted, nucleotides encoding the "signal sequence" may be inserted upstream into the gene's coding sequence. For expression towards a control sequence, the appropriate gene for treatment according to the invention is linked to the control sequence in an appropriately read structure. A sequence promoter may be incorporated into the plasmid vector, and may support the transcription of the subtilase mutating gene, including but not limited to the prokaryotic β-lactamase promoter. (yiHa-Kamaroff, et al. (1978) Proc. Natl. Acad. Sci. USA 75 3727-3731) and the tac promoter (DeBoer, et al. (1983) Proc. Natl. Acad. Sci. USA 80 21-25 ). Further references may be found in "Useful proteins from recombinant bacteria" in Scieniific American (1980) 242 74-94.

According to one embodiment, B. subtilis is transformed with an expression vector carrying mutant DNA. If expression takes place in a secreting microorganism such as B. subtilis, the signal sequence can complement the translations of the initial signal and produce the appropriate DNA sequence. The signaling sequence acts to transfer the expression product to the cell wall where it is detached from the product during secretion. The term "control sequence" as defined above is intended to include the signal sequence, if any.

Other host systems known to those skilled in the art are also suitable for the expression and production of the protease variants of the invention. This arrangement of fungi, including filamentous fungi, plants, avian and mammalian cells, as well as others.

Materials and methods

Strains

B. subtilis309 and 347 are variants of BamlIuc ic8 ^ / ^ sι, deposited with NCIB under NCIB Nos. 10309 and 10147, respectively, and described in US-A-3 723 250, incorporated herein by reference.

E. coli MC 1000 1M.J. CaJabCaaa aS.N. Cohen OeSO ^ y. Moth. Biol. U38 179-2179, made r + m + by conventional methods and also described in US-A-039 298.

Protenhtic activity

In the context of the present invention, the proteolytic activity is expressed in Kilo NOVO Proteaze Units KNPU. Activity is relative to a standard enzyme (SAWINASE) and determination is based on digestion of the casein solution (DMC) by a proteolytic enzyme under standard conditions, e.g. 50 ° C, pH 8.3; reaction time 9 minutes, measuring time 3 minutes. The folder AF 220/1 is available from Novo Nordisk A / S, Denmark, which folder is included in this application as a reference.

GU is a glycine unit defined as the proteolytic activity of the enzyme, under standard conditions, during a 15 minute incubation at 40 ° C with N-acetyl casein as substrate, producing an amount of NH 2 groups equivalent to 1 µmol of glycine.

Enzymatic activity can also be measured using the PNA assay, according to reaction with a succinate-aljwinj-jljwin-prnlin-phenyl-aljwinα-paranitrnfennl substrate solution, which is described in the Journal of American Oil Chemists Society, Rothgeb, TM, Goodljwder, B.D. Garrison, P.H. and Smith L.A. (1988).

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Examples

For the determination of the enzyme variants, the same materials and methods were used as described, inter alia, in: WO-A-89/06279 (Novo Nordisk A / S), EP-A-130756 (Genentech), EP-A-479870 (Novo Nordisk A / S), EP-A-214435 (Henkel), WO-A-87/04461 (Amgen), WO-A-87/05050 (Genex), application EP-87/303761 (Genentech), EP-A-260105 ( Genencor), WO-A-88/06624 (Gist-Brocades NV), WO-A-88/07578 (Genentech), WO-A-88/08028 (Genex), WO-A-88/08033 (Amgen), WO-A-88/08164 (Genex), Thomas et al. (1985) Nature 318 375-376; Thomas et al. (1987) J. Mol. Biol. 193 803-813; Russel and Fersht (1987) Nature 328 496-500. Other methods well known in the art may also be used.

Example 1

Construction and expression of enzyme variants

A vector was constructed which is capable of encoding gene synthesis for subtilase 309 and its mutants. This is essentially the pUC19 plasmid [Yanish-Perron and Messing (1985) Gene 33 103-119] in which many cloning centers have been replaced by the restriction site junctions used to separate the five subfragments making up the gene. The new junction was introduced into EcoRI-HindIII cleaved by pUC19 which destroyed these centers. Details of the construction are described in WO 92/19729 pages 25-26 and figure 1 (sheets 1 / 7-7 / 7), the contents of the publication are incorporated herein by reference.

Each subfragment contained 6 to 12 oligonucleotides-. Oligonucleotides were synthesized on an automatic DNA synthesizer using phosphoramiditin on a controlled glass substrate [Beaucage and Carruthers (1981) Tetrahedron Letters 22 1859-1869].

Five subfragments were isolated on a 2% agar gel and inserted into pSX191. The sequence was verified by sequencing with a dideoxynucleotide. Fragments A-E were isolated and ligated together with KpnI-BamHI cut with pSX191. The ligation mixture was used to transform the appropriate E. coli MC 1000 r ', m + selected for ampicillin resistance. The 850 bp KpnI-BamHI fragment consisting of part of the gene encoding subtilisin 309 of the mature part of the enzyme was used to replace the wild-type pSX212 growth-producing gene with pSX222, which was then transformed into the appropriate B. subtilisis strain. After fermentation of the transformed strain and purification of the enzyme, it was found that it was indistinguishable from the wild type product.

Protease variants derived from the synthetic gene were generated by using altered oligonucleotides at the site (s) where mutation is desired (e.g. in the sequences provided below) and mixing them with the rest of the oligonucleotides intended for the synthetic gene. Assembly of the gene variant is carried out with the variant materials in a manner analogous to that described above. Further information on general gene synthesis is available in Agarval et al. (1970) Nature 227 27-34.

The KpnI center was introduced into the initial fragment of the synthetic 309 subtilase gene encoding the mature portion of the enzyme. The method used was called oligonucleotide direct double-strand breakage repair mutagenesis and described by Mandecki (1986) Proc. Nat. Acad. Sci. USA. 83 7177-7181. pSX172 is opened by NcoI at the beginning of the mature part of the subtilase 309 gene and is mixed with oligonucleotide NOR 789 (cf. WO-A-92/19729), heated to 100 ° C, cooled to 0 ° C, and transformed into E. coli. After retransformation, recombinants can be tested for hybridization colonies using 32-P-labeled nOr 789. Recombinants were changed to positive During the test, the KpnI site was introduced directly to the start of NcoI by changing the two bases without changing the amino acid sequence. pSX172 is described in EP 405 901. The KpnI center thus produced is inserted into pSX120 in the 400-bp PvuI-NheI fragment, giving rise to pSX212. pSX120 is also described in EP-A-405 901.

A synthetic gene is inserted between KpnI and BamHI on pSX212, giving rise to pSX222.

184 399

Examples of mutations and corresponding oligonucloeotide sequences are as follows: Rm! (fragment D1)

5'- AATTCAGGTGCAGGCTCAATCAGCTATCCGGCGCTCTAT -3

11111111111111 and 111111111111111 * Μ 11 5 '- GTCCACGTCCGAGTTAGTCGATAGGCCX5CX3AGATACGCTTG - 3

RHH (fragment D1)

5'- AATTCAGGTGCAGGCTCAATCAGCTATCCGGCX3ATCTAT - 3 'lllll! Llllllllll! Llllllllllll ** llll

5- GTCCACGTCCGAGTTAGTCGATAGGCCGCTAGATACGCTTG -3 '

S57P (fragment B1)

5 '- AGCTTTGTACCAGGGGAACCGCCGACTCAAGATGGG - 3' lllllllllllllllll * lllllllllllllll

3 '- AACATGGTCCCCTTGGCGGCTGAGTTCTACCCTTACCC - 5'

These oligonucleotides were linked to the rest of the oligonucleotides from the synthetic gene that was not altered.

Example 2

Purification of enzyme variants:

The procedure relates to the 10 liter purification of fermentation of the Subtilisin 147 enzyme, the Subtilisin m enzyme or their mutants.

About 8 liters of the fermentation solution was centrifuged at 5000 rpm in a 1 liter beaker. The filtrates were adjusted to pH 6.5 with 10% acetic acid and filtered on Seitz Supra S100 filter plates.

The filtrates were concentrated to approximately 400 mL using an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The concentrated UF was centrifuged and filtered before absorption at room temperature on a Bacitracin affinity column at pH 7. The protease was eluted from the Bacitracin column at room temperature using 25% 2-propanol and 1 M sodium chloride in a solution buffered with 0.01 M dimethylglutaric acid , 0.1M boric acid and 3I332M potassium chloride adjusted to pH 7.

The active protease fractions from the Batricin column purification step were pooled and applied to a 750 ml Sephadex G25 column (5 cm diameter) equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.2 M boric acid and 0.002 M potassium chloride adjusted to pH 6.5.

Fractions with protease activity from the Sephadex G25 column were pooled and applied to a 150 ml CM Sepharose CL 6B cation exchange column (5 cm diameter) equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.2 M boric acid and 0.1002 M potassium chloride adjusted to pH 6 , 5.

The protease was eluted with a linear gradient of 0-0.1 M sodium chloride in 2 liters of the same buffer (3-3.2 M sodium chloride for Subtilisin 147).

In the final purification step, fractions containing protease from the CM Sepharose column were pooled in an Amicon ultrafiltration chamber equipped with a GR81PP membrane (from Danish Sugar Factories hic.)

The following Stibilisin 309 variants were prepared and isolated using the technique of Example 1 for the construction and the isolation procedure described above:

184 399

A G159I

B S164I

C Y167I

D R170I

E R170L

F R170M

G R170F

H G195F

And S57P + R170L

J S57P + N218S

K S57P + R170L + N218S

L R170L + N218S + M222A

M S57P + R170L + S188P + A994P

N Y167I + R170L

About S57P + R170L + Q206E P R170L + Q206E

Q Y167I + R170L + Q206E

R Y167I + R170L + A194P S Y167I + R170L + N288S

T Y167I + R170L + A194P + N228S

U Y167I + Y17II

V R170G W R170C Example 3

Detergent compositions containing variant enzymes

Example D1:

A (isotropic) aqueous detergent liquid according to an embodiment of the invention was prepared containing:

Ingredient % NaLAS 8.0 Neodol 25-9 8.0 AES 25-3S 14.0 Na citrate · 2H2O 5.0 Propylene glycol 5.0 Sorbitol 4.5 Dye F Tinopal UNPA-GX 0.15 Lytron 614 opacifier 0.03 Kathon the preservative 0.0003 Cyan acid 80 0.00117 Violet acid 48 0.0033 Savinaza 16L 0.25 100L lipolase 0.70 Smell 0.15 Water to 100.0

The pH was adjusted to 7.1.

184 399

Table III

Residual enzyme activity (in percentage of the initial activity) after storage at 37 ° C for example D1 containing BLS309 variant S57P + R170L + N218S

Storage time (days) Wild type S57P + R170L + N218S 0 100 100 3 44 74 7 11 50 10 5 36 14 7 27

It is evident from Table III that the S57P + R170L + N5I8S variant shows a significant improvement in the stability of this type of detergent. Moreover, the S57P + R170L + N218S variant has excellent lipase compatibility.

Table IV

Residual lipase activity (in percent of the initial activity) after storage at 37 ° C for Example D1 containing BLS309 variant S57P + R170L + N218S and Lipolazy ™

Storage time (in days) Lipolaza plus: Wild type S57P + R170L + N218S 0 100 100 3 38 67 7 24 44 10 22 33 14 21 27

It can be seen from the above Table IV that in addition to protease stability, the compatibility of the protease with Lipolase is also improved.

Example d2:

A non-aqueous liquid detergent according to the invention was prepared with 38.5% C 1 -C 4 linear primary alkoxylated alcohol 4.9 mol / mole ethylene oxide and 2.7 mol / mole propylene oxide, 5% triacetin, 30% sodium triphosphine, 4% ash. soda ash, 15.5% sodium perborate monohydrate containing small amounts of 4% TAED, 0.25% EDTA, of which 0.1% is phosphoric acid, Aerosil 0.6%, SCMC 1% and 0.6% protease. The pH was adjusted to a value between 9 and 10, e.g. about 9.8.

Example D3:

The structured liquid detergent may for example contain, in addition to the protease described herein, 2-15% non-ionic surfactant, 5-40% total surfactants, including non-ionic and optionally anionic surfactants, 5-35% phosphate-containing or non-phosphate builder, 0.2-0.8% polymeric thickener, e.g. cross-linked acrylic polymer with molecular weight above 10 ⁶ , at least 10% sodium silicate, e.g. as neutral waterglass, bases (e.g. potassium-containing base) to adjust to appropriate The pH, preferably in the range of 9-10 or greater, e.g. greater than 11, with a sodium cation: silica anion (as free silica) ratio (by weight) less than 0.7: 1, and a viscosity of 0.3-30 Pas (in 20 ° C and 20I7).

The samples were about 5% of a nonionic surfactant C ₁₃ _ ₁₅ alcohol eIksksyIowenego about 5 EO groups and at moi of about 2.7 PO groups moi, 15-23%

184,399 neutral waterglass with a 3.5 weight ratio between silica and sodium oxide, 13-19% KOH, 8-23% STPP, 0-11% sodium carbonate, 0.5% Carbopol 941 ™.

Protease may be included in an amount of 0.5%, for example.

Example D4: (liquid deflocating polymer)

Priolene 6907 4.5

KOH 10

Ethoxylated alcohol 7EO (Synperonic A7) 4.5

Ethoxylated alcohol 3EO (Synperonic A3) 4.5

Zeolite 4A 15

Fluorescer Tinopal CBS-X 0.08

Narlex DC1 1

Citric acid 8.23

DB100 silicone antifoam 0.3

LAS acid 11.5

Odor 0.0

Water up to 110%

Table V

Residual enzyme activity (in percentage of initial activity) after storage at 37 ° C for Example D4 containing the R ^ L BLS309 variant

Storage time (days) R170L Wild type 0 100 100 2 98 73 4 96 66 10 94 46 33 87 8 81 78 2.1 101 71 0

It is evident from Table V that the R 1 L modification shows a significant improvement in the stability of this type of detergent.

Example D5: (liquid deflocating polymer)

Priolene 6907 4.5

KOH 10

Ethoxylated alcohol 7EO (Synperonic A7) 4.5

Ethoxylated alcohol 3EO (Synperonic A3) 4.5

Zeolite 4A 15

Fluorescer Tinopal CBS-X 0.08

NarlexDC1 1

Citric acid 8.23

DB100 silicone antifoam 0.3

LAS acid 11.5

Lipolase 1000 0.6

Smell 0.5

Water up to 100%

184 399

Table VI

Residual enzyme activity (in percent of initial activity) after storage at 37 ° C for Example D5 containing BLS309 variant S57P + R170L + N218S.

Time storage (days) Residual activity Proteases Residual activity Lipases S57P + R170L + N218S wild type wild type S57P + R170L + N218S 0 100 100 100 100 2 - 75 41 94 5 97 50 15 76 8 87 thirty 7 71 12 91 twenty 12 78 28 100 18 12 70

It is evident from Table VI that modification S57P + R170L + N218S shows a significant improvement in the stability of this type of detergent. Moreover, the S57P + R170L + N218S variant has excellent lipase compatibility.

Example D6:

Soap bars were prepared containing 49.7 wt% 80/20 tall oil / coconut soap, 49.0% water, 20% sodium citrate, 1.0% citric acid and 0.031% protease. After production, the soap bars were stored at room temperature and after a specified period of time, samples were taken and protease activity was measured. The stability data are given in Table VII below:

Table VII

Storage time (days) W.T. R170L R170L + N218S + S57P R170L + Y167I 0 100 100 100 100 1 50 100 97 94 2 25 91 100 83 3 100 94 80 6 98 89 90 10 0 100 94 71 17 93 80 73 27 95 86 70

It is evident from Table VII that the subtilase variant R170L, S57P + R170L + N218S and R170L + Y167I show a significant improvement in the stability of this type of detergent.

Example D7:

Soap bars were prepared containing 63.88% 80/20 tall / coconut oil soaps, 1% coconut fatty acid, 25.1% water, 10% sodium citrate and 0.021% protease. The laundry soap bars were stored at 37 ° C and after a specified period of time, samples were taken and the protease activity was measured.

184 399

Table VIII Stability Data

Storage (days) W.T. R170O + N218S + S57P 0 100 100 10 90.1 14 81.5 10 10 twenty 0 91.4 31 72.8 35 79 45 78

From the above Table VIII, it is evident that the variant subtilase R1N0L + N318S + S57P shows a significant improvement in the stability of this type of detergent.

Example 4

Washing performance of detergent laundry compositions containing enzyme variants. The following examples illustrate the results of several washing tests carried out under the following conditions.

Table IX

Conditions for conducting research on the Subtilisin varieties 309

Detergent Protease model detergent '95 Detergent dose 3 g / l pH 9.5 Washing time 15 minutes Temperature 15 ° C Water hardness 9 ° dH 1.61 mMCa3 ⁺ / Mg3 ⁺ Enzymes Subtilisin 309 varieties listed below Enzyme concentration 0; 0.1; 0.2; 0.3; 0.4; 0.5; 1.0; 2.0; 3.0 mg / l Test method mini wash * (SOP DEF-SM-OO3D.O1 / 01) * Fabric swatch / volume 5 fabric samples (Φ 2.5 cm) / 50 ml Material tested grass on cotton (rinsed in water); * DF-4417N12 *

The above detergent model is a simple detergent formulation. The most characteristic thing is that STP is used as a filler and the content of anionic surfactants (LAS) is quite high. Furthermore, the pH is adjusted to 9.5 which is low for powder detergents.

The composition of the model detergent was as follows:

184 399

Table X

25.0% STP (Na5P3O10) 25.0% Na2SO4 10.0% For ₂ CO3 20.0% FOREST (Nansa 80S) 5.0% non-ionic (Dobanol 25-7) 5.0% Na2Si ₂ O5 0.5% carboxymethyl cellulose (CMC) 9.5% water dose 3 g / l pH set to 9.5

Measurements of the remission (R) of the test material were carried out at 460 nm using an Elrepho 2000 photometer (no UV). The measurement results were put into the following expression:

a AR,

AR _m ax + ac

The improvement factor was calculated using the initial slope of the curve IF = a / are

Δ R - enzyme washing effect in reemission units, a - initial curve slope (c - 0). a ^ - initial slop = dirty water for enzyme ref c - enzyme concentration in mg / l.

ΔΚ ^, χ - eeoeetycrny maximatoy ffek-memory eizymui weednoscce eecH ^ - (L -> oo ..

Table XI

Variations and enhancement factors for Subtilisin 309

Choice Variety IF 1 2 3 S003 * R170Y 2.8 S004 * R170Y + G195E 2.6 S012 * R170Y + G195E + K251E 1.6 G. R170F 3.3 E. R170L 3.8 F. R170M 2.4 D R170I 4.1 AND S57P + R170L 3.9 J. S57P + N218S 1.6 K. S57P + R170L + N218S 2.3 n Y167I + R170L 6.2 P. R170L + Q206E 2.6 V R170G 2.0

184 399

Table XI - continued

1 2 3 in R170C 3.4 about S57P + R170L + Q206E 2.9 Q Y167I + R170L + Q206E 2.4

* described in WO-A-91/00345

As can be seen from Table XI, all 25 Suetilisin 309 varieties according to the invention show improvements in washing performance.

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Claims (10)

Patent claims A liquid detergent composition containing a subtilase variant, characterized in that, in addition to the usual components, it comprises a subtilase, in which one or more amino acid residues located in or adjacent to the hydrophobic parent domain of the subtilase has been substituted by an amino acid residue more hydrophobic than the original residue, the variant being the subtilase is R170L or R170I. 2. A composition according to claim 1 The composition of claim 1, wherein the parental subtilase is selected from the group consisting of ABSS168, BASBPN, BSSDY and BLSCAR. 3. A composition according to p. A subtilase as claimed in claim 1 or 2, the parent subtilase of which is selected from the subgroup I-S2. 4. A composition according to p. The method of claim 3, wherein the parent subtilase is selected from the group consisting of BLS147, BLS309, BAPB92 and BYSYAB. 5. A composition according to p. A subtilase according to claim 4, the parent of which is TVTHER. 6. A composition according to p. A variant as claimed in claim 1 or 2, characterized in that it comprises a subtilase which is a variant combined with further substitutions, deletions and / or insertions in any one or more of positions: 36, 57, 76, 218, 222 and 224. 7. A composition according to p. The composition of claim 6, characterized in that it comprises a subtilase belonging to the subgroup I-S2 with a further change selected from the group consisting of * 36D, S57P, N76D, N218S, M222S, M222A and T224S. 8. A composition according to p. The method of claim 1, comprising a subtilase variant selected from the group of variants including: a) S57P + R170L a ') S57P + R170I b) R170L + N218S b ') R170I + N218S c) S57P + R170L + N218S c) S57P + R170I + N218S c) S57P + V104Y + R170L + N218S c) S57P + V104Y + R17 ^ I + N218S d) R170L + N218S + M222A d ') R170I + N218S + M222S e) S57P + R170L + S188P + A194P e ') S57P + R170I + S188P + A194P f) Y167L + R170L f) Y167L + R170I g) Y1671 + R170L g ') Y1671 + R170I h) N76D + R170L + N218S h ') N76D + R170I + N218S i) S57P + N76D + R170L + N218S i ') S57P + N76D + R1701 + N218S j) N76D + R170L + N218S + M222A j ') N76D + R170I + N218S + M222S j) N76D + R170L + N218S + M222A j) N76D + R170L + N218S + M222S k) S57P + R170I + S188P + A194P + N218S k ') S57P + R170L + S188P + A194P + N218S 184 399 l) * 36D + N76D + H120D + R170L + G195E + K235L l ') * 36D + N76D + H120D + R170I + G195E + K235L m) N76D + H120D + R170L + G195E + K235L m ') N76D + H120D + R170I + G195E + K235L n) * 36D + G97N + V104Y + H120D + R170L + A194P + G195E + K235L n ') * 36D + N97N + V104Y + H120D + R1701 + A194P + G195E + K235L o) S57P + R170L + Q206E o ') S57P + R170I + Q206E p) R170L + Q206E p ') R170I + Q206E q) Y167I + R170L + Q206E q ') Y167I + R170I + Q206E r) Y167F + R170L r0 Y167F + R170I t) Y167I + R170L + A194P V) Y167I + R170I + A194P u) Y167I + R170L + N218S u ') Y167I + R170I + N218S v) Y167I + R170L + A194P + N218S V) Y167I + R170I + A194P + N218S x) R170L + P131V x ') R170I + P131V y) * 33D + Y167I + R170L /) * 36D + Y1677 + R170I aa) Y167V + R170L aa ') Y167V + R170I 9. A composition according to p. The method of claim 1 or 2, further comprising a deflocating polymer. 10. The composition of claim 1 The method of claim 9, wherein the subtilase variant is S57P + V104Y + R170L + N218S or S57P + V104Y + R170I + N218S.