JPH11505275A - Subtilisin mutant - Google Patents

Abstract

  (57) [Summary] A detergent composition containing a subtilase variant, wherein in the subtilase variant, one or more amino acid residues located in or near the hydrophobic domain of the parent subtilase have an amino acid residue that is more hydrophobic than the original residue. This hydrophobic domain contains residues P129, P131, I165, Y167, Y171 of BLS309 (based on the numbering of BASBPN) and the neighboring residues are E136 of BLS309. , G159, S164, R170, A194 and G195 residues (based on BASBPN numbering), except for the R170M, R170I and R170V variants of BABP92. You. These enzymes show improved stability and / or cleaning performance in detergents, especially liquid detergents and soap bars, as compared to their wild-type parent enzymes. These enzymes are very suitable for use in liquid detergent compositions and bar soaps.

Description

DETAILED DESCRIPTION OF THE INVENTION                            Subtilisin mutant Technical field   The present invention is useful for formulating detergent compositions and maintaining their cleaning performance. A new mutant enzyme that exhibits improved storage stability while maintaining or improving Or enzyme variants; cleaning and detergent compositions containing these enzymes; Mutations that, when inserted into cells or organisms, encode the expression of these enzymes A gene; as well as being transformed with such a gene and expressing this variant of the enzyme. Said host cells.Background of the Invention   In the detergent industry, enzymes have been incorporated into detergent compositions for over thirty years. This Enzymes used in compositions such as proteases, lipases, amylase , Cellulases, and other enzymes, or mixtures thereof. Commercially heaviest What is important are proteases.   Proteases have been used in the detergent industry for more than 30 years, but these enzymes How the substrate interacts with other substances present, for example, in the detergent composition For more information Not known. Specific properties related to specific residues, such as oxidation and thermal stability Some factors affecting quality have been largely identified, but many are not. Absent. Also, any physical or chemical characteristics may be good for a particular detergent composition. Still knowing exactly what will result in good cleaning performance or protease performance Not in.   Most of the proteases currently in use are derived from nature. It has been found by isolating and testing them in detergent compositions.Protease   Enzymes that break amide bonds in protein substrates are proteases or (mutually Classified as peptidases (Walsh, 1979, Enzymatic Reacti on Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3, see). Ba Bacteria of the genus cillus are two extracellular protease species, neutral proteins. And metalloproteases, and are functionally serine endopeptidases. It always secretes an alkaline protease called subtilisin. These proteins The secretion of enzymes is associated with the bacterial growth cycle, and during sporulation, during the stationary phase. Also Maximal expression of the Rotase occurs. Joliffe et al. (1980) Bacteriol 141 1199- 1208 shows that Bacillus proteases function in cell wall turnover Suggested.Subtilase   Serine proteases catalyze the hydrolysis of peptide bonds and are essential for the active site. An enzyme with a serine residue (White, Handler and Smith, 1973 “Principles of Biochemistry ”, 5th edition, McGraw-Hill Book Company, NY, pp. 271-272) .   Bacterial serine proteases range in molecular weight from 20,000 to 45,000. With quantity. They are inhibited by diisopropyl-fluorophosphate. So They hydrolyze simple terminal esters and produce true serine proteases. Similar in activity to nuclear chymotrypsin. Sub-group, more narrow sense Alkaline proteases are optimal for some serine proteases at high pH, pH 9.0 to 11.0 (for review, Priest (1977) Bacteriolo) gical Rev. 41 711-753).   A subgroup of serine proteases, tentatively named subtilases, en et al., Protein Engng. 4 (1991) 719-737 More presented. They are a kind of cellulase previously called subtilisin-like proteases. Defined by homology analysis of 40 or more amino acid sequences of proteases. Subtilisin is a serine protein produced by Gram-positive bacteria or fungi. It was previously defined as a subsequence, but now according to Siezen et al., Subtilases Is a subgroup of A wide variety of subtilisins have been identified and The amino acid sequence of such subtilisins has been determined. These include Bacill us subtilisins derived from six strains, namely subtilisin 168, subtilisin Ricin BPN ', Subtilisin Carlsberg, Subtilisin Y , Subtilisin amylosaccharides, and mesenteric peptidases (Ku rihara et al. Biol. Chem. 247 5629-5631; Wells et al. (1983) Nucleic A cids Res. 117911-7925; Stahl and Ferrari (1984) J. Bacteriol. 159 811-819 Jacobs et al. (1985) Nucl. Acids Res. 13 8913-8926; Nedkov et al. (1985) Biol. Chem. Hoppe-Seyler 366 421-430, Svendsen et al. (1986) FEBS Lett. 196 228-23 2), one kind of subtilisin derived from actinomycetes, namely, Thermoactinomyces vulgaris Thermistase (Meloun et al. (1985) FEBS Lett. 198 195-200), And a protein derived from a fungal subtilisin, namely Tritirachium album Nase K (Jany and Mayer (1985) Biol. Chem. Hoppe-Seyler 366 584-492) No. For further reference, Table I of Siezen et al.   Subtilisins are well characterized physically and chemically. these In addition to knowledge of the primary structure (amino acid sequence) of the enzyme, substrate binding, transition state, generation Exhibit at least three different protease inhibitors, and More than 50 high-resolution X-ray structures of subtilisins that define the structure have been determined (Kraut (1977) Ann. Rev. Biochem. 46 331-358).   As used herein, a substrate undergoes hydrolysis by a subtilisin protease. Most broadly including compounds containing at least one peptide bond Should be.   The expression “product” is used in the present invention to refer to a subtilisin protease. Should be construed as including the products of hydrolysis reactions involving. The product is May be a substrate for the subsequent hydrolysis reaction.   One subgroup of subtilases, I-SI, comprises subtilisin 168, subtilisin. Ricin BPN ', Subtilisin Carlsberg And "classical" subtilisins.   A further subgroup of subtilases, IS2, is described by Siezen et al. Is recognized. Subgroup I-S2 proteases are highly alkaline Subtilisin And enzymes such as alkaline elastase YaB.   In the present invention, a subtilase mutant or a mutated subtilase is Produces this mutated subtilisin protease when expressed in a suitable host. The original or parent gene mutated to produce the resulting mutant gene Expressing a mutant gene derived from a parent microorganism having the corresponding parent enzyme and producing Subtilase produced by a living organism.   Both random and site-specific mutations in the subtilase gene Based on knowledge of the physical and chemical properties of the enzyme, Has provided information on media activity, substrate specificity, tertiary structure, etc. (Wells et al. (1987) Pr. oc. Natl. Acad. Sci. U.S.A. 84; 1219-1223; Wells et al. (1986) Phil. Trans. R. Soc. Lon d. A. 317 415-423; Hwang and Warshel (1987) Biochem. 26 2669-2673; Rao et al. , (1987) Nature 328 551-554).   More recent publications across this region include cleavage of specific target sequences in substrates (Positions 24 and 64) Carter et al. (1989) Proteins 62 40-248; Considers a number of previously published results Graycar et al. (1992) Annals of  the New York Academy of Sciences 672 71-79; and also review previous results Takagi (1993) Int. J. Biochem. 25 307-312.   In particular, site-directed mutagenesis of the subtilisin gene has attracted much attention. Various mutations are described in the following patent applications and patents:   Site-specific or random abruptness in "carbonyl hydrolases" Mutation, and k_cat/ K_mRatio, pH-activity profile, and oxidation stability EP-for subsequent screening of mutant enzymes for various properties such as A-130 756 (GENENTECH) (US Patent No. 34,606 (GENENCOR) Corresponding). This published specification allows for site-specific mutations That a particular specified position, ie,^-1Tyr,³²Asp,¹⁵⁵Asn,¹⁰⁴Ty r,²²²Met,¹⁶⁶Gly,⁶⁴His,¹⁶⁹Gly,¹⁸⁹Phe,³³Ser,²²¹ Ser,²¹⁷Tyr,¹⁵⁶Glu or¹⁵²Subtilisin BPN 'in Ala Mutations provide an enzyme that exhibits altered properties. position All of these positions except -1 were involved in the function of the enzyme prior to the filing of this patent application. This application is known to have the desired Solve the problem of deciding where to introduce mutations in order to obtain enzymes with properties Not very helpful in deciding.   Cloning and subcloning of subtilisin Carlsberg and its two mutants And EP-A-214 435 for expression (HENKEL). In this application,¹⁵⁸As from p¹⁵⁸To Ser, and¹⁶¹From Ser¹⁶¹Reasons for mutation to Asp Is not provided at all.   In WO 87/04461 (AMGEN), improved pH and thermal stability Number of Asn-Gly sequences present in parent enzyme to obtain qualitative mutant enzyme It has been proposed in this application that the subtilisin BPN '^{Ten 9} Asn and²¹⁸Emphasis has been placed on removing, mutating or modifying Asn residues. No examples are provided to delete or modify the Gly-residue.   WO 87/05050 (GENEX) improves after random mutation A large number of subtilisin BPN 'mutants for the identified properties It is disclosed to do. In this application,²¹⁸Asn,¹³¹Gly,²⁵⁴Thr,^{1 66} Gly,¹¹⁶Ala,¹⁸⁸Ser,¹²⁶Leu, and⁵³Sudden change in Ser position Differences are noted.   In EP-A-251 446 (GENENCOR), points at the primary and tertiary structure level How homologous amino acid residues are conserved by considering homology It is stated whether to do. Tertiary of our subtilisin BPN ' This information, along with structural knowledge, should not be expected to yield mutants with altered properties. This makes it possible to select a large number of positions susceptible to mutation. Like this The defined position is:¹²⁴Met,²²²Met,¹⁰⁴Tyr,¹⁵²Ala,¹⁵⁶Glu,^{1 66} Gly,¹⁶⁹Gly,¹⁸⁹Phe,²¹⁷Tyr. Also,¹⁵⁵Asn,^{twenty one}Ty r,^{twenty two}Thr,^{twenty four}Ser,³²Asp,³³Ser,³⁶Asp,⁴⁶ Gly,⁴⁸Ala,⁴⁹Ser,⁵⁰Met,⁷⁷Asn,⁸⁷Ser,⁹⁴Lys,⁹⁵ Val,⁹⁶Leu,¹⁰⁷Ile,¹¹⁰Gly,¹⁷⁰Lys,¹⁷¹Tyr,¹⁷²Pro ,¹⁹⁷Asp,¹⁹⁹Met,²⁰⁴Ser,²¹³Lys, and²²¹The position of Ser It has been identified as being expected to affect various properties of the enzyme. Also Several mutations are exemplified to support these proposals. these In addition to a single mutation at a position, we also have several multiple mutations went. Further, the present inventors have²¹⁵Gly,⁶⁷His,¹²⁶Leu,¹³⁵Leu, And segments 97-103, 126-129, 213-215, and 1 Although the amino acid residues within 52-172 have been identified as of interest, these Mutations at any position are not exemplified.   Of particular interest for the purposes of the present invention is the inventor of EP-A-251 446. Etc.¹⁷⁰Suggests replacing Lys (subtilisin BPN ', type I-S1) In particular, they have introduced Glu or Arg instead of the original Lys. And suggests. A Glu mutant was created, which was very autolytic Apparently, it seems to be clear [for example, 48, 121, 123 pages (Table XXI contains obvious errors, but has an autolysis half-life of 86 minutes. 13 min) and FIG. 32].   EP-A-260 105 (GENENCOR) has three residues involved in catalytic activity ( Select an amino acid residue within about 15Å from the catalytic triad, and select the remaining amino acid residue. Contains three residues involved in catalytic activity by replacing the group with another residue Modifications of certain properties of the enzyme are described. Subtila described in this specification -Type enzymes belong to a class of enzymes containing three residues involved in catalytic activity It states in particular. Positions 222 and 217 of the subtilisins are substituted It is shown to be the preferred position.   In addition, subtilisin BPN '⁹⁹Asp⁹⁹When converted to Ser, this enzyme Changes in pH dependence may be due to Thomas, Russell, and Fersht (1985) Nature 3. 18 375-376.   Later literature (1987) Mol. Biol. In 193 803-813, the same authors, And¹⁵⁶Instead of Glu¹⁵⁶We are considering replacing Ser. These bumps Both mutations are active What⁶⁴It is within a distance of about 15 ° from His.   In Nature 328 496-500 (1987), Russel and Fersht reported that Considering the results, by mutating the enzyme to obtain a change in surface charge, A set of rules for altering the H-activity profile is presented.   WO-A-88 / 08028 (Genex) and WO-A-88 / 08033 (A mgen) are both amino acid residues at the calcium binding site of subtilisin BPN ' The modification of The enzyme replaces the original residue with a more negatively charged residue It is said that this will stabilize.   In WO-A-89 / 06279 (NOVO NORDISK A / S), the position 170 is interesting. And suggests replacing the existing residue with Tyr. You. However, no data is given for such a mutant. W The same suggestion was made in OA-91 / 00345 (NOVO NORDISK A / S). The Tyr mutant at position 170 of subtilisin 309 (type IS2) has about 8 Shows improved cleaning performance in detergents at pH (Tables III, IV, V, VI, VII Mutant S003 in I and X). Combined with other substitutions at other positions The same substitutions made by the superordinate concepts of this application are all improved cleaning performance (same table) And S004, S011-S014, S022-S024, S0 in Table VII 19, S020, S203, S225, and S227).   In EP-A-525 610 (SOLVAY), the hydrophobicity of that particular surface area Enzyme by reducing ionic tensides Improves the stability of IS2-type subtilase, which is closely related to butyricin PB92 It has been suggested that That is, the numbering at position 164 (BPN ' It has been suggested to replace 170) Arg with Gln if used. this No variants containing substitutions are disclosed in this application.   In WO-A-94 / 02618 (GIST-BROCADES N.V.), the type I-S2 At position 164 of the ricin PB 92 (170 if BPN 'numbering is used) Several variants have been described. Original Arg is Met, Val, Tyr, I An example is provided that replaces le. Cleaning performance test of powder detergents of those variants Shows only a slight improvement. In particular, cleaning performance of Ile mutant against cacao In testing, about 20-30% improvement It is shown. No stability data is provided.Industrial application of subtilases   Proteases such as subtilisins are useful for removing proteinaceous stains. As a result, a great deal of utility has been found in this industry, especially in detergent compositions. I have been.   At present, at least the following proteases are commercially available Known, many of them are marketed in large quantities in many countries of the world:   Subtilisin BPN 'or Novo, for example, SIGMA, St. Louis enters from U.S.A. Possible.   Subtilisin Carlsberg by NOVO NORDISK A / S (Denmark) And both belong to subtilase subgroup I-S1.   Among the subgroups I-S2 of subtilases, the following are commercially available It is known:   Bacillus lentus subtilisin, subtilisin 309, It is commercially available.   Subtilisin PB92, Gist-Brocades N.V. Marketed by Enzyme.   Bacillus lentus subtilisin, subtilisin 147,   However, to be effective, such enzymes are active under wash conditions. Not only show, but also need to be compatible with other detergent ingredients during detergent manufacture and storage .   For example, subtilisins are combined with other enzymes that are active on other substrates. The subtilisins selected can be used to enhance stability against such enzymes. The selected subtilisin should preferably have the degradation of other enzymes Should not be catalyzed. Also, the selected subtilisins are bleaching agents, oxidizing agents, etc. Should be resistant to the action of other ingredients in such detergent compositions, in particular, Enzymes used in food are detergents during storage The oxidizing power and calcium binding provided by non-enzymatic components in the It should be stable with respect to integration properties and pH conditions.   For example, it catalyzes the degradation of various natural substrates present on the items to be washed during washing. Often the ability of an enzyme to be cleaned is determined by its cleaning ability, detergency, be called. In the specification of the present application, the term cleaning performance includes this property. Used for   Other detergent compositions before use (usually by adding water during the washing process) The ability of an enzyme to retain activity in the presence of a component is usually determined by storage stability or shelf life. Called. It is often the half-life t_1/2Is measured as The present inventors In this specification, the expression "storage stability" is used when referring to this property. Is used.   Naturally occurring subtilisins are washed under varying parameters such as pH. Has been found to have a very variable nature with respect to power or ability . Some of the commercially available detergent proteases described above were in fact about 20 years ago. It has better performance than those on the market, but for optimal performance Means that each enzyme is formulated and washed under conditions such as pH, temperature, ionic strength (= I) , Active systems (surfactants, surfactants, bleaches, etc.), builders, etc. Has its own specific requirements.   Therefore, enzymes having desirable properties at low pH and low I are more allylic. Under sexual conditions and high I, it is unattractive and also good at high pH and high I. It can be seen that enzymes exhibiting favorable properties are not attractive at low pH and low I conditions.   It has also been found that the storage stability differs between enzymes, Elemental parameters include several parameters such as pH, pI, bleaching system, surfactants, etc. And the physical state of the detergent composition, which may be in powder, dust, or liquid form. It has been found that storage stability varies greatly for various detergent compositions. I have. Furthermore, it can be concentrated or diluted.   The advent and development of recombinant DNA technology has had a profound impact on the field of protein chemistry.   It is now possible to construct enzymes with desired amino acid sequences through the application of this technology. Or is possible, as shown above, considerable A number of studies have been devoted to the design of subtilisins with altered properties.   Among the proposals, EP-A-130 756 (GENENTECH) (US Patent No. 34,606 (GENENCOR)) and International Publication No. 87/05050 (GE Produce and screen a large number of mutant enzymes as described in NEX) The technique represents, to a considerable extent, the classical method of isolating the native enzyme, These can be used for classical mutagenesis (using radiation or chemical mutagens). And screen for their properties. Replace at specific position Through the knowledge of the existence of numerous mutant enzymes, these methods have become more effective and There is a difference in that there is.   Subtilisin enzymes typically consist of about 275 amino acid residues. It Each residue can be one of the 20 possible natural amino acids. Therefore, the hand One very significant drawback in the method is that some preliminary measures to determine their properties Generating a large number of mutations that need to be screened. You.   The approaches outlined in these patent applications have resulted in many years Only slightly better than the traditional random mutation method known for It is.   Other known techniques include oxidative stability, thermal stability, Ca stability, transesterification and And hydrolysis rate (EP-A-260 105 (GENENCOR)), pH-activity Feel (Thomas, Russell, and Fersht, supra), and substrate specificity (international Certain properties can be altered, such as published 88/07578 (GENENTECH). And about. These publications relate to changes in the cleaning performance or storage stability of enzymes. Not something.   In International Patent Application No. PCT / DK88 / 000002 (NOVONORDISK A / S) To obtain the desired change in wash performance, which amino acid is selected for mutation Use the concept of homology comparisons to determine which amino acids to replace at these positions. It has been proposed to use.   By using such a method, the number of mutants generated is considerably small This significantly reduces the screening effort, but this approach can Enzymes exhibiting useful properties of both enzymes and enzymes used for comparison It is only foreseen.   Thus, as indicated above, well-defined properties of enzymes such as those described above The relationship between and the cleaning performance and storage stability of enzymes in various detergent compositions is still Not determined at all.   The problem is that much research has been directed at elucidating the mechanism of enzyme activity. But most of its characteristics, especially when the enzyme is present in the complex mixture. Structure and amino acid residues to determine enzyme properties such as storage stability of detergents The fact that very little is known about factors in group combinations It is considered that   Therefore, further improvements and modifications of the enzyme to the detergent system, as well as the detergent or On the action of proteases and the mechanism of degradation in practical use of detergent compositions There is still a need for better understanding. Such understanding is of considerable degree. Reliably, improved storage stability under the conditions specified in the detergent composition and / or Or because it is applied to select mutations that give rise to performance enzymes Would be able to lead to a rule that canSummary of the Invention   Surprisingly, improved storage stability and / or improved storage stability in detergent compositions. Alternatively, a subtilase variant with improved performance may be substituted for the hydrophobic domain of the parent subtilase. One or more amino acid residues located at or near the main are more hydrophobic than the original residues By replacing it with higher amino acid residues, the hydrophobic region Residues P129, P131, I165, Y167 and Y171 of LS309 (BAS (Based on BPN numbering), and its residues in the vicinity are BL E136 of S309, G159, S164, S164, R170, A194 and And 195 residues (based on BASBPN numbering), provided that , BABP92 R170M. . This excludes the R170I and R170V variants. And found.   The invention therefore relates, in its first aspect, to detergents, especially liquid detergents, in particular Improved stability and / or detergency in concentrated liquid detergents and bar soaps The present invention relates to an enzyme mutant showing an ability.   In its second aspect, the invention subsequently evolves the subtilisin enzyme of the first aspect. When inserted into a host cell to be expressed by an appropriate method, the enzyme of the first aspect is expressed. DNA constructs that can be used.   In a third aspect, the invention relates to the use of a DNA construct according to the second aspect in a suitable host. And culturing the host to express the desired subtilase enzyme, and The present invention relates to the production of the subtilisin enzyme of the present invention by collecting a substance.   The invention is not limited in part to, but is not limited to: Genes expressing butyrase subgroup I-S2 enzyme and subsequent enzyme variants With respect to the mutant.   Other subtilase gene variants encompassed by the present invention are improved in detergents. Subtilase subgroup I-S1, showing improved stability and / or cleaning performance, eg Examples include the subtilisin BPN 'and subtilisin Carlsberg genes. Ensuing mutant subtilisin BPN ', proteinase K and And subtilisin Carlsberg enzymes.   Still further subtilase gene variants encompassed by the present invention are For example, proteinase K and other proteins exhibiting improved stability and / or performance Subtilase enzyme, and subsequent mutant proteinase K and other subtilases It is a mutant of the enzyme.   Other examples of parent subtilase enzymes that can be modified according to the present invention are listed in Table I Raise it.   In addition, the present invention relates to the use of mutant enzymes in cleaning compositions and to the mutations. CLEANING COMPOSITIONS CONTAINING HETEROGENIC ENZYMES, ESPECIALLY DETERGENT COMPOSITIONS CONTAINING THE MUTANT SUBTILISIN ENZYME About adult. Specifically, the present invention relates to a liquid detergent set comprising such an enzyme variant. The invention relates to products, in particular concentrated liquid detergents and soap bars.                             Abbreviation description amino acid A = Ala = alanine V = Val = Valine L = Leu = leucine I = Ile = isoleucine P = Pro = proline F = Phe = phenylalanine W = Trp = Tryptophan M = Met = methionine G = Gly = glycine S = Ser = Serine T = Thr = Threonine C = Cys = cysteine Y = Tyr = Tyrosine N = Asn = asparagine Q = Gln = Glutamine D = Asp = aspartic acid E = Glu = glutamic acid K = Lys = Lysine R = Arg = arginine H = His = histidineNucleic acid base A = Adenine G = guanine C = cytosine T = thymine (only for DNA) U = uracil (only for RNA)Mutant   In describing the various enzyme variants produced or intended according to the present invention, The following nomenclature is adapted for easy reference: Sent: Original amino acid-position-substituted amino acid.   According to this, replacing the glycine at position 195 with glutamic acid is:         Gly 195 Glu or G195E Deletion of glycine at the same position:         Gly 195 * or G195 * And insertion of additional amino acid residues such as lysine:         Gly 195 GlyLys or G195GK It is called.   The deletion is in subtilisin as shown in Table I or not shown in Table I. If present, the insertion of such a position is: Insertion of the aspartic acid at position 36         Indicated as * 36Asp or * 36D.   Multiple mutations are separated by a plus, ie: Arginine and glycine were replaced with tyrosine and glutamic acid, respectively Mutations at positions 170 and 195   Arg170Tyr + Gly195Glu or R170Y + G195E Represented byposition   In describing the variants in the present application specification and the appended claims, the aforementioned Sie An aligned sequence of various subtilases such as zen is used. Others related to subtilases Use other aligned sequences or numbering of particular enzymes. Honcho Determining the location of specific residues by numbering used in the textbook is routine for the skilled worker. That is what. Also, an alignment of residues relevant to the present invention from a number of subtilases Reference is made to FIG. 1, which shows the sequence. Also, there are many subtilases related to the present invention. Reference is made to Table I of WO-A-91 / 00345 which shows the aligned sequence of the residues.                         References used in Table I (In parentheses): ARB172 Kamekura and Seno, (1990) Biochem. Cell Biol. 68         352-359 (amino acids of mature protease residues 1-35)         Sequencing; residue 14 not determined). BSS168 Stahl. And Ferrari. (1984) J.C. Bacteriol. 158,         411-418 (KO1988). (1488) J. Yoshimoto, Oyama, et al.         Biochem. 103, 1060-1065 (B. subtilis var.         Mature subtilisin from amylosacchariticus is         It differs in having T130S and T162S).         (1986) FEBS Lett. 196, 228-232 (PIR A23624;         No acid sequencing; Mature alkali from mesentericus         Sex mesenteric peptidases are S85A, A88S, S89A,         It differs in having S183A and N259S). BASBPN Wells et al. (1983) Nucl. Acids Res. 11 7911-7925         (X00165). Vasantha et al. (1984) J. Am. Bacteriol. 159         811-814 (K02496). (1983) Hoppe-Seyler's Z. BSSDY Nedkov et al. Physiol. Chem.         364 1537-1540 (PIR A00969; amino acid sequencing). BLSCAR Jacobs et al. (1985) Nucleic Acids Res. 13 8913-8926         (X03341). (1968) J. Smith et al. Biol. Chem. 243         2184-2191 (PIR A00968; amino acid sequencing; mature pro         Thease sequences are T103S, P129A, S158N, N161S and         And S212N). BLS147 Hastrup et al. (1989) Published July 13, 1989.         PCT Patent Application WO 8906279 (via B. lentus)         Microbiol. Biotechnol., 33 519-523 (Bacillus sp.         no. Mature alkaline protease residue from AH-101         1-20 amino acid sequence determination; this sequence contains N11S         Is different from BLS147). BABP92 van der Laan et al. (1991) Appl. Environ. Microbiol.         Patent Application WO 890 published on July 13, 2008         6279 (subtilisin 309 from B. lentus)         Godette et al. (1991) (5th Symposium of Protein Society)         Abstracts, June 6, Baltimore: Summary M8 (B.         The highly alkaline protease from lentus is N87S,         With S99D, S1O1R, S103A, V104I and G159S         Is different). BDSM48 Rettenmaier et al. (1990) Published April 19, 1990         PCT Patent Application WO 90/04022. (1989) J. BYSYAB Kaneko et al. Bacteriol. 171 5232-5236         (M28537). (1988) J. BSEPR Sloma et al. Bacteriol. 170 5557-5563         (M22407). Bruckner (1990) Mol. Gen. Genet. 221         486-490 (X53307). BSBPF Sloma et al. Bacteriol. 172 1470-1477         (M29035; amendment). (1990) J. Wu et al. Biol. Chem. 265         6845-6850 (J05400; this sequence contains A169V and frame         Has less than 586 C-terminal residues due to mishift         Is different). (1986) J. BSISP1 Koide et al. Bacteriol. 167 110-116         (M13760). BSIA50 Strongin et al. Bacteriol. 133 1401-1411         (Amino acid sequence determination of mature protease residues 1-54         Residues; residues 3, 39, 40, 45, 46, 49 and 5         0 is not determined).         BTFINI Chestukhina et al. (1985) Biokhimiya 50         1724-1730 (from B. thuringiensis var. Israeliensis         Native mature protease residues 1-14 and         residues 1-16 and 223- from var.finitimus         243). Kunitate et al. (1989)         Agric. Biol. Chem. 53 3251-3256 (var.kurstaki         Amino acid sequence of mature protease residues 6-20 derived from         Decision. BTKURS). BCESPR Chestukhina et al. (1985) Biokhimiya 50 1724-1730         (Amino acids of mature residues 1-16 and 223-243         Sequencing). NDAPII Tsujibo et al. (1990) Agric. Biol. Chem. 54 2177-         2179 (Amino acid residue 1-26 amino acid sequencing). TVTHER Meloun et al. (1985) FEBS Lett. 183 195-200 (PIR         A00973; Amino acids of mature protease residues 1-274         Sequencing). EFCYLA Segarra et al. (1991) Infect. Immun. 59 1239-1246. SEEPIP Schnell et al. (1991) Personal communication         (Siezen et al. (Ibid.)). (1990) J. SPSCPA Chen et al. Biol. Chem. 265 3161-3167         (JO5224). DNEBPR Kortt et al. (1991) 5th Protein Society Symposium Abstract         Vol., June 22-26, Baltimore, Abstract S76. LLSK11 Vos et al. Biol. Chem. 264 13579-13585         (J04962). Kok et al. (1988) Appl. Environ. Microbiol.         54 231-238 (M24767; sequence from strain Wg2         44 positions including 18 differences in the thease domain,         And deletion of residues 1617-1676).         (1989) Mol. Microbiol .. 3 359-369         (X14130; sequence from strain NCD0763 is a protease         46 positions, including 22 of the domain, and residue 1617         -1676 deletions). XCEXPR Liu et al. (1990) Mol. Gen. Genet. 220 433-440. SMEXSP Yanagida et al. Bacteriol. 166 937-994         (M13469). TAAQUA Terada et al. Biol. Chem. 265 6576-6581         (J054I4). TRT41A McHale et al. (1990) 5th European Biotechno         Gee Conference Abstracts, Christiansen, Munck and         Villadsen (Editor), Munksgaard Int. Publishers,         Penhagen. (1989) Gene 76 281-288 (M25499). SRESPD Lavrenova et al. (1984) Biochemistry USSR. 49 447-         454 (amino acid sequencing of residues 1-23; residue 13,         18 and 19 were not determined). AVPRCA Maldener et al. (1991) Mol. Gen. Genet. 225 113-120         (Published sequence is due to misreading of reading frame         28 unspecified residues near the 200-210 location         Group). TAPROK Gunkel and Gassen (1989) Eur. J. Biochem. 179         185-194 (X14688 / XI4689). Jany et al. Biol.         Chem. Hoppe-Seyler 367 87 (PIR A24541; amino acid sequence         Sequence determination; mature proteases are S745G, SILST204-         The difference is that it has 208DSL and VNLL264-267FNL.) TAPROR Samal et al. (1990) Mol. Microbiol. 4 1789-1792         (X56116). TAPROT Samal et al. (1989) Gene 85 329-333. AOALPR Tatsumi et al. (1989) Mol. Gen. Genet. 219 33-38.         Cheevadhanarah et al. (1991) EMBL Data Library         (X54726). MPTHMY Gaucher and Stevenson (1976) Methods Enzymol. 45         415-433 (having residues 1-28 and active site serine         Hexapeptide LSGTSM amino acid sequencing). ACALPR Isogai et al. (1991) Agric. Biol. Chem. 55 471-477.         (1986) Int. J. Biochem. 18 369-375         (Amino acid sequence determination of residues 1-27: mature protein         Ze has a point with H13 [1] Q, R13 [2] N and S13 [6] A         Is different). KLKEX1 Tanguy-Rougeau, Wesolowski-Louvel and Fukuhara         (1988) FEBS lett. 234 464-470 (X07038). SCKEX2 Mizuno et al. (1988) Biochem. Biophys. Res. Commun.         156 246-254 (M24201). SCPRB1 Moehle et al. (1987) Mol. Cell. Biol. 7 4390-4399         (M18097). YLXYPR2 Davidow et al. Bacteriol. 169 4621-4629         (M17741). Matoba et al. (1988) Mol. Cell Biol. 8         4904-4916 (M23353). CEBL14 Peters and Rose (1991) The Worm Breeder's         Gazette 11 28. DMFUR1 Roebroek et al. (1991) FEBS Lett. 289 133-137         (X59384). DMFUR2 Roebroek et al. (1992) 267 17208-17215. CMCUCU Kaneda et al. Biochem. 95 825-829 (active part         Octapeptide NIISGT with SerineSM amino         Acid sequencing). HSFURI van den Ouweland et al. (1990) Nucl. Acids Res. 18         664 (X04329) (Mouse furin sequence is a catalytic domain         5 of the following: A15E, Y21F, S223F, A232V and N258 [2] D         At 51 positions including). Misumi et al. (1990)         Nucl. Acids Res. 18 6719 (X55660: Rat pretend         Sequence of three catalytic domains: A15E, Y21F, H24R         At 49 positions including). HSIPC2 Smeekens and Steiner (1990) Biol. Chem. 265         2997-3000 (J05252). Seidah et al. (1990) DNA Cell         Biol. 9 415-424 (mouse pituitary PC2 protea         The sequence of the enzyme consists of seven protease domains: I4F,         S42 [2] Y, E45D, N76S, D133E, V134L and G239 [1] D         At 23 positions, including). MMPPC3 Smeekens et al. (1991) Proc. Natl. Acad. Sci. USA 88         340-344 (M58507). (1990) DNA Cell Biol.         9 415-424 (M55668 / M55669; partial sequence). HSTPP Tomkinson and Jonsson (1991) Biochemistry 30 168-         174 (J05299).                             BRIEF DESCRIPTION OF THE FIGURES In the drawings, FIG. 1 shows an aligned sequence of a number of subtilases described in Table I. FIG. 2 shows some of the hydrophobic domains replaced by the present invention and their vicinity. 3 is a three-dimensional display of subtilisin 309 showing the positions of amino acid residues.Detailed description of the invention   Surprisingly, residues P129, P131, I165, Y16 of subtilisin 309 7. Make the amino acid residues located near the hydrophobic domain containing Y171 more hydrophobic Washing with Found that the storage stability and / or performance of subtilase in the preparation is improved did. The residues in question are, in particular, E136, G159, S164, R170, A19 4 and G195.   FIG. 2 shows some of the residues in order to increase the hydrophobicity of the hydrophobic domain. At and near the hydrophobic domain of subtilisin 309 in which Shows neighboring residues. This involves replacing non-hydrophobic residues with hydrophobic residues and / or This can be achieved by substituting residues so that they are more hydrophobic than the enzyme. Wear.   The same principle, its identification is within the skill of the average person working in this field Applied to the corresponding hydrophilic domains of other subtilases. Schematic as in Figure 2 Expressions can be made for other subtilases to identify target residues to be replaced according to the invention. Can be determined.   This number is shown in Table II below: Table II was created using the alignment shown in FIG. Cover other subtilases It is easy for those skilled in the art to easily create similar or larger tables. it is obvious.   Therefore, the present invention provides that the residue at that position is Are also hydrophobic and include relatively long hydrophobic chains such as Ile, Leu, and Val. 129, 131, 165, 167, 17 Amino acid residues at positions 1, 136, 159, 164, 170, 194 and 195 It is desirable to modify the codon (parent enzyme or gene) that leads to the expression of the group, Thus, when the mutated gene is expressed, the hydrophobicity of its domain Amino acid residues have been replaced by more hydrophobic residues, Subtilase whose amino acid sequence has been changed through mutation of lysine enzyme gene For mutants.   The hydrophobic amino acid residues are usually: Val (V), Ile (I), Leu (L), Met (M), Phe (F), Pro (P) and Trp (W) It is. Of these, Val, Ile and Leu are preferred. See Table II, And by applying the principles of the present invention, a large number of replacement candidates become apparent.   For both BASBPN and BLSCAR, 136, 159, 164, 16 Substitutions at positions 7, 170 and 195 may be appropriate. BLS At 309, the positions of 136, 164, 167 and 170 are the first choices. 159 And positions 195 would also be a second choice. In BLS147, 136, 16 Positions 7, 170, and 195 are the first choice, while 159 and The position at 164 is the second choice. Finally, in TVTHER, 136, 16 Positions 7 and 194 are the first selection, and 164 is the second selection.   According to the present invention, the Gly residues of the hydrophobic domain are reduced to bulkier and more hydrophobic residues. Substitution to a group would necessarily be advantageous.   Such an increase in hydrophobicity means that it is considered to be advantageous. Think of a hydrophilic or hydrophobic residue that can occupy any of the above positions Apply. This includes, for example, the charged residues Agr (R), Asp (D), Gl Very hydrophilic residues such as u (E) or Lys (K) Means that it can be replaced by a different residue. Less hydrophilic Are the residues Gly (G), Cys (C), Ser (S), Ala (A), T hr (T), Tyr (Y), Gln (Q), His (H) or Asn (N) Including.   Similar considerations apply to other subtilisers that have a hydrophobic domain in this part of the enzyme surface. It can be applied to mosses.   In the context of the present invention, the subtilase is Etc. More narrowly, it applies to many embodiments of the invention. Possible subtilases of interest belong to subgroups IS1 and IS2. Things. In a more particular sense, many of the embodiments of the present invention have a primary structure Serine protein of a gram-positive bacterium capable of providing substantially distinct homology to With respect to theases, these subtilases are listed in Table I above.   The present invention also relates to any other substitution, deletion or deletion of the amino acid sequence of the parent enzyme. Or one or more permutations of the above positions in combination with additions. In particular, Combination with other substitutions known to provide improved properties for the enzyme Is foreseen.   Such combinations are available at positions: 222 (improved oxidative stability), 218 ( Qualitative), a substitution at the Ca binding site that stabilizes the enzyme, eg at position 76 , And many others apparent from the prior art. Further, EP-A-405   Combinations with the variants set forth in 901 are also specifically contemplated.Mutant A: Single mutant: Subtilisin BPN ', Subtilisin Carlsberg, Carlsberg Syn-168 and subtilisin DY mutants:         K136V, K136I, K136L, K136M, K136F,         S159V, S159I, S159L, S159M, S159F,         T164V, T164I, T164L, T164M, T164F,         K170V, K170I, K170L, K170M, K170F,         E195V, E195I, E195L, E195M, E195F, Thermitase mutant:         Q136V, Q136I, Q136L, Q136M, Q136F,         T159V, T159I, T159L, T159M, T159F,         A164V, A164I, A164L, A164M, A164F,         Y167V, Y167I, Y167L, Y167M, Y167F,         Y170V, Y170I, Y170L, Y170M, Y170F,         S194V, S194I, S194L, S194M, S194F, Subtilisin 309, Subtilisin 147, and Bacillus PB92 protein Ze mutant:         E136V, E136I, E136L, E136M, E136F,         G159V, G159I, G159L, G159M, G159F,         G164V, G164I, G164L, G164M, G164F, (BLS147)         S164V, S164I, S164L, S164M, S164F, (BLS309 and BAPB92)         Y167V, Y167I, Y167L, Y167M, Y167F,         R170V, R170I (discard both for PAPB92),         R170L, R170M (discarded for PAPB92), R170F, R170G, R170C,         A194V, A194I, A194L, A194M, A194F, (BLS309 and BAPB92)         P194V, P194I, P194L, P194M, P194F, (BLS147)         E195V, E195I, E195L, E195M, E195F, (BLS147)         G195V, G195I, G195L, G195M, G195F, (BLS309 and BAPB92) B: Combination mutant:   Some of the above variants are 27, 36, 57, 76, 101, 104, 123, 218, 222, 224 and 274 It is expected that an advantage will be shown when combined with the mutant.   In particular, the following BLS309 variants were combined: K27R, * 36D, S57P, N76D, S101G, V104A, V104 N, V104Y, N123S, A194P, Q206E, N218S, M222 S, M222A, T224S and T274A are considered appropriate.   Also, in combination with one or more other substitutions, deletions and / or insertions described above, One or two of X167L, X167I, X170L, and / or X170I Such variants containing two substitutions are also advantageous.   Further, a modification in combination with one or more of the above substitutions, deletions and / or insertions. V104N + S101G, K27R + V104Y + N123S + T274A , Or mutants containing either N76D + V104A show improved properties It is recognized that. Specific combinations are listed below: 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 d '') R170L + N218S + M222A d '' ') R170I + N218S + M222S e) S57P + R170L + S188P + A194P e ') S57P + R170I + S188P + A194P f) Y167L + L170L 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) 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 + R170I + S188P + A194P + N218S 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 + G97N + V104Y + H120D + R170I + 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 r ') Y167F + R170I t) Y167I + R170L + A194P t ') Y167I + R170I + A194P u) Y167I + R170L + N218S u) Y167I + R170I + N218S v) Y167I + R170L + A194P + N218S v ') Y167I + R1701 + A194P + N218S x) R170L + P131V x ') R170I + P131V y) * 36D + Y167I + R170L y ') * 36D + Y167I + R170I z) Y167I + Y171I aa) Y167V + R170L aa ') Y167V + R170I Detergent compositions containing mutant enzymes   The invention also relates to the use of the mutant enzymes according to the invention in cleaning and detergent compositions. As well as such compositions containing mutant subtilisin enzymes. this Such cleaning and detergent compositions can in principle be in any physical form. However, the subtilase variants are preferably liquid, exhibiting improved enzyme stability. Detergent compositions or detergents for direct application in the form of sticks, tablets, sticks, etc. Formulated in the composition.   Among the detergent compositions of the present invention, the first is a liquid detergent, in particular, for example, a homogeneous physical Aqueous liquid detergents having the characteristics, for example, they are continuous aqueous phases, so-called It may consist of a micellar solution of a surfactant in an isotropic liquid.   Alternatively and preferably, they can have a heterogeneous physical phase, For example, it can consist of a dispersion of lamellar droplets in a continuous aqueous phase, for example EP-A-346 995 (Unilever), which is incorporated herein by reference. ), A hydrophilic backbone and at least one hydrophobic side chain May be included. These latter liquids are heterogeneous Yes, for example, the types of Suspended solid particles, such as particles of rudder material, can be included.   Preferably, the liquid cleaning and detergent composition of the present invention comprises EP-A-328 17 7, EP-A-359 308, EP-A-328176, EP-A-346 It should have a high electrolyte concentration, as described in 995 (all Unilever).   Such a composition comprises one or more subtilis according to any of the foregoing aspects of the invention. In addition to the lysine enzyme variant alone, or to such compositions well known to those of skill in the art. It comprises in combination with any of the usual components included.   Such components include builders such as phosphate or zeolite builders, Such as anionic, cationic, nonionic or zwitterionic surfactants Polymers such as surfactants, acrylic polymers or equivalent Such as bleach precursors or activators containing perborates or amino acids Bleaching, structurant such as silicate structuring agent, for adjusting pH Includes alkali or acids, wetting agents, and / or neutral inorganic salts.   Furthermore, A. Any co-surfactant B. Tartrate succinate builder C. Neutralization system D. Foam inhibitor E. FIG. Other enzymes F. Other optional ingredients Some optional ingredients are usually present in the compositions of the present invention.   The weight ratio of synthetic anionic surfactant to ethoxylated nonionic surfactant is: 1: 1 to 5: 1. The composition is a 10% by weight aqueous solution at 20 ° C. from 7.0 At a pH of 9.0, a critical micelle concentration below 200 ppm, and at this critical micelle concentration It has an air / water interfacial tension of 32 dynes / cm or less at 35 ° C. in distilled water. Composition Is preferably clear, homogeneous and phase stable, has good cleaning performance and enzyme stability. It is qualitative.Various ingredients : 1. Synthetic anionic surfactant The liquid detergent compositions of the present invention comprise from about 10% to about 50%, preferably about 15% by weight. % To about 50%, more preferably about 20% From 40%, and most preferably from 20% to about 30% of a natural or synthetic anion. A surfactant. Suitable natural or synthetic anionic surfactants include, for example, Soaps and US-A-4 285 841 and US-A-3 929 678.   Useful anionic surfactants include about 10 to about 10 in their molecular structure. An alkyl group having 20 carbon atoms and a sulfonic or sulfuric ester group Water-soluble salts of organic sulfuric acid reaction products, especially alkali metals, ammonium and And alkylol ammonium (for example, monoethanol ammonium or triethanolamine). Ethanolammonium) salt. (The term “alkyl” includes Includes the alkyl portion of the reel group. Examples of this group of synthetic surfactants include sulfuric acid Reduction of alkyls, especially glycerides in tallow or coconut oil Higher alcohols (C₈-C₁₈Carbon atom) Wherein the alkyl group has about 9 to about 15 carbon atoms in a straight chain. Or alkyl benzene sulfonates having a branched chain structure, for example, US Pat. No. 2,220,099 and No. 2,477,383. There are types. Of particular value is the average number of carbon atoms in the alkyl group. Linear alkylbenzene sulfonates of about 11 to 14.   Other anionic surfactants herein have from 8 to about 24 (preferably about Paraffin sulfonates containing carbon atoms of 12 to 18); Seryl ether sulfonates, especially C₈-C₁₈Alcohols (for example, tallow And those derived from coconut oil); from 1 to about 4 units per molecule Containing from 8 to 12 carbon atoms in the ethylene oxide and alkyl groups of Alkyl phenol ethylene oxide ether sulfate; and per molecule 1 to 4 units of ethylene oxide and 10 to 20 carbon atoms in the alkyl group Water-soluble salts of alkyl ethylene oxide ether sulfates containing nitrogen atoms You.   Other useful anionic surfactants include 6-20 carbons in the fatty acid groups. Α-sulfone containing from 1 to 10 carbon atoms in the atoms and ester groups Water-soluble salts of esters of unsaturated fatty acids; from 2 to 9 carbon atoms in the acyl group And 2-acylio containing 9 to 23 carbon atoms in the alkane moiety Water-soluble salts of xy-alkane-1-sulfonic acids; 12 to 24 carbon atoms Water-soluble salts of olefin sulfonates containing: and 1 in the alkyl group Contains from 2 to 3 carbon atoms and from 8 to 20 carbon atoms in the alkane moiety Β-alkyloxyalkanesulfonates.   Preferred anionic surfactants include an average of 4 or less acids per mole of alkyl sulfate. C containing ethylene fluoride units_Ten-C₁₈Alkyl sulfates and alkyl ethoxy Sulfates, C₁₁-C₁₃Linear alkyl benzene sulfonates, and their salts Is a mixture of 2. Ethoxylated nonionic surfactants   The second optional ingredient is from 2% to 14% by weight, preferably from 2% to 8%, up to Also preferred is 3% to 5% of an ethoxylated nonionic surfactant. Synthetic shade The weight ratio of ionic surfactant (on an acid basis) to nonionic surfactant is 1: 1 To 5: 1, preferably 2: 1 to 5: 1, most preferably 3: 1 to 4: 1. is there. This ensures the formation and adsorption of surfactants of sufficient hardness at the air / water interface. Indeed, it will provide good greasy / greasy soil removal.   The general formula of the ethoxylated nonionic surfactant is R¹(OC_TwoH_Four)_nOH (Where R¹Is C_Ten-C₁₆Alkyl group or C₈-C₁₂With an alkylphenyl group And n is from 3 to 9), this nonionic surfactant is preferably from 6 to 14, Or HLB (hydrophilic-lipophilic balance) of 10 to 13. These worlds Surfactants are US-A-4 285 841 and US-A-4 284 53 2 specifically. Particularly preferred are C₁₂-C_Fifteenalcohol Condensation products of alcohols with 3 to 8 moles of ethylene oxide per mole of alcohol, eg For example, C condensed with about 6.5 moles of ethylene oxide per mole of alcohol₁₂− C₁₃Alcohol. Other nonionic surfactants to be mentioned are APG, EG E and glucamide surfactants. 3. Detergency builder   Conventional detergent components that can be present in conventional amounts in the detergent compositions of the present invention include: And the following: The composition may be structured (built), It may be unstructured and may be of the zero-P type (i.e., any phosphorus-containing building (Also does not contain a binder). Therefore, the present composition comprises For example, 1-50% by weight, for example at least about 5%, often about 35-40% or less of one or more organic and / or inorganic builders Good. Representative examples of builders include those already described above, and Include alkali metal ortho, pyro, and tripoly-phosphates, alkali metal charcoal Acid salts, alone or calcite, alkali metal citrates, alkali metal nitrite Rotriacetates, carboxymethyloxysuccinates, zeolites, polya Any of mixtures with cetal carboxylate and the like can be mentioned.   More specifically, the compositions herein have 10 to 18 carbon atoms. Fatty acids and / or polycarboxylates, zeolites, polyphosphones 5% by weight, which may be a phosphate and / or polyphosphate builder From 20%, preferably from 10% to 15% of detergency builders. Preferred 12 are 0 to 10% (more preferably 3 to 10%) by weight% 0 to 10% by weight, with saturated fatty acids having from 14 to 14 carbon atoms, Preferably 2% to 8%, most preferably 2% to 5% polycarboxylate Builder, most preferably citric acid, in a weight of 1: 1 to 3: 1 They are mixed in a ratio.   When the ratio of builder to water hardness is close to 1, the proteolytic enzyme herein Is considered to provide optimal storage stability benefits compared to other enzymes The composition is preferably 2 to 10, preferably 3 to 8 grams per gallon. Contains enough builder to sequester the lane.   Suitable saturated fatty acids include plant or animal esters (eg, palm kernel oil, par Oil and coconut oil) or can be obtained synthetically. (Eg, via oxidation of petroleum or Fischer-Tropsi) (By hydrogenation of carbon monoxide via heat treatment). Suitable saturated fats for the composition of the invention Examples of fatty acids include capric acid, lauric acid, myristic acid, coconut and coconut. Nucleus fatty acids. Preferred are saturated coconut fatty acids; lauric acid And a 5: 1 to 1: 1 (preferably about 3: 1) weight ratio mixture of and myristic acid; Mixtures of the above with small amounts (eg, 1% -30% of total fatty acids) of oleic acid And palm kernel fatty acids.   Further, the composition of the present invention is preferably described in US-A-4 284 532. Polycarboxylate, polyphosphone And polyphosphate builder. Water-soluble polycarboxylate bi Luders, especially citrates, are a preferred member of this group. The right political Various aminopolycarboxylates and boxylate builders can be used. Loalkane polycarboxylates, ether polycarboxylates, alk Polycarboxylates, epoxy polycarboxylates, tetrahydroph Orchid polycarboxylates, benzene polycarboxylates and polya Cetal polycarboxylates are exemplified.   Examples of such polycarboxylate builders include ethylenediamine Sodium and potassium tetraacetate; sodium and potassium nitrilotriacetate Water-soluble salts of phytic acid, for example as disclosed in US-A-1 739 942 Sodium and potassium phytate, US-A-3 364 103, Polycarboxylate materials; and US-A-3 308 067 Water soluble salts of the stated polycarboxylate polymers and copolymers You.   Other useful detergency builders include the following structural and physical characteristics: (a) acid A minimum molecular weight of about 350 calculated in form; (b) 50-80 equivalent weights, calculated in acid form; (c) alternating with no more than 2 carbon atoms At least 45 models with at least two carboxyl groups separated by Percent of the monomeric species; (d) Carboxy-containing group polys which are separated by no more than 3 carbon atoms along the rimer chain Water-soluble salts of high-molecular aliphatic polycarboxylic acids having a mer chain binding site . Specific examples of such builders include itaconic acid, aconitic acid, maleic Acid, mesaconic, fumaric, methylenemalonic, and citraconic acid polymers And copolymers.   Other suitable polycarboxylate builders include melitic acid, citric acid, Pyromellitic acid, benzenepentacarboxylic acid, oxydiacetic acid, carboxymethyl Xsuccinic acid, carboxymethyloxymalonic acid, cis-cyclohexanehexa Of carboxylic acids, cis-cyclopentanetetracarboxylic acids and oxydisuccinic acids Water-soluble salts, especially sodium and potassium salts.   Other polycarboxylates include US-A-4 144 226, and U.S. Pat. Polyacetate according to SA-4146,495 There are tar carboxylates.   Other detergency builders are described in U.S. Pat. No. 4,405,483. Zeolites such as aluminosilicate ion exchange materials.   Other preferred builders have the general formula R-CH (COOH) CH_Two(COOH) Or a derivative of succinic acid (where R is C_Ten-C₂₀Alkyl or a Lucenyl, preferably C₁₂-C₁₆And R is hydroxyl, sulfo, sulfo Xy or sulfone substituents). These succinate buildings Dars are preferably sodium, potassium and alkanol ammonium Used in the form of their water-soluble salts, including salts. Specific of succinic acid builders Examples are: lauryl succinate, myristyl succinate, palmityl succinic acid Salts, 2-dodecenyl succinate and the like. 4. Protease   The enzymes of the present invention can be used in standard amounts well known in detergent compositions. The amount is Very broadly, for example, about 0.0002-0.1 a / g of detergent composition. Nson units, for example in the range of about 0.005-0.05. Another simple Represent by position And the protease is present in about 0.1 to 100 GU / mg detergent composition (eg, 1 -50, especially 5-20 GU / mg) or per gram of detergent composition. Or about 0.01-4, for example, a wide range of quantities centered on 0.1-0.4 KNPU Can be included in the present composition.   For example, this corresponds to an enzyme activity on the order of 0.08 KNPU per liter. Use the enzyme at a rate of about 0.25 mg of enzyme protein per liter of washing solution May be preferred. Corresponding detergent compositions are, for example, 0.1-0.4 KNPU / G of the present enzyme.   In other words, the composition of the present invention comprises the protease of the present invention in an amount of about It contains 0.01% to about 5%, preferably about 0.1% to about 2%.   The proteolytic enzymes described are preferably present in an amount of from 0.05 to about 1.0 mg, more preferably about 0.1 to 0.75 mg, and most preferably about 0. Included in an amount sufficient to provide 125 to about 0.5 mg of activity. 5. Enzyme stabilization system   The liquid detergent according to the invention comprises calcium ions, boric acid, Including enzyme stabilization system containing lenglycol and / or short-chain carboxylic acids be able to. The enzyme stabilizing system comprises from about 0.5% to about 15% by weight of the composition. Including.   The composition preferably contains about 0.01 calcium ion per liter. From about 50 mmol, preferably from about 0.1 to about 30 mmol, more preferably about Contains 1 to 20 mmol. The amount of calcium ions depends on the builders in the composition. Always take into account the minimum level available for the enzyme after taking into account complexation with Should be chosen to be Calcium chloride, calcium formate, and vinegar Use any water-soluble calcium salt, including calcium acid, as a source of calcium ions Can be. Also, due to calcium in the enzyme slurry and formulation water, The amount of calcium ions, usually about 0.05 to 0.4 mmol per liter, , Often present in the composition. About 0.03% to about 0.6% calcium formate Is preferred.   The second preferred enzyme stabilizer contains only carbon, hydrogen and oxygen atoms Polyols. These preferably have 2 to 6 carbon atoms and 2 Contains from 1 to 6 hydroxy groups. For example, propylene glycol (special , Preferred 1,2-propanediol), ethylene glycol, glycerol, sorbitol Mannitol, and glucose. Polyols are usually heavy From about 0.5% to 15%, preferably from about 1.5% to about 8% by weight of the composition. Hit. Preferably, the weight ratio of polyol to any added boric acid is low. It is at least 1, more preferably at least 1.3.   In addition, the composition is preferably a water-soluble solution described in US-A-4 318 818. It also contains a short-chain carboxylate. These formate salts are present in weight percent of the composition. About 0.05% to about 5%, preferably about 0.2% to about 2%, most preferably 0% . It can and preferably is used in an amount of 4% to 1.5%. Sodium formate is preferred Good.   Also, the compositions herein may optionally comprise from about 0.25% to about 5% by weight, most It preferably contains about 0.5% to about 3% boric acid. Boric acid is used as boric acid in the composition. Can be formed by a compound capable of forming do not do. Boron oxide, borax and other alkali metal borates (e.g., ortho, Tar and sodium pyroborate, and other compounds such as sodium pentaborate) While boric acid is preferred, boric acid is preferred. [Substituted boric acids such as phenylboronic acid, butaneboronic acid, and p- Bromophenylboronic acid) can also be used in place of boric acid. 6. water   The liquid detergent composition of the present invention may be an aqueous liquid or a non-aqueous liquid. aqueous When liquid, about 15% to about 60% by weight, preferably about 25% to about 4% by weight. Contains 5% water.Further optional ingredients A. Any co-surfactant   An optional co-surfactant for use with the ethoxylated non-ionic surfactant described above; Amides of the following general formula (Where R¹Is an alkyl containing 8 to 20 carbon atoms, a hydroxy An alkyl or alkenyl group;^TwoAnd R^ThreeIs hydrogen, methyl, ethyl, Propyl, isopropyl, 2-hydroxyethyl, 2-hydroxypropyl, 3 Selected from the group consisting of -hydroxypropyl; Containing a Tylene unit, provided that R^TwoAnd R^ThreeIn At least one contains a hydroxyl group).   Preferred amides are those in which each alkylol group contains 1 to 3 carbon atoms. Which can contain up to two ethylene oxide units in₈-C₂₀Fatty acid al These are chlorolamides. Particularly preferred are C₁₂-C₁₆Fatty acid monoethano And diethanolamides.   When used, the amides are preferably the ethoxylated nonionic surfactants described above. Agent and amide surfactant from 4: 1 to 1: 4, preferably 3: 1 to 1: 3 Is present in an amount such that the weight ratio is   Preferred optional co-surfactants used in amounts of 0.15% to 1% are US- A-4507 The quaternary ammonium, amine and amine oxide domains described in 219 It is a surfactant.   In the above, C_Ten-C₁₄Alkyltrimethylammonium salts such as decyl Trimethyl ammonium methyl sulfate, lauryl trimethyl ammonium Chloride, myristyltrimethylammonium bromide, and coconut Trimethyl ammonium chloride and methyl sulfate are preferred. 0. 2% to 0.8% monoalkyltrimethylammonium Nium chloride is preferred. B. Tartrate succinate builder   The compositions herein are preferably from 0 to about 10%, preferably by weight, on an acid basis. 0 to about 6% of a tartrate succinate builder selected from the group consisting of: Contains material: i) (Where X is a salt-forming cation); ii) (Where X is a salt-forming cation); and iii) their mixtures.   Tartaric acid succinic acid compounds used herein are described in US-A-4 663 071. Are listed. C. Neutralization system   Further, the composition of the present invention may optionally contain monoethanolamine, diethanolamine. , Triethanolamine, and mixtures thereof. Alkanolamine selected from the group consisting of About 0 to 0.04 mol, preferably about 0.01 to 0.035 mol, more preferably Or about 0.015 to about 0.03 mol. Low level arca Nolamines, especially monoethanolamine, provide product stability, It is preferable to increase the energy. However, the amount of alkanolamine is best Should be minimized for chlorine bleach compatibility.   In addition, the composition may be sufficient to neutralize anionic species and provide a desired product pH. It contains sodium ions, and preferably potassium ions, in amounts. D. Foam inhibitor   Other optional ingredients used in the liquid detergent herein are from 0 to about 1.5% by weight, Preferably from about 0.5% to about 1.0% of a silicone-based suds suppressor.   Silicones are widely known and taught for use as very effective foam control agents Have been. For example, US-A-3 455 839 discloses a composition and a small amount of A method for eliminating bubbles in an aqueous solution by including a polydimethylsiloxane liquid therein. I do.   Useful foam control silicones are described, for example, in German Patent Application DE-A-2 124   A mixture of silicone and silanized silica as described in US Pat. .   Silicone defoamers and foam control agents are disclosed in US Pat. No. 3,933,672 and No. 4,652,392, a granular detergent composition is maintained from a detergent surfactant. Has been successfully incorporated into it.   Preferred silicone-based suds suppressors for use herein are essentially (I) about 20 cs. From about 1500 cs. Polydimethyl having a viscosity of Siloxane liquid; (Ii) about 5 to about 50 parts by weight per 100 parts by weight (i) of about 0.6: 1 About 1.2: 1 (CH_Three)_ThreeSiO_1/2Units vs SiO_TwoIn the unit ratio (CH_Three)_ThreeSi O_1/2Unit and SiO_TwoA siloxane resin consisting of units; (Iii) about 1 to about 20 parts by weight of solid silica gel per 100 parts by weight of (i) And a foam-inhibiting amount of a foam control agent.   The “foam inhibiting amount” is defined by the formulator of the composition as desired. It means that the amount of this foam regulator that regulates the foam can be selected. Foam control The amount of agent will vary with the detergent surfactant selected. For example, a well-bubble interface Surfactants have relatively more foam control compared to less foaming surfactants Use the agent to achieve the desired foam control. E. FIG. Other enzymes   The detergent composition of the present invention can also contain additional enzymes.   For example, lipase is a carrier substance (eg, as described in EP-A-258 068). (Novo Nordisk A / S) containing lipolytic enzyme (particulate composition (or) It can be conveniently added in liquid or slurry form.   The amount of lipase added can be within wide limits, e.g., for surfactant systems or detergent compositions. 50 to 30,000 LU / g per ram, for example, often at least 100 LU / g, very usefully at least 500 LU / g, sometimes preferably 1000 More than 2000 LU / g or more than 4000 LU / g and therefore very often For example, in the range of 50-4000 LU / g, or in the range of 200-1000 U / g. You can choose within. This specification In this document, lipase units are defined as in EP-A-258 068. You.   The lipolytic enzyme can be selected from a wide range of lipases. In particular, the example For example, the following patent specifications: EP-A-214 761 (Novo Nordisk A / S), EP-A-214761 Lipases according to A-258 068, and especially Thermomyces lanugiosus A Lipases exhibiting anti-serum and immune cross-reactivity against lipase derived from TCC 22070, EP-A-205 208 and EP-A-206 390, and especially Chromo against lipase from bacter viscosum var lipolyticum NRRLB-3673 or Anti-lipase from Alcaligenes PL-679, ATCC 31371 and FERM-P 3783 Lipases exhibiting immune cross-reactivity with serum, also described in specification WO 87/0859 ( Gist-Brocades) and EP-A-204 284 (Sapporo Breweries) Lipases. Particularly suitable Novo Nordisk A / S, Amano lipases CE, P, B, AP, M-AP, AML, Lipozym, SP225, SP285 (all Novo Nordisk), Saiken lipase, Enzeco lipase , Toyo Jozo lipase and Diosynth lipase (trademarks) Lipases as described in N.V.), and WO-A-94 / 03578 (Unilever). Kind.   The amylase can be present, if desired, for example, from about 1 to about 1 per gram of the detergent composition. 100 MU (maltose units) (or 0.014-1.4, for example 0.07 It can be used in amounts ranging from -0.7 KNU / g (Novo units). Suitable Ami Lases Cellulases may be present, if desired, for example, from about 0.3 to about 0.3 per gram of the detergent composition. Used in an amount in the range of about 35 CEVU units   Other enzymes contemplated for use in the present invention are oxidases and peroxygens. It is a sidase. F. Other optional ingredients   Other optional ingredients for the liquid detergent herein include soil removers, soil release polish. Anti-redeposition agents such as limers and tetraethylenepentamine ethoxylate ( About 0.5% to 3%, preferably about 1% to about 3% by weight), foam control agents, Thorium Kume Hydrotropes such as sulfonates, opacifiers, antioxidants, fungicides , Dyes, fragrances, and clarifiers known in the art. like this The optional ingredients are usually less than about 15%, preferably about 0.5% by weight of the composition. % To 10%, more preferably from about 1% to about 10%.   The composition comprises from 0% to about 8%, preferably from 0% to about 5%, by weight of C₁₂-C₁₄ It may contain alkenyl succinic acid or a salt thereof. These substances are General formula R-CH (COOX) CH_Two(COOX) (where R is C₁₂-C₁₄Al Each X is H or sodium, potassium, ammonium, Or alkanol ammonium (eg, mono-, di-, or tri-ethano A suitable cation, such as ammonium chloride). A specific example is , 2-dodecenyl succinate (preferred) and 2-tetradecenyl succinate There is a   The compositions herein enhance cleaning performance when pre-treating textiles. Optionally, from about 0.1% to about 1% by weight, preferably from about 0.2% to about 0%. . 6% ethylenediaminetetramethylenephosphonic acid, diethylenetriamine Ntamethylene Phosphonic acid, ethylenediaminetetraacetic acid (preferred), Contains a water-soluble salt of amine pentaacetic acid (most preferred).   In addition, the detergent composition may contain 1-35% bleach or bleach precursor or bleach. Containing systems comprising whitening agents and / or precursors and their activators Can be.   Further optional ingredients include foaming enhancers, corrosion inhibitors, soil suspending agents, metals Blocking agents, stain reattachment preventing agents, and the like.   The compositions herein preferably contain no more than about 10% ethanol. G. FIG. Other properties   The composition is typically present in a 10% by weight aqueous solution at 20 ° C., from about 7.0 to 9.0, preferably Or about pH 8.0 to about 8.5.   The composition also has a critical micelle concentration (CMC) of 200 ppm or less, Less than 32 dynes per centimeter in CMC at 35 ° C. in distilled water, preferably Has an air / water interfacial tension of 30 dynes or less. These measurements are described in ″ Measurem ent of Interfacial Tension and Surface Tension-General Review for Practi cal 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 Effec ts ”, CA Miller and P. Neogi, Chapter 1, pages 29-36 (1985), Marcel Dekker, I nc. It is listed in New York.   The composition of the present invention may be a fibrous material, especially but not limited to cotton and cotton. Can be used for cleaning fibers and polyester-based fibers and their mixtures. You. For example, about 60-65 ° C or less, for example, about 30-35 ° C or less. Washing performed at the following temperatures is particularly preferred. If desired, of course, Lower or higher concentrations can be used, but may require, for example, about 0.4 Use of the composition in a ratio sufficient to provide -0.8 g / l of surfactant It can be very suitable. Without limitation, for example, the detergent formulation The use rate of 1 to 10 g / l, for example about 3-6 g / l, means that the composition is substantially It can be said that it is suitable for use when it is as in the examples.   In this aspect, the invention particularly provides a) anionic surfactants, nonionic surfactants, wetting agents, An aqueous detergent solution comprising an organic acid, a caustic alkali and a pH adjusted to a value of 9 to 10; Prescription detergent composition b) a liquid nonionic interface consisting essentially of a linear alkoxylated primary alcohol Activator, triacetin, sodium triphosphate, caustic, perborate monohydrate Whitening agent precursor, and tertiary amine bleach activator, p adjusted to a value of 9 to 10 Detergent composition formulated as a non-aqueous detergent liquid comprising H c) When used in a proportion corresponding to 0.4-0.8 g / l surfactant, the washing solution is Enzyme-containing liquid detergent composition formulated to have a pH of 9 or less d) When used at a ratio corresponding to 0.4-0.8 g / l surfactant, the washing solution is Enzyme-containing liquid detergent composition formulated to have a pH of 8.5 or more e) When used in a proportion corresponding to 0.4-0.8 g / l surfactant, the washing solution is Enzyme formulation formulated to have an ionic strength of 0.03 or less, for example 0.02 or less Liquid detergent composition f) When used in a proportion corresponding to 0.4-0.8 g / l surfactant, the washing solution is Enzyme formulation formulated to have an ionic strength of 0.01 or more, for example 0.02 or more Liquid detergent composition About.   In addition, the subtilase variants of the present invention may be used on textiles, hard surfaces or any other For direct application to surfaces, in detergent compositions in the form of sticks, tablets, sticks, etc. Have been found to be usefully formulated. In particular, these are notable enzyme stable It can be formulated into a soap or soap / synthetic composition in the form of a bar, exhibiting properties.   Detergent compositions in the form of sticks, tablets, sticks, etc. for direct use include, for example, See, for example, South African Patent No. 93/7274, incorporated herein by reference. ing. Thus, preferred rods according to the present invention include, in addition to subtilase variants,   i) with soap or a mixture of soap and synthetic cleaning activator which is regarded as anhydrous From 25% to 80%, most preferably from 25% to 70% by weight Cleaning activator;   ii) 0 to 50% by weight and most preferably 10 to 30% water;   iii) 0 to 35% by weight and most preferably 0.1 to 30% by weight of charge Including filler.   Usually, the subtilase modification contained in such compositions of the present invention. The amount of the variant may be from 0 to GU / mg glycine units per milligram of the composition. . 1 to 100 GU / mg, preferably 0.5 to 20 GU / mg, most preferred Or one that is consistent with a proteolytic activity of 1.0 to 10 GU / mg.How to make mutations in a subtilase gene   Numerous methods for inducing mutations in a gene are well known in the art. Zubuchila After a brief description of the cloning of the protease gene, any part of the subtilase gene Methods for mutagenizing both positions and specific sites are described.Cloning of subtilase gene   The gene encoding subtilase can be prepared by various methods well known in the art, as shown in Table I. Cloned from any of the organisms listed above, especially Gram-positive bacteria or fungi Can be. First, the chromosome from the organism producing the subtilase to be studied Using DNA or messenger RNA, the genomic and / or cD An NA library needs to be constructed. Then, the amino acid sequence of subtilase If known, a homologous labeled oligonucleotide probe is synthesized and From genomic or cDNA libraries of bacterial DNA using The obtained clone encoding subtilisin can be identified. Or, A labeled oligonucleotide containing a sequence homologous to a subtilase from another strain of bacteria or organism. Oligonucleotides as probes, with lower stringency Cloning of subtilase using hybridization and washing conditions. Can be identified.   Yet another method of identifying subtilase-producing clones is to use plasmids. Insertion of genomic DNA fragment into an expression vector such as genomic DN Transformation of protease negative bacteria using the A library and then Of transformed bacteria on agar containing substrates for subtilases such as milk Including plating. These containing plasmids that produce subtilase Bacteria are converted to clear agar due to digestion of skin milk by secreted subtilase. Make a colony surrounded by circles.Creation of random mutations in the subtilase gene   Once the subtilase gene has been cloned into a suitable vector, such as a plasmid, If so, several methods can be used to introduce random mutations into the gene. it can.   One method is to use a portion of the recoverable vector in a mutator strain of E. coli. Is to incorporate the cloned subtilase gene.   Other methods generate a single stranded form of the subtilase gene, and then DNA fragment containing ze gene and part of subtilase gene remain single-stranded Annealing with such other DNA fragments. This separate single-stranded region Is then, but not limited to, sodium bisulfite, hydro Some mutagenesis involving xylamine, nitrous acid, formic acid or hydralazine It can be contacted with any of the agents. Of this method of inducing random mutation Specific examples are described in Shortle and Nathans (1978, Proc. Natl. Acad. Sci. U.S.A., 7 5 2170-2174). According to the method of Shortle and Nathans, Plasmids containing the butyrase gene are reduced by restriction enzymes that cut within the gene. Is locked. This nick is used for the exonuclease action of DNA polymerase. And expand into a gap. The resulting single-stranded gap was then Mutagenesis can be induced using any one of the mutagenic agents.   Alternatively, a Bacillus sp. Containing natural promoter and other regulatory sequences Subinfection of subtilisin gene with helper phage IR1 For the preparation of single-stranded plasmid DNA by E. coli and B. subtilis for both A plasmid containing a replicon, a selectable phenotypic marker and an M13 origin of replication. It can be cloned into a sumid vector. Cloned subtilisin inheritance Isolated single-stranded plasmid DNA containing the subtilisin coding region Gaps as a result of annealing with a DNA sequence containing no DNA fragment A chain molecule is obtained. With either sodium bisulfite, nitrous acid or formic acid, Or subtilisin by replication in a mutator strain of E. coli as described above. Induce a mutation in the gene. Sodium bisulfite is used in single-stranded DNA Because it reacts exclusively with tosin, the mutations created using this mutagen Is restricted to only the code region. Reaction times and bisulfite concentrations are on average Various experiments to create 1 to 5 mutations per butylicin gene Change with. 10 μg gap in 4 M sodium bisulfite, pH 6.0 Reaction volume of double-stranded DNA with lipase at 37 ° C for 9 minutes 400 µl Incubation deaminates about 1% of cytosine in the single-stranded region. The coding region for mature subtilisin contains approximately 200 cytosines, depending on the DNA strand. Have. Advantageously, the reaction time is reduced to about 4 minutes (about 1 mutation frequency per 200 cells). To about 20 minutes (about 5 per 200).   After mutagenesis, the gapped molecule was replaced with DNA polymerase I (Klenow fragment). ) In vitro to generate a complete double-stranded molecule and confirm the mutation. Compilation The tent E. coli was then transformed with the mutagenized DNA to obtain the mutant Create an amplified library of subtilisin. Amplified mutant library Lee believes that the range of mutations is limited by the error-prone DNA polymerase. Growth of plasmid DNA in increasing E. coli Mut D strains Can be created.   Mutagenic nitrite and formic acid also used to generate mutant libraries Can be. These chemicals are more sensitive to single-stranded DNA than sodium bisulfite. And not specific, the mutagenesis reaction is performed by the following method. Zubu Cloning of the coding portion of the thyricin gene into M13 phage by standard methods To prepare single-stranded phage DNA. Single stranded DNA was then added to 1M nitrous acid Acid pH 4.3 and 23 ° C for 15-60 minutes, or 2.4M formic acid and 23 ° C 1- Incubate for 5 minutes. These reaction times range from 1 out of 1000 to 1000 Results in a mutation frequency of 5. After mutagenesis, the universal primer was replaced with M13 Anneals with DNA to make the subtilisin gene coding region completely double-stranded. As described above, this mutagenized single-stranded DNA was used as a template to prepare a double-stranded DN. A is synthesized. At this point, the coding region is cut from the M13 vector with restriction enzymes. However, the non-mutagenized expression vector should be used so that the mutation occurs only in the restriction fragment. Can be connected in the reactor. (Myers et al., Science 229 242-257 (1985)) .Creating site-specific mutations in the subtilase gene   Once the subtilase gene is cloned, the desired site is Once the residue to be substituted for the original residue is determined, the synthetic oligonucleotide Can be used to introduce these mutations. These oligonucleotides The tide contains the nucleotide sequence adjacent to the desired mutation site; Nucleotide nucleotides during oligonucleotide synthesis You. In a preferred method, a single-stranded DNA strand that crosslinks the subtilase gene The vector is created in a vector having the subtilase gene. Then, the desired bump Anneal the synthetic nucleotide having the mutation with a homologous portion of the single-stranded DNA. You. Fill remaining gaps with DNA polymerase I (Klenow fragment) This construct is then ligated using T4 ligase. A specific example of this method is Mori (1984, Biotechnology 2 646-639). Morinaga etc. According to this, fragments within the gene are removed using restriction endonucleases. Now gi The capsulated vector / gene is then denatured and the gap-containing Instead, use another restriction endonuclease at a site outside the area where the gap is associated. Hybridize with the cut vector / gene. Single-stranded region of this gene Is then useful for hybridization with the mutated oligonucleotide. The remaining gap is used for the Klenow fragment of DNA polymerase I. The insert is ligated with T4 DNA ligase and, after one cycle of replication, A double-stranded plasmid having the above mutation is produced. This Morinaga method is Eliminates the need for further steps to construct new restriction sites, and Facilitates the creation of mutations at a number of sites. Issued May 10, 1994 US Patent No. 34,606 by Estell et al. Discloses minor modifications to cassettes. Can induce oligonucleotides with multiple mutations. However, it is necessary to introduce a large number of oligonucleotides of various chain lengths. Therefore, it is possible to introduce various mutations at once by Morinaga method. Can be.Expression of subtilase mutant   According to the present invention, the method described above, or any alternative known in the art, The resulting mutated subtilase gene is expressed in an enzyme form using an expression vector. Can be expressed. Expression vectors are typically representative cloning vectors Components, i.e., enable autonomous replication of vectors in microorganisms independent of the genome of the microorganism. Elements to enable, and whether they contain one or more phenotypic markers for selection Thus, expression vectors usually fall under the definition of cloning vectors. Expression vector Are promoters, operators, ribosome binding sites, translation initiation signals, And, optionally, co-repressor genes or various activator genes. Contains regulatory sequences to be loaded. Expressed protein In order to allow white matter secretion, a "signal sequence" must precede the coding sequence for this gene. The nucleotides encoding the sequence "can be inserted. Under the direction of regulatory sequences For expression, the target gene to be treated according to the invention is transformed into a suitable reading frame. Operably couple to a knot array. Mutant incorporated into a plasmid vector As a promoter sequence capable of supporting transcription of the subtilase gene, Prokaryotic β-lactamase promoter (Villa-Kamaroff et al. (1978) Proc. Natl. A cad. Sci. U.S.A. 753 727-3731) and the tac promoter (DeBoer et al. (1983) ) Proc. Natl. Acad. Sci. U.S.A. 80 21-25) but are limited to these Not necessarily. For further reference, see Scientific American (1980) 242 74-94. "Useful proteins from recombinant bacteria".   According to one embodiment, the expression vector carrying the mutated DNA . Transform subtilis. B. Expression occurs in secretory microorganisms such as subtilis In this case, the signal sequence follows the translation initiation signal and the target DNA sequence It is possible to precede a column. The signal sequence is cut from the product during secretion. Acts to transport expression products to the disrupted cell wall You. “Regulatory sequences” as defined above, when present, include signal sequences. It is intended to   For the expression and production of the protease variant of the present invention, other Host systems are also contemplated. Such host systems include fungi, including filamentous fungi, plants, birds and And mammalian cells and others.                              Materials and methods Strain: B. subtilis 309 and 147 have accession numbers NCIB 10309 and 10 US-A, deposited with the NCIB at 147 and incorporated herein by reference. Bacillus lentus as described in US Pat.   E. coli MC1000 (MJ Casadaban and SN Cohen (1980); J. Mol. Biol . 138 179-207) is r^-, M⁺This is a US patent application No. 039,298.Proteolytic activity   In the context of the present invention, proteolytic activity is measured in kiloNOVO protease units. (KNPU). Activity is enzyme standard (SAVINASE), which is determined under standard conditions, ie, 50 ° C. pH 8.3, 9 minutes reaction time, 3 minutes measurement time, dimethyl by protease Based on digestion of casein (DMC) solution. Folder AF220 / 1 is This folder is available upon request to Novo Nordisk A / S, Denmark. -Are included in this specification for reference.   GU was prepared under standard conditions at 40 ° C. with N-acetylcasein as substrate. During the 1 minute incubation, an amount of NH equivalent to 1 μmol glycine was added._Two-Group It is a glycine unit defined as the proteolytic enzyme activity produced.   Enzyme activity was determined by the Journal of American Oil Chemists Society, Rothgeb, T.M., G oodlander, B.S. D., Garrison, P.H., and Smith, L.A., (1988) Soluble substrate succinyl-alanine-alanine-proline-phenyl-alanine- It can also be measured using PNA analysis by reaction with paranitrophenol .                                  Example   For the production of enzyme variants according to the invention, WO-A-89 / 06279 (Novo  Nordisk A / S), EP-A-130 756 (Genentech), EP-A-479 870 (Novo Nordisk A / S), EP-A- 214-435 (Henkel), WO-A-87 / 04461 (Amgen), WO-A -87/05050 (Genex), EP application 87373061 (Genentech), EP-A-260 105 (Genencor), WO-A-88 / 06624 (Gist-B rocades 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. (19 87) J. Mol. Biol., 193, 803-813; Russel and Fersht (1987) Nature 328 496-50. Same materials and methods as described in 0. Other methods well established in the industry Can be used.Example 1 Construction and expression of enzyme variants:   Vector suitable for synthetic gene encoding subtilase 309 and mutants thereof Was built. This is essentially the five subfragments that make up this gene. Linkers containing restriction sites used to separate The pUC19 plasmid in which the roning site has been replaced [Yanish-Perron And Messing (1985) Gene; 33 103-119]. A new linker, thereby Insertion into EcoRI-HindIII cut pUC19 which destroys these sites Was. Details of this construction can be found in WO 92/19729, pages 25-26 and its This is described in FIG. 1 (sheets 1 / 7-7 / 7), and the contents thereof are referred to in this specification. Shall be included.   Each subfragment was made from 6 to 12 oligonucleotides. Oligonucleotides use phosphoramidite chemistry on a controlled glass support Synthesized on an automated DNA synthesizer [Beaucage and Carruthers (1981); Tet rahedron Letters 22 1859-1869].   The five subfragments were isolated on a 2% agarose gel and loaded into pSX191. Inserted. The sequence was confirmed by dideoxynucleotide sequencing. fragment AE was isolated and ligated with KpnI-BamHI cut pSX191. Competite E. coli MC1000r^-, M⁺To select for ampicillin resistance The ligation mixture was used to transform. Then, subtilis encoding the mature part of the enzyme The 850 bp KpnI-BamHI fragment constituting the Syn 309 gene part was Competent in B. subtilis on pSX212 producing pSX222, transformed into a strain Used to replace the wild-type gene. Fermentation of transformed strains and enzymatic purification After production, this product was shown to be indistinguishable from the wild-type product.   Protease variants derived from synthetic genes change where you want to mutate Oligonucleotides having the following sequence (for example, having the sequence shown below) The oligonucleotide and the remaining oligonucleotides suitable for the synthetic gene. It is made by mixing with tide. Mutant gene assembly, mutant material In the same manner as described above. Updates on synthetic genes More information is available in Agarval et al. (1970); Nature; 227 27-34.   At the start of the subtilase 309 synthetic gene encoding the mature part of this enzyme A KpnI site was introduced. The method used was oligonucleotide-specific double-strand breaks. This is called repair mutagenesis and is described in Mandecki (1986) Proc. Nat. Acad. Sci. USA 83 7177-7181. pSX172 was purified from subtilase 309 Cleavage with NcoI at the start of the mature part of the gene to open the oligonucleotide NOR 789 (WO-A-92 / 1972) 9), heated to 100 ° C, cooled to 0 ° C and transformed into E. coli. . After retransformation, colony hybridization using 32-P-labeled NOR789 Recombinants can be screened by the method. screening Recombinants that were found to be positive in the KpnI site introduced just before NcoI by changing three bases did. pSX172 is described in EP Patent Application Publication No. 405 901 . The KpnI site thus created was placed on a 400-bp PvuI-NheI fragment. Insert into pSX120 to generate pSX212. pSX120 is also EP-A -405 901.   The synthetic gene was inserted between KpnI and BamHI on pSX212 to create pS Generate X222.   Examples of mutations and corresponding oligonucleotide sequences are as follows: :R170L (Fragment D1) R170I(Fragment D1) S57P(Fragment B1) These oligonucleotides are the remaining oligonucleotides from the unaltered synthetic gene. Mixed with nucleotides.Example 2 Purification of enzyme variants:   This technique involves subtilisin 147 enzyme, subtilisin 309 enzyme or their For purification of a 10 liter scale fermentation of the mutant.   Approximately 8 liters of fermentation broth is transferred to a 1 liter beaker at 5000 rpm for 35 minutes. Centrifuged for minutes. The supernatant was adjusted to pH 6.5 using 10% acetic acid, and Seitz Supr a Filtered on S100 filter plate.   The filtrate was loaded on an Amicon CH2A UF unit equipped with an Amicon SIY10 UF cartridge. And concentrated to about 400 ml. UF concentrate Centrifuge, prior to adsorption at room temperature on a bacitracin affinity column at pH 7. Stand and filter. 0.01 dimethyl glutaric acid adjusted to pH 7, 0.1 M boron 25% 2-propano in buffer with acid and 0.002M calcium chloride And a 1M sodium chloride solution at room temperature from the bacitracin column. Ease was eluted.   The protease activity-containing fractions obtained in the bacitracin purification step were combined, and the mixture was adjusted to pH 6.5. 0.01M dimethylglutaric acid, 0.2M boric acid and 0.002M adjusted to 750 ml of Sephadex G2 equilibrated with a buffer containing calcium chloride The sample was placed on five columns (diameter: 5 cm).   The fractions having proteolytic activity obtained from the Sephadex G25 column are combined, and p 0.01 M dimethyl glutaric acid, 0.2 M boric acid and H . 150 ml CM cell equilibrated with buffer containing 002M calcium chloride It was mounted on a Pharos CL 6B cation exchange column (5 cm in diameter).   A linear gradient of 0-0.1 M sodium chloride in 2 liters of the same buffer (sub In the case of thyricin 147, 0-0.2 M sodium chloride) Was eluted.   In the final purification step, the protease-containing fraction obtained from the CM Sepharose column was used. Combine and equip with GR81PP membrane (Danish Sugar Factories Inc.). And concentrated in an Amicon ultrafiltration cell.   Using the method of Example 1 for construction and the isolation procedure described above, A butyricin 309 mutant was created and isolated: A: G159I B: S164I C: Y167I D: R170I E: R170L F: R170M G: R170F H: G195F I: S57P + R170L J: R170L + N218S K: S57P + R170L + N218S L: R170L + N218S + M222A M: S57P + R170L + S188P + A194P N: Y167I + R170L O: S57P + R170L + Q206E P: R170L + Q206E Q: Y167I + R170L + Q206E R: Y167I + R170L + A194P S: Y167I + R170L + N218S T: Y167I + R170L + A194P + N218S U: Y167I + Y171I V: R170G W: R170CExample 3 Detergent compositions containing enzyme variants Example D1:   According to an embodiment of the present invention, the (isotropic) aqueous detergent liquid may contain: Prescribe to:   From Table III, the mutant S57P + R170L + N218S was identified as a detergent of this type. It is evident that it shows a significantly improved stability in such a case. Furthermore, mutant S57P + R170L + N218S has excellent compatibility with lipases. From Table IV above, it can be seen that in addition to protease stability, It is evident that the compatibility of Ze is also improved.Example D2: According to an embodiment of the present invention, the non-aqueous detergent liquid is mixed with 4.9 mol / mol ethylene oxide and % Of C13 alkoxylated with 2.7 mol / mol of propylene oxide -C15 linear primary alcohol, 5% triacetin, 30% sodium triphosphate 15.5% sodium perborate monohydrate with 4% soda ash, small amount of oxoborate Japanese, 4% TAED, 0.25% EDTA, 0.1% of which is phosphonic acid, A with erosil 0.6%, SCMC 1%, and 0.6% protease To prescribe. pH is Adjust between 9 and 10, for example, about 9.8.Example D3:   Structured liquid detergents may be used, for example, as described herein. In addition to Rotase, 2-15% non-ionic detergent, non-ionic and optional A total of 5-40% of surfactants, optionally containing anionic surfactants, 5-35% A phosphate-containing or non-phosphate-containing builder, 0.2-0.8% of a polymeric thickener, For example, 10⁶Crosslinked acrylic polymer with a higher molecular weight, at least 10% Sodium silicate, for example, neutral water glass, the desired pH, preferably 9-10 An alkali (eg, potassium) for adjusting to the above range, for example, a pH of more than 11 Containing alkali), sodium cation: silicate anion (as free silica) Of less than 0.7: 1 (by weight), and 0.3-30 Pas (20 ° C. and 2 0^s-1At) viscosity.   A suitable example is about 5 EO groups per mole and 2.7 PO groups per mole. About 5% of a nonionic surfactant of alkoxylated C13-15 alcohol, Weight of silica and sodium oxide 15-23% neutral water glass with a quantitative ratio of 3.5, 13-19% KOH) 8-23 % STPP, 0-11% sodium carbonate, 0.5% Carbopol 9 41 (TM).   Proteases can be included, for example, at 0.5%.Example D4: (Decoupling polymer liquid) Priorene 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 8.23 citric acid Antifoam silicone DB100 0.3 LAS acid 16.5 Spice 0.5 Up to 100% water   From Table V, the R170L mutant shows significantly improved stability with this type of detergent. It is clear thatExample D5: (Decoupling polymer liquid) Priorene 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 DC 1 8.23 citric acid Antifoam silicone DB100 0.3 LAS acid 16.5 Lipolase 100L 0.6 Spice 0.5 Up to 100% water   From Table VI, the mutant S57P + R170L + N218S was found to be effective in this type of detergent. And significantly improved stability. Furthermore, the mutant S57P + R 170L + N218S has excellent compatibility with lipases.Example D6:   49.7% by weight 80/20 tallow / coconut soap, 49.0% Water, 20% sodium citrate, 1.0% citric acid and 0.031% A bar soap containing protease was produced. After preparing the bar soap, store at room temperature. After a specified time interval, samples were taken and protease activity was measured. Stability The data are shown in Table VII below:   From Table VII above, the subtilase mutants R170L, R170L + N218S + S57P and R170L + Y167I are significantly improved in this type of detergent It is evident that it exhibits the provided stability.Example D7:   63.88% 80/20 tallow / coconut soap, 1% coconut fatty acid, 25 . 1% water, 10% sodium citrate and 0.021% protease A bar soap containing it was produced. Store laundry bar soap at 37 ° C for a specific time After separation, samples were taken and protease activity was measured.   From Table VIII above, the subtilase mutant R170L + N218S + S57P However, it is clear that this type of detergent shows significantly improved stability.Example 4 Detergent performance of detergent compositions comprising enzyme variants   The following examples provide the results of several cleaning tests performed under the indicated conditions.   The above model detergent is a simple detergent formulation. The most unique feature is the STP Used as a builder, said that the content of anionic surfactant (LAS) is quite high That is. Further, the pH is adjusted to 9.5 which is low as a powder detergent.   The composition of the model detergent is as follows:   The measurement of the remmision (R) on the test substance is carried out by means of an Elrepho 2000, photometer ( (No UV) at 460 nm. Formula the measured value: Apply to   The improvement factor is calculated by using the initial slope of the curve: ΔR is the cleaning effect of the enzyme in terms of the specific reflectance. a is the initial slope (c → 0) of the fitted curve. a_refIs the initial slope of the reference enzyme. c is the enzyme concentration in mg / l. △ R_maxIs the theoretical maximum cleaning effect of the enzyme in the specific reflectance unit (c → ∞) ).   As can be seen from Table XI, all subtilisin 309 variants of the invention were washed Indicates an improvement in performance.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), UA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AL, AM, AT, AU, AZ , BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, I S, JP, KE, KG, KP, KR, KZ, LK, LR , LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, S D, SE, SG, SI, SK, TJ, TM, TR, TT , UA, UG, UZ, VN (72) Inventor Von del Osten, Claus             Denmark, Day Car-2800 Ring             Bey, Bow Leverden 14, El 66 (72) Inventor Cielkstra, Laurens Nikolaas             N.L.-2623 N.A., The Netherlands             Le Delft, Talinghof 10 (72) Inventor Baudets, Peter             Denmark, Day Co-2100 Copen             Hagen, Lillesogiase 18, Fie             Rule

Claims (1)

[Claims] 1. A detergent composition comprising a subtilase variant, wherein the detergent composition comprises a subtilase variant. In the variant, one located in or near the hydrophobic domain of the parent subtilase The above amino acid residues have been replaced with amino acid residues that are more hydrophobic than the original residues, The hydrophobic domains are P129, P131, I165, Y16 of BLS309. 7, containing the Y171 residue (based on BASBPN numbering) Are the E136, G159, S164, R170, and A194 of BLS309. And residues corresponding to G195 residues (based on BASBPN numbering) Provided that R170M, R170I and R170V mutants of BABP92 The above-described composition, wherein 2. The original amino acid residue is Val (V), Ile (I), Leu (L) , Met (M), Phe (F), and Trp (W) Group, preferably a subtilase modification substituted with Val, Ile, or Leu. The composition of claim 1, comprising a variant. 3. The parent subtilase is selected from subgroup I-S1; The composition according to claim 1. 4. The parent subtilases are ABSSS168, BASPN, BSSDY, and 4. The composition of claim 3, wherein the composition is selected from the group consisting of BLSCAR. 5. 3. The parent subtilase is selected from subgroup IS2. A composition according to claim 1. 6. The parent subtilase is BLS147, BLS309, BAPB92, and The composition according to claim 5, wherein the composition is selected from the group consisting of BYSYAB. 7. 6. The composition of claim 5, wherein the parent subtilase is TVTHER. 8. The substitution is in combination with a substitution, insertion or deletion at any other position. A composition according to any of the preceding claims. 9. The substitution at any of positions 36, 222, 218, 76; 9. The composition of claim 8, wherein the composition is in combination with an insertion or deletion. 10. Less than:         K136V, K136I, K136L, K136M, K136F,         S159V, S159I, S159L, S159M, S159F,         T164V, T164I, T164L, T164M, T164F,         K170V, K170I, K170L, K170M, K170F,         E195V, E195I, E195L, E195M, E195F,         Q136V, Q136I, Q136L, Q136M, Q136F,         T159V, T159I, T159L, T159M, T159F,         A164V, A164I, A164L, A164M, A164F,         Y170V, Y170I, Y170L, Y170M, Y170F,         S194V, S194I, S194L, S194M, S194F,         E136V, E136I, E136L, E136M, E136F,         G159V, G159I, G159L, G159M, G159F,         G164V, G164I, G164L, G164M, G164F,         S164V, S164I, S164L, S164M, S164F,         R170V, R170I, R170L, R170M, R170F,         A194V, A194L, A194L, A194M, A194F,         P194V, P194I, P194L, P194M, P194F,         E195V, E195I, E195L, E195M, E195F,         G195V, G195I, G195L, G195M, G195F, The composition according to any one of claims 1 to 9, which is one of the following. 11. The variants have positions 36, 57, 76, 218, 222, and 22 Combined with further substitutions, deletions and / or insertions at any one or more of positions 4 11. The composition according to any of the preceding claims, wherein the composition is in contact. 12. The subtilase belongs to the IS2 subgroup, and the further change is * 36D, S57P, N76D, N218S, M222S, M222A, and The composition of claim 11, wherein the composition is selected from the group consisting of T224S. 13. The mutant is 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 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) 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 + R170I + S188P + A194P + N218S 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 + G97N + V104Y + H120D + R170I + 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 r ') Y167F + R170I t) Y167I + R170L + A194P t ') 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) * 36D + Y167I + R170L y ') * 36D + Y167I + R170I z) Y167I + Y171I aa) Y167V + R170L aa ') Y167V + R170I 13. The composition according to claim 12, wherein the composition is selected from the group of variants consisting of: 14． The subtilase variant is R170L or R170I, and the composition is 14. The composition according to any of claims 1 to 13, which is a liquid detergent and comprises a deflocculating polymer. Stuff. 15. The subtilase mutant was S57P + V104Y + R170L + N218 15. S or S57P + V104Y + R170I + N218S. A composition as described. 16. 14. The composition according to any of the preceding claims, which is in the form of a solid.