WO2004041979A2 - Subtilase variants - Google Patents

Subtilase variants Download PDF

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Publication number
WO2004041979A2
WO2004041979A2 PCT/DK2003/000756 DK0300756W WO2004041979A2 WO 2004041979 A2 WO2004041979 A2 WO 2004041979A2 DK 0300756 W DK0300756 W DK 0300756W WO 2004041979 A2 WO2004041979 A2 WO 2004041979A2
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WO
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Prior art keywords
variant
subtilase
variants
s99sd
enzyme
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PCT/DK2003/000756
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French (fr)
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WO2004041979A3 (en
Inventor
Henriette Dragborg
Peter Kamp Hansen
Mads Eskelund BJØRNVAD
Mads Nørregaard-Madsen
Mikael Mikkelsen
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Novozymes A/S
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Publication date
Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to EP03769258A priority Critical patent/EP1563064A2/en
Priority to JP2005502097A priority patent/JP4694964B2/en
Priority to AU2003277836A priority patent/AU2003277836B2/en
Priority to EP09172028.4A priority patent/EP2138574B1/en
Priority to KR1020127003715A priority patent/KR101394294B1/en
Priority to KR1020057007858A priority patent/KR101293498B1/en
Priority to MXPA05004755A priority patent/MXPA05004755A/en
Priority to KR1020177008215A priority patent/KR20170037684A/en
Priority to CA2503965A priority patent/CA2503965C/en
Priority to BR0315916-7A priority patent/BR0315916A/en
Priority to KR1020137035040A priority patent/KR20140021051A/en
Publication of WO2004041979A2 publication Critical patent/WO2004041979A2/en
Publication of WO2004041979A3 publication Critical patent/WO2004041979A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

  • the present invention relates to novel subtilase variants exhibiting alterations relative to the parent subtilase in one or more properties including: Wash performance, thermal stability, storage stability or catalytic activity.
  • the variants of the invention are suitable for use in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions.
  • the present invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention. Further, the present invention relates to cleaning and detergent compositions comprising the variants of the invention.
  • Enzymes used in such formulations comprise proteases, lipases, amyiases, cellulases, as well as other enzymes, or mixtures thereof. Commercially the most important enzymes are proteases.
  • proteases protein engineered variants of naturally occurring wild type proteases, e.g. Durazym ® , Relase ® , Alcalase ® , Savinase ® , Primase ® , Duralase ® , Esperase ® , Ovozyme ® and Kannase ® (Novozymes A/S), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect OxPTM, FN2TM, FN3TM and FN4TM (Genencor International, Inc.). Further, a number of protease variants are described in the art. A thorough list of prior art protease variants is given in WO 99/27082.
  • an object of the present invention is to provide improved subtilase variants for such purposes.
  • the present invention relates to a subtilase variant comprising at least a) an insertion, substitution or deletion of one of the amino acid residues K,H,R,E,D,Q,N,C,V,L,I,P,M,F,W,Y,G,A,S,T in one or more of the positions
  • N173D A174V; M175L,I,V,A,S,T; N183D; N184D.S; N185S.D; R186L,C,H; S188G; S190A;
  • modification(s) comprise(s): deletion, insertion and/or substitution of an amino acid residue selected from the group consisting of K,H,R,E,D,Q,N,C,V,L,I,P, ,F,W,Y,GAS and T.
  • the present invention relates to a subtilase variant comprising a) the combination of one or more of the modifications X62D,XD,XE,XG,DE
  • the present invention relates to a subtilase variant comprising at least one of the alterations disclosed in Table I below:
  • subtilase variants of the inventions having one or more of the alterations:
  • each position corresponds to a position of the amino acid sequence of subtilisin B PN', shown in Figure 1 and SEQ ID NO: 1.
  • the present invention relates to an isolated polynucleotide encoding a subtilase variant of the invention.
  • the present invention relates to an expression vector comprising the isolated polynucleotide of the invention.
  • the present invention relates to a microbial host cell transformed with the expression vector of the invention.
  • the present invention relates to a method for producing a subtilase variant according to the invention, wherein a host according to the invention is cultured under conditions conducive to the expression and secretion of the variant, and the variant is recovered.
  • the present invention relates to a cleaning or detergent composition, preferably a laundry or dish wash composition, comprising the variant of the invention.
  • the present invention relates to a subtilase variant comprising at least one of the alterations disclosed in Table II below:
  • subtilase variants of the inventions having one or more of the alterations:
  • each position corresponds to a position of the amino acid sequence of subtilisin B PN', shown in Figure 1 and SEQ ID NO: 1.
  • the present invention relates to a subtilase variant comprising one of the alterations N252D and N252M.
  • the present invention relates to a subtilase variant comprising one or more of the alterations M119L, I, V, A, S; M175L, I, V, A, S and M222L, I, V, A, S in combination with the subtilase variants listed in tables I and II above.
  • Fig. 1 shows an alignment between subtilisin BPN'(a) (BASBPN) and subtilisin 309 (b) (BLSAVI). This alignment is in this patent application used as a reference for numbering the residues.
  • a frame of reference is first defined by aligning the isolated or parent enzyme with subtilisin BPN' (BASBPN).
  • Another method is to use known recognized alignments between subtilases, such as the alignment indicated in WO 91/00345. In most cases the differences will not be of any importance.
  • subtilisin 309 SEQ ID NO.2
  • subtilisin 309 SEQ ID NO.2
  • Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3).
  • subtilase BPN subtilase BPN' (BASBPN) sequence.
  • BPN' sequence see Fig. 1 , SEQ ID NO:1 or Siezen et al., Protein Engng. 4 (1991) 719-737.
  • a serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 "Principles of Biochemistry," Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).
  • the bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease.
  • alkaline protease covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753).
  • subtilases A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. (1997).
  • subtilisin 168 subtilisin 168
  • subtilisin BPN subtilisin BPN
  • subtilisin Carlsberg ALCALASE ®
  • NOVOZYMES A/S subtilisin Carlsberg
  • subtilisin DY subtilisin DY
  • subtilases I-S2 or high alkaline subtilisins
  • Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprises enzymes such as subtilisin PB92 (BAALKP) (MAXACAL ® , Genencor International Inc.), subtilisin 309 (SAVINASE ® , NOVOZYMES A/S), subtilisin 147 (BLS147) (ESPERASE ® , NOVOZYMES A S), and alkaline elastase YaB (BSEYAB).
  • SAVINASE® is marketed by NOVOZYMES A/S. It is subtilisin 309 from B. Lentus and differs from BAALKP only in one position (N87S). SAVINASE® has the amino acid sequence designated b) in Fig. 1 and in SEQ ID NO:2.
  • parent subtilase describes a subtilase defined according to Siezen et al. (1991 and 1997). For further details see description of "Subtilases” above.
  • a parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modifications have been made while retaining the characteristic of a subtilase.
  • a parent subtilase may be a subtilase which has been prepared by the DNA shuffling technique, such as described by J.E. Ness et al., Nature Biotechnology, 17, 893-896 (1999). Alternatively the term "parent subtilase” may be termed "wild type subtilase".
  • Modification(s) of a subtilase variant The term "modification(s)" used herein is defined to include chemical modification of a subtilase as well as genetic manipulation of the DNA encoding a subtilase.
  • the modification(s) can be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.
  • subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host.
  • GCG package version 9.1 can be applied (infra) using the same settings.
  • the output from the routine is besides the amino acid alignment the calculation of the "Percent Identity" between the two sequences.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3" untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
  • the term "an isolated polynucleotide” may alternatively be termed "a cloned polynucleotide”.
  • the term “isolated” indicates that the protein has been removed from its native environment.
  • the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. "homologous impurities” (see below)).
  • An isolated protein is more than 10% pure, preferably more than 20% pure, more preferably more than 30% pure, as determined by SDS-PAGE. Further it is preferred to provide the protein in a highly purified form, i.e., more than 40% pure, more than 60% pure, more than
  • isolated protein may alternatively be termed “purified protein”.
  • homologous impurities means any impurity (e.g. another polypeptide than the subtilase of the invention), which originate from the homologous cell where the subtilase of the invention is originally obtained from.
  • the term "obtained from” as used herein in connection with a specific microbial source means that the polynucleotide and/or subtilase produced by the specific source, or by a cell in which a gene from the source has been inserted.
  • substrate used in connection with a substrate for a protease should be interpreted in its broadest form as comprising a compound containing at least one peptide (amide) bond susceptible to hydrolysis by a subtilisin protease.
  • product used in connection with a product derived from a protease enzymatic reaction should, in the context of the present invention, be interpreted to include the products of a hydrolysis reaction involving a subtilase protease.
  • a product may be the substrate in a subsequent hydrolysis reaction.
  • wash performance is used as an enzyme's ability to remove proteinaceous or organic stains present on the object to be cleaned during e.g. wash or hard surface cleaning. See also the wash performance test in Example 3 herein.
  • Fig. 1 shows an alignment between subtilisin BPN' (a) and Savinase®(b) using the GAP routine mentioned above.
  • the present invention relates to novel subtilase variants exhibiting alterations relative to the parent subtilase in one or more properties including: Wash performance, thermal stability, storage stability or catalytic activity.
  • variants which are contemplated as being part of the invention are such variants where, when compared to the wild-type subtilase, one or more amino acid residues has been substituted, deleted or inserted, said variants comprising at least
  • modification(s) comprise(s): deletion, insertion and/or substitution of an amino acid residue selected from the group consisting of K,H,R,E,D,Q,N,C,V,L,I,P,M,F,W,Y,G,A,S and T.
  • variants of the present invention comprises at least one or more of the alterations indicated in Table I and II, wherein
  • a subtilase variant of the first aspect of the invention may be a parent or wild-type subtilase identified and isolated from nature. Such a parent wild-type subtilase may be specifically screened for by standard techniques known in the art.
  • One preferred way of doing this may be by specifically PCR amplify conserved DNA regions of interest from subtilases from numerous different microorganism, preferably different Bacillus strains.
  • Subtilases are a group of conserved enzymes, in the sense that their DNA and amino acid sequences are homologous. Accordingly it is possible to construct relatively specific primers flanking the polynucleotide sequences of interest.
  • subtilase variants of the invention is predominantly a variant of a parent subtilase.
  • a subtilase variant suitable for the uses described herein may be constructed by standard techniques known in the art such as by site-directed/random mutagenesis or by DNA shuffling of different subtilase sequences. See the "Material and Methods" section and Example 1 herein (vide infra) for further details.
  • variants described herein may comprise one or more further modifications, in particular one or more further substitutions or insertions.
  • the variants described herein may encompass mutation at more than just one position.
  • the variant according to the invention may contain mutations at one position, two positions, three positions or more than three positions, such as four to eight positions.
  • the parent subtilase belongs to the subgroups I-S1 or I-S2, especially subgroup I-S2, both for enzymes from nature or from the artificial creation of diversity, and for designing and producing variants from a parent subtilase.
  • subtilase In relation to variants from subgroup I-S1 , it is preferred to select a parent subtilase from the group consisting of BSS168 (BSSAS, BSAPRJ, BSAPRN, BMSAMP), BASBPN, BSSDY, BLSCAR (BLKERA, BLSCA1 , BLSCA2, BLSCA3), BSSPRC, and BSSPRD, or functional variants thereof having retained the characteristic of sub-group I-S1.
  • subtilase In relation to variants from subgroup I-S2 it is preferred to select a parent subtilase from the group consisting of BSAPRQ, BLS147 (BSAPRM, BAH 101 ), BLSAVI (BSKSMK, BAALKP, BLSUBL), BYSYAB, BAPB92, TVTHER, and BSAPRS, or functional variants thereof having retained the characteristic of sub-group I-S2.
  • the parent subtilase is BLSAVI (Savinase®, NOVOZYMES A/S), and a preferred subtilase variant of the invention is accordingly a variant of Savinase®.
  • the present invention also encompasses any of the a bove m entioned s ubtilase variants i n combination with any other modification to the amino acid sequence thereof. Especially com- binations with other modifications known in the art to provide improved properties to the enzyme are envisaged.
  • the art describes a number of subtilase variants with different improved properties and a number of those are mentioned in the "Background of the invention" section herein (vide supra). Those references are disclosed here as references to identify a subtilase variant, which advantageously can be combined with a subtilase variant described herein.
  • subtilase variant described herein may advantageously be combined with one or more modification(s) in any of the positions:
  • a particular interesting variant is a variant, which, in addition to modifications according to the invention, contains the following substitutions:
  • subtilase variants of the main aspect(s) of the invention are preferably combined with one or more modification(s) in any of the positions 129, 131 and 194, preferably as 129K, 131H and 194P modifications, and most preferably as P129K, P131H and A194P modifications. Any of those modification(s) are expected to provide a higher expression level of the subtilase variant in the production thereof.
  • the wash performance of a selected variant of the invention may be tested in the wash performance test disclosed in Example 3 herein.
  • the wash performance test may be employed to assess the ability of a variant, when incorporated in a standard or commercial detergent composition, to remove proteinaceous stains from a standard textile as compared to a reference system, namely the parent subtilase or a similar subtilase exhibiting an even better wash performance (incorporated in the same detergent system and tested under identical conditions).
  • the enzyme variants of the present application were tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA test the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined rapidly. Using this test, the wash performance of a selected variant can be initially investigated, the rationale being that if a selected variant does not show a significant improvement in the test compared to the parent subtilase, it is normally not necessary to carry out further test experiments.
  • AMSA Automatic Mechanical Stress Assay
  • variants which are particularly interesting for the purposes described herein are such variants which, when tested in a commercial detergent composition such as a US type detergent, an Asian type, a European type or a Latin American type detergent as described in the wash performance test (Example 3), shows an improved wash performance as compared to the parent subtilase tested under identical conditions.
  • a commercial detergent composition such as a US type detergent, an Asian type, a European type or a Latin American type detergent as described in the wash performance test (Example 3)
  • the improvement in the wash performance may be quantified by calculating the so-called intensity value (Int) defined in Example 3, herein.
  • the variant of the invention when tested in the wash performance test has a Performance Score (S) of at least 1 , preferably a
  • S (1) variant performs better than the reference at one or two concentrations.
  • the variant of the invention fulfils the above criteria on at least the stated lowest level, more preferably at the stated highest level.
  • subtilase genes Many methods for cloning a subtilase and for introducing substitutions, deletions or insertions into genes (e.g. subtilase genes) are well known in the art.
  • subtilase variant of the invention In general standard procedures for cloning of genes and introducing mutations (random and/or site directed) into said genes may be used in order to obtain a subtilase variant of the invention.
  • suitable techniques reference is made to Example 1 herein (vide infra) and (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular Biology”. John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus”. John Wiley and Sons, 1990), and WO 96/34946.
  • subtilase variant may be constructed by standard techniques for artificial creation of diversity, such as by DNA shuffling of different subtilase genes (WO 95/22625; Stemmer WPC, Nature 370:389-91 (1994)). DNA shuffling of e.g. the gene encoding Savinase® with one or more partial subtilase sequences identified in nature, will after subsequent screening for improved wash performance variants, provide subtilase variants suitable for the purposes described herein.
  • a recombinant expression vector comprising a DNA construct encoding the e nzyme o f t he invention may be any vector that may conveniently be subjected to recombinant D NA p rocedures.
  • the vector may be an autonomously replicating vector, i.e. a vector that exists as an extra- chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one that on introduction into a host cell is integrated into the host cell genome in part or in its entirety and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is operably linked to additional segments required for transcription of the DNA.
  • the expression vector is derived from plasmid or viral DNA, or may contain elements of both.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the enzyme.
  • the promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha- amylase gene, the Bacillus amyloliquefaciens alpha-amylase gene, the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, or the phage Lambda P R or PL promoters or the E. coli lac, trp or tac promoters.
  • the DNA sequence encoding the enzyme of the invention may also, if necessary, be operably connected to a suitable terminator.
  • the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, or the like, or resistance to heavy metals or herbicides.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
  • the secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame.
  • Secretory signal s equences a re commonly positioned 5' to the DNA sequence encoding the enzyme.
  • the secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.
  • the DNA sequence encoding the present enzyme introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment.
  • the term "homologous” is intended to include a DNA sequence encoding an enzyme native to the host organism in question.
  • heterologous is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence m ay b e f rom a nother o rganism, o r it may b e a synthetic sequence.
  • the host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell that is capable of producing the present enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells including plants.
  • Examples of bacterial host cells which, on cultivation, are capable of producing the enzyme of the invention are gram-positive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium or ⁇ . thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gram-negative bacteria such as Escherichia coli.
  • Bacillus such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium or
  • the transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
  • the enzyme When expressing the enzyme in bacteria such as E. coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells a re lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme.
  • the enzyme When expressing the enzyme in gram-positive bacteria such as Bacillus or Streptomyces strains, the enzyme may be retained in the cytoplasm, or may be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme may be recovered from the medium as described below.
  • the present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
  • the medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question.
  • the expressed subtilase may conveniently be secreted into the culture medium and may be recovered there-from by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • the enzyme of the invention may be added to and thus become a component of a detergent composition.
  • cleaning and detergent compositions are well described in the art and reference is made to WO 96/34946; WO 97/07202; WO 95/30011 for further description of suitable cleaning and detergent compositions.
  • the detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general h ousehold h ard s urface cleaning operations, or be formulated for hand or machine dishwashing operations.
  • the invention provides a detergent additive comprising the enzyme of the invention.
  • the detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
  • enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidas
  • the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
  • proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included.
  • the protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease.
  • alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 a nd s ubtilisin 168 (described in WO 89/06279).
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
  • useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.
  • Preferred commercially available protease enzymes include Durazym ® , Relase ® , Alcalase ® , Savinase ® , Primase ® , Duralase ® , Esperase ® , Ovozyme ® and Kannase ® (Novozymes A S), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect OxPTM, FN2TM, FN3TM and FN4TM (Genencor International, Inc.).
  • Lipases S Amble I ipases i nclude t hose o f b acterial o r f ungal o rigin. C hemically m odified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.
  • Humicola semomyces
  • H. lanuginosa T. lanuginosus
  • Pseudomonas lipase e.g. from P. alcaligenes or P. pseudoalcaligenes (EP
  • cepacia EP 331 376
  • P. stutzeri GB 1 ,372,034
  • P. fluorescens Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002)
  • P. wisconsinensis WO 96/12012
  • Bacillus lipase e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131 , 253-360), B. stearothermophilus (JP 64/744992) or ⁇ . pumilus (WO 91/16422).
  • lipase variants such as those described in WO 92/05249, WO 94/01541 , EP 407 225, EP 260 105, WO 95/35381 , WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
  • Lipex ® Lipex ®
  • Lipolase ® Lipolase Ultra ®
  • Amyiases include those of bacterial or fungal origin.
  • Amyiases include, for example, ⁇ -amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in GB 1 ,296,839.
  • amyiases examples include the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181 , 188, 190, 197, 202, 208,
  • amyiases are Duramyl ® , Termamyl ® , Fungamyl ® and BAN ®
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits.
  • cellulases examples include cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
  • cellulases include Celluzyme ® , and Carezyme ® (Novozymes A/S), ClazinaseTM, and Puradax HATM (Genencor International Inc.), and KAC-500(B)TM (Kao Corporation).
  • Peroxidases/Oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include Guardzyme ® (Novozymes A/S).
  • the detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
  • a detergent additive of the invention i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc.
  • Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
  • Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • the detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid.
  • a liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non-aqueous.
  • the detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic.
  • the surfactants are typically present at a level of from 0.1% to 60% by weight.
  • the detergent When included therein the detergent will usually contain from about 1 % to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, a lkyl s ulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, a lkyl s ulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • the detergent When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanol- amide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides").
  • a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanol- amide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).
  • glucamides N-acyl N-alkyl derivatives of glucosamine
  • the detergent may contain 0-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylene- diaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenyl-succinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylene- diaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenyl-succinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • the detergent may comprise one or more polymers.
  • examples are carboxymethyl-cellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinyl-pyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as poly-acrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the detergent may contain a bleaching system which may comprise a H 2 O 2 source such as perborate or percarbonate which may be combined with a p eracid-forming b leach a ctivator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
  • a bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.
  • the detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • Detergent Examples 1 and 2 provide ranges for the composition of a typical Latin American detergent and a typical European powder detergent respectively.
  • Detergent Example 1 Typical Latin American detergent composition.
  • the enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
  • a polyol such as propylene glycol or glycerol
  • a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid
  • any single enzyme in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-200 mg of enzyme protein per liter of wash liqour, preferably 0.05-50 mg of enzyme protein per liter of wash liqour, in particular 0.1-10 mg of enzyme protein per liter of wash liqour.
  • the enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.
  • Standard textile pieces are obtained from EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland. Especially type EMPA116 (cotton textile stained with blood, milk and ink) and EMPA117 (polyester/cotton textile stained with blood, milk and ink).
  • Bacillus lentus strain 309 is deposited with the NCIB and accorded the accession number NCIB 10309, and described in US Patent No. 3,723,250 incorporated by reference herein.
  • the parent subtilase 309 or Savinase® can be obtained from Strain 309.
  • the expression host organism is Bacillus subtilis.
  • the plasmid pSX222 is used as E. coli - B. subtilis shuttle vector and B. subtilis expression vector (as described in WO 96/34946).
  • Fermentations for the production of subtilase enzymes are performed at pH 7.3 and 37°C on a rotary s haking table at 225 rpm. i n 50 ml tubes containing 15 ml double TY media for 2-3 days.
  • the s ubtilase variant s ecreted from the h ost cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • WASH PERFORMANCE TEST In order to asses the wash performance of selected subtilase variants in detergent compositions, washing experiments are performed. The enzyme variants of the present application is tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA test the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined.
  • the AMSA plate has a number of slots for test solutions and a lid firmly squeezing the textile swatch to be washed against all the slot openings. During the washing time, the plate, test solutions, textile and lid are vigorously shaken to bring the test solution in contact with the textile and apply mechanical stress.
  • WO 02/42740 especially the paragraph "Special method embodiments" at page 23-24.
  • Detergents for wash performance tests of the subtilase enzymes of the invention can be obtained by purchasing fully formulated commercial detergents at the market and subsequently inactivate the enzymatic components by heat treatment (5 minutes at 85°C in aqueous solution). Moreover a commercial detergent base without enzymes can be purchased directly from the manufacturer. Further a suitable model detergent can be composed according to the provisions at page 19-24 herein and used for wash performance tests.
  • Subtilisin 309 (Savinase ® ) site-directed variants of the invention comprising specific insertions/deletions/substitutions are made by traditional cloning of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) produced by PCR with oligos containing the desired mutations.
  • the template plasmid DNA may be pSX222, or an analogue of this containing a variant of subtilisin 309. Mutations are introduced by oligo directed mutagenesis to the construction of variants.
  • the subtilisin 309 variants are transformed into E. coli.
  • DNA purified from an over night culture of t hese t ransformants is transformed i nto ⁇ . subtilis by restriction endonuclease digestion, purification of DNA fragments, ligation, transformation of B. subtilis. Transformation of B. subtilis is performed as described by Dubnau et al., 1971 , J. Mol. Biol. 56, pp. 209-221.
  • SITE-DIRECTED MUTAGENESIS IN ORDER TO INTRODUCE MUTATIONS IN A SPECIFIC REGION
  • Mutagenic primers are synthesized corresponding to the DNA sequence flanking the sites of mutation, separated by the DNA base pairs defining the insertions / deletions / substitutions.
  • the resulting mutagenic primers are used in a PCR reaction with the modified plasmid pSX222.
  • the resulting PCR fragment is purified and extended in a second PCR- reaction, the resulting PCR product is purified and extended in a third PCR-reaction before being digested by endonucleases and cloned into the E. coli - B. subtilis shuttle vector pSX222.
  • the PCR reactions are performed under normal conditions.
  • the plasmid DNA is transformed into E. coli by well-known techniques and one E. coli colony is sequenced to confirm the mutation designed.
  • subtilase variants of the invention can be constructed as described above.
  • the pSX222 expression plasmid comprising a variant of the invention was transformed into a competent ⁇ . subtilis strain and fermented as described above.
  • HCIC Hydrophobic Charge Induction Chromatography
  • the HCIC uses a cellulose matrix to which 4-Mercapto-Ethyl-Pyridine (4-MEP) is bound.
  • Beads of the cellulose matrix sized 80-100 ⁇ m are mixed with a media containing yeast extract and the transformed ⁇ . subtilis capable of secreting the subtilisin variants and incubated at pH
  • the concentration of the purified subtilisin enzyme variants is assessed by active site titration (AST).
  • the purified enzyme is incubated with the high affinity inhibitor CI-2A at different concentrations to inhibit a varying amount of the active sites.
  • the protease and inhibitor binds to each other at a 1 :1 ratio and accordingly the enzyme concentration can be directly related to the concentration of inhibitor, at which all protease is inactive.
  • a substrate 0.6 mM Suc-Ala-Ala-Pro-Phe-pNA in Tris/HCI buffer
  • a substrate 0.6 mM Suc-Ala-Ala-Pro-Phe-pNA in Tris/HCI buffer
  • p NA paranitrophenol
  • the performance of the enzyme variant is measured as the brightness of the colour of the textile samples washed with that specific enzyme variant. Brightness can also be expressed as the intensity of the light reflected from the textile sample when luminated with white light.
  • the intensity of the reflected light can be used to measure wash performance of an enzyme variant.
  • Colour measurements are made with a professional flatbed scanner (PFU DL2400pro), which is used to capture an image of the washed textile samples.
  • the scans are made with a resolution of 200 dpi and with an output colour dept of 24 bits.
  • the scanner is frequently calibrated with a Kodak reflective IT8 target.
  • a special designed software application is used (Novozymes Color Vector Analyzer).
  • the program retrieves the 24 bit pixel values from the image and converts them into values for red, green and blue (RGB).
  • RGB red, green and blue
  • T he intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:
  • wash performance (P) of the variants was calculated in accordance with the below formula:

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Abstract

The present invention relates to novel subtilase variants exhibiting alterations relative to the parent subtilase in one or more properties including: Wash performance, thermal stability, storage stability or catalytic activity. The variants of the invention are suitable for use in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions. The subtilase variant has a modification in at least one of the positions 62, 68, 97, 98, 99, 106, 131, 170, 245 and/or 252 in combination with one or more modifications in other positions (159 specified positions), or one or more modification in position 68 (no combination). Further claimed are 56 subtilase variants not linked to the variants specified above.

Description

SUBTILASE VARIANTS
TECHNICAL FIELD
The present invention relates to novel subtilase variants exhibiting alterations relative to the parent subtilase in one or more properties including: Wash performance, thermal stability, storage stability or catalytic activity. The variants of the invention are suitable for use in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions. The present invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention. Further, the present invention relates to cleaning and detergent compositions comprising the variants of the invention.
BACKGROUND OF THE INVENTION
In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amyiases, cellulases, as well as other enzymes, or mixtures thereof. Commercially the most important enzymes are proteases.
An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild type proteases, e.g. Durazym®, Relase®, Alcalase®, Savinase®, Primase®, Duralase®, Esperase®, Ovozyme® and Kannase® (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, FN3™ and FN4™ (Genencor International, Inc.). Further, a number of protease variants are described in the art. A thorough list of prior art protease variants is given in WO 99/27082.
However, even though a number of useful protease variants have been described, there is still a need for new improved proteases or protease variants for a number of industrial uses such as laundry or hard surface cleaning. Therefore, an object of the present invention is to provide improved subtilase variants for such purposes.
SUMMARY OF THE INVENTION
Thus, in a first aspect the present invention relates to a subtilase variant comprising at least a) an insertion, substitution or deletion of one of the amino acid residues K,H,R,E,D,Q,N,C,V,L,I,P,M,F,W,Y,G,A,S,T in one or more of the positions
62, 68, 97, 98, 99, 106, 131 , 170, 245, 252 in combination with at least one of the following modifications
*0AQSVPWG; A1T.V; Q2L; S3T,A,L; V4L.A; I8V,T; S9G,D,R,K,LN; R10H.K; V11A; Q12D;
A13V; P14S,T,D,A,MN,K,Q,L,H,R,I; A15M.T; A16P; H17R; Ν18S.H; R19W,K,L,F,G,I;
G20*,R,A; L21 F,LP,LW,LA,LG; T22S,A,K,TV,TG,TL,TW,TV,G,L,TY; G23S; S24P; K27R, V28I; V30I; I35T,V;T38S; P40L; N43D; R45H.K; G46D; A48T; S49N; F50S; V51A,I,D; P52V.A;
P55S,A; S57P; G61E,D,S,R,GP; N62D,ND,NE,DE,NG,E,S; V68A,S,L,I; T71A; I72V; L75I;
N76S.D; N77S; S78T; V81A; A85T; S87C; A88V.T; E89G; K94N; V95C.T; L96LA.LG;
G97E,D,W,A,GG,GA,GV,N,GS; A98S,D,E,T,AS,AD,AV,AE,AH,Q,N,M,L,G,RN,S;
S99D,L,A,AD,SD,SM,SG,DA,P,G,N,C,MN,I; G100S,GE,C; S101SA, SK; G102D.S; S103D,E,Y,L,Q,H,T; V104T,S,R,I,Ν,M,L,D; S106D,E,T,M,G,A,L,F,I; I107TN.M; A108V,T,S;
L111 IN; A114V; Ν116S.D; G118D; M119L,IN,A,S; H120Ν,D,Q,K,E,Y,S; V121A; L124C;
L126I; G127E; S128N,I,G,C; P129PSN,T,E,D,S,N,A; S130P,T,C,*;
P131 M,F,W,L,A,H,T,*,PA,S,Q,R,E,G,D,C; S132G.T; A133ASA; T134A; Q137H,E,D; A138GN;
V139L.I; Ν140D, K; T143A; S144D, N,P; R145G; V150I; A151V.G; A152P; A158TN,C,E,L,D, M; G160A,D; S163G,C,Ν,A; Y167K,A,I; A168G; A169G; R170C,S,H,L; Y171C; A172V;
N173D; A174V; M175L,I,V,A,S,T; N183D; N184D.S; N185S.D; R186L,C,H; S188G; S190A;
Y192H; G195F.E; V203S,A,L,Q,M, F,l; N204T,D,S; Q206L; Y209C.H; G211 D; S212N,L;
T213A; Y214C.H; A215D.T; N218D.S; M222L,IN,A,S; A223G; T224A.S; A228T; A230V;
A232S,L,T,P; V234I; Q236A,L,D,T,C,M,F,S; K237R; Ν238D; P239T.S; S240F; S242T; V244I.M.A; Q245R,K,E,D,T,F,NN,W,G,I,S,C,LΛM; N248P,D,S; K251E.R;
N252G,H,D,V,M,S,T,E,Y,S,Q,KAL; A254S; T255A.S; S256N,R,G; L257G; G258K,
S259A,N,G; T260A.R; N261D; L262S, Q,V; Y263H.F; G264E; S265G,R,N; V268L,I; N269T;
N296K; E271A; T274S,L,A,R or
b) one of the following combination variants
A108T+L111V; L124I+S125A; P129S+S130AT; L96LA+A151G+V203A; S49N+V203L+N218D; S3T+A16P+R45C+G100S+A230V; I8V+R19K+V139I; N76D+A174AL+A194P+A230V; N185R; N62NE; H120Q+Q137E, G61GE, G61GS, G100L, A133D, V68A, N123D, L111F+Y263H, V11A+G61GE+V227A+S240F, A133E+S144K+N218D, S128A+P129S+S130SP, S9R+A15T+T22TQ+S101P, S9R+A15T+H120R+Q137D+N173S, G97E, Q245W, S9R+A15T+L96LG+Q137E+Y209H, S9R+A15T+L111V+Q137E+G211D, S9R+A15T+L1111+Q137E, S9R+A15T+L1111+H120N+Q137E, S9R+A15T+L96LG+H120Q+Q137E, S9R+A15T+T260M, S9R+A15T, Q245I, S9R+A15T+H120G+Q137E+N218D, S9R+A15T+S130P, Q245F, S9R+A15T+N218D, G63E+N76D+A194P+A230V, S9R+A15T+T224A, G100S, S9R+A15T+D60DG, A138V+V139I+A194P+N218D+A230V, A108V+A169G+R170A+Y171 H, I8V+P14L+R19L+V30I+I35V+S57P+P129S+Q137D+S144D+S256N, A133D+T134S+Q137A, Q137D, A98AH, V51 D, Q12E+P14L+A15T, G63E+N76D+A194P+A230V, Q12E+P14L+A15T, G97GS or
c) one or more modifications in position 68, wherein said modification(s) comprise(s): deletion, insertion and/or substitution of an amino acid residue selected from the group consisting of K,H,R,E,D,Q,N,C,V,L,I,P, ,F,W,Y,GAS and T.
In a second aspect the present invention relates to a subtilase variant comprising a) the combination of one or more of the modifications X62D,XD,XE,XG,DE
X68A,S,L,I
X97E,D,W,A,N,XG,XA,XV,XS
X98S,D,E,T,XS,XD,XV
X99D,L,A,P,G,N,AD,XD,X ,XG,DA X106D,E,T,M,GΛ ,F,I
X131 M,F,W,L,A,H,T,*,S,Q,R,E,G,XA
X170C,S,H
X245R,K,E,D,T,F,NN,W,G,I,S,C,L,A
X252G,H,D,V,M,S,T,E,Y,S,Q,K
with at least one of the following modifications
*0AQSVPWG; A1TN; Q2L; S3T,A,L; V4L.A; I8V.T; S9G,D,R,K,LN; R10H,K; V11A; Q12D;
A13V; P14S,T,D,A,MN,K,Q,L,H,R,I; A15M,T; A16P; H17R; Ν18S.H; R19W,K,L,F,G,I;
G20*,R,A; L21 F,LP,LW,LA,LG; T22S,A,K,TV,TG,TL,TW,TV,G,L,TY; G23S; S24P; K27R, V28I; V30I; I35T,V;T38S; P40L; N43D; R45H,K; G46D; A48T; S49N; F50S; V51A,I,D; P52V,A;
P55S.A; S57P; G61 E,D,S,R,GP; N62D,ND,NE,DE,NG,E,S; V68A,S,L,I; T71A; I72V; L75I;
N76S.D; N77S; S78T; V81A; A85T; S87C; A88V,T; E89G; K94N; V95C.T; L96LA.LG;
G97E,D,W,A,GG,GA,GV,N,GS; A98S,D,E,T,AS,AD,AV,AE,AH,Q,N, ,L,G,RN)S;
S99D,LΛAD,SD,SM,SG,DA,P,G,Ν,C,MN,I; G100S,GE,C; S101SA, SK; G102D.S; S103D,EN,L,Q,H,T; V104T,S,R,I,N,M,L,D; S106D,E,T,M,G,A,L,F,I; I107TN.M; A108V,T,S;
L111 IN; A114V; Ν116S.D; G118D; M119L,IN,A,S; H120Ν,D,Q,K,E,Y,S; V121A; L124C;
L126I; G127E; S128N,I,G,C; P129PSN,T,E,D,S,N,A; S130P,T,C,*;
P131 M,F,W,L,A,H,T,*,PA,S,Q,R,E,G,D,C; S132GJ; A133ASA; T134A; Q137H,E,D; A138GN;
V139L.I; Ν140D, K; T143A; S144D, N,P; R145G; V150I; A151V.G; A152P; A158TN,C,E,L,D, M; G160A.D; S163G,C,N,A; Y167K,A,I; A168G; A169G; R170C,S,H,L; Y171C; A172V;
N173D; A174V; M175L,IN,A,S,T; Ν183D; N184D,S; N185S,D; R186L,C,H; S188G; S190A;
Y192H; G195F.E; V203S,A,L,Q, , F,l; N204T,D,S; Q206L; Y209C,H; G211 D; S212N.L; T213A; Y214C.H; A215D.T; N218D.S; M222L,I,V,A,S; A223G; T224A.S; A228T; A230V; A232S,L,T,P; V234I; Q236A,L,D,T,C,M,F,S; K237R; N238D; P239T,S; S240F; S242T; V244I,M,A; Q245R,KIE)D,T,F,NN,W,G,I,S,C,L,A,M; N248P,D,S; K251E.R; N252G,H,DN,M,S,T,EN,S,Q,K,A,L; A254S; T255A.S; S256Ν,R,G; L257G; G258K, S259A,N,G; T260A.R; N261D; L262S, QN; Y263H.F; G264E; S265G,R,Ν; V268L.I; N269T; N296K; E271A; T274S,L,A,R.
In a third aspect the present invention relates to a subtilase variant comprising at least one of the alterations disclosed in Table I below:
Table I, subtilase variants of the inventions having one or more of the alterations:
Figure imgf000005_0001
Figure imgf000006_0001
Figure imgf000007_0001
P52V+S78T+S99SD S3A+V30I+S99SD+S106G+N248S
A15M+S99SD+V268I I8V+A15T+S99SD
S99G+S128N+N183D+A232L+ S3T+S9R+P14H+A15M+R19L+S99SD+ Q236T+Q245R V139I
S99R+S101SA S9R+A15T+G97GG+H 120D+Q137E
L96LA+A98T+P131AA S9R+A15T+G20A+G97GV+H120D+P131 H
A98E+S99P S163N+A232L+Q236A+Q245G
V28I+S99AD+P131 F N173D+A232L+Q236A+Q245N
S9R+A15T+G97GV+Q137H P55S+V68A+S106M+A108T+P129T
V81A+P131T+A133S+Q137E K27R+V68L+G118D+A158E
N43D+V68A+S106F+N238D A98E+S99A+S101SK
V68A+V203F V68A+N140D+T143A+S144N
V68A+S106E N62D+N 140K+T143A+S144D
V68A+S106I S9F+P14T+R19L+A98AD
V68A+A158M+R170C S9V+P14R+R19F+A98AD
V68A+P129T+N218D S99A+S99SD+G258K+L262Q
V68S+P129E S87C+S99SA+S99D+P131A
V68S+P129D S99A+S99SD+G258K+L262Q
V68L+P129E+N261 D V28l+S99A+*99aD+P131 F
G97GV+H120D A85T+G102D+S106T+K237R
P131A+A133ASA V68A+T71A
L111 F+Y263H G61GS
V11A+G61GE+V227A+S240F G100L
A133E+S144K+N218D A133D
S128A+P129S+S130SP V68A
G61GE N123D
S9R+A15T+T22TW+N204D+Q245I Q245W+N252V
S9R+A15T+G97GG+P131 S+Q137H R45H+Y171 C+Q245W+N252S
S9R+A15T+T22TG+N62D+V139L+Q245G G20R+A48T+R170C+Q245W+N252Q
S9R+A15T+T22TL+N62D+I107V+V139L+ S9R+A15T+A16P+G97GA+P131S+Q137D+ Q245W N204S
S9G+A15T+G97GA+Q137H N218D+Q245W+N252E
S9R+A15T+V68A+Q245R G20R+R170C+Q245R+N252V
S9R+A15T+G97GA+H120N+S212L S9R+P14I+R19K+A98AD+T274S
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
wherein
(a) the variant of Table I exhibits protease activity, and
(b) each position corresponds to a position of the amino acid sequence of subtilisin B PN', shown in Figure 1 and SEQ ID NO: 1.
In a fourth aspect the present invention relates to an isolated polynucleotide encoding a subtilase variant of the invention.
In a fifth aspect the present invention relates to an expression vector comprising the isolated polynucleotide of the invention.
In a sixth aspect the present invention relates to a microbial host cell transformed with the expression vector of the invention.
In a seventh aspect the present invention relates to a method for producing a subtilase variant according to the invention, wherein a host according to the invention is cultured under conditions conducive to the expression and secretion of the variant, and the variant is recovered.
In an eighth aspect the present invention relates to a cleaning or detergent composition, preferably a laundry or dish wash composition, comprising the variant of the invention.
In a ninth aspect the present invention relates to a subtilase variant comprising at least one of the alterations disclosed in Table II below:
Table II, subtilase variants of the inventions having one or more of the alterations:
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
wherein
(a) the variant of Table II exhibits protease activity, and
(b) each position corresponds to a position of the amino acid sequence of subtilisin B PN', shown in Figure 1 and SEQ ID NO: 1.
In a tenth aspect the present invention relates to a subtilase variant comprising one of the alterations N252D and N252M.
In an eleventh aspect the present invention relates to a subtilase variant comprising one or more of the alterations M119L, I, V, A, S; M175L, I, V, A, S and M222L, I, V, A, S in combination with the subtilase variants listed in tables I and II above.
Concerning alignment and numbering, reference is made to Fig. 1 which shows an alignment between subtilisin BPN'(a) (BASBPN) and subtilisin 309 (b) (BLSAVI). This alignment is in this patent application used as a reference for numbering the residues.
DEFINITONS
Prior to discussing this invention in further detail, the following terms and conventions will first be defined.
For a detailed description of the nomenclature of amino acids and nucleic acids, we refer to
WO 00/71691 beginning at page 5, hereby incorporated by reference.
NOMENCLATURE AND CONVENTIONS FOR DESIGNATION OF VARIANTS
In describing the various subtilase enzyme variants produced or contemplated according to the i nvention, the following nomenclatures and conventions have been adapted for ease of reference:
A frame of reference is first defined by aligning the isolated or parent enzyme with subtilisin BPN' (BASBPN).
The alignment can be obtained by the GAP routine of the GCG package version 9.1 to number the variants using the following parameters: gap creation penalty = 8 and gap extension penalty = 8 and all other parameters kept at their default values.
Another method is to use known recognized alignments between subtilases, such as the alignment indicated in WO 91/00345. In most cases the differences will not be of any importance.
Thereby a number of deletions and insertions will be defined in relation to BASBPN (SEQ ID NO.1). In Fig. 1 , subtilisin 309 (SEQ ID NO.2) has 6 deletions in positions 36, 58, 158, 162, 163, and 164 in comparison to BASBPN. These deletions are in Fig. 1 indicated by asterixes
For a detailed description of the nomenclature of modifications introduced in a polypeptide by genetic manipulation we refer to WO 00/71691 page 7-12, hereby incorporated by reference.
Proteases
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3).
Numbering of amino acid positions/residues
If nothing else is mentioned the amino acid numbering used herein correspond to that of the subtilase BPN' (BASBPN) sequence. For further description of the BPN' sequence, see Fig. 1 , SEQ ID NO:1 or Siezen et al., Protein Engng. 4 (1991) 719-737. Serine proteases
A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 "Principles of Biochemistry," Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272). The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753).
Subtilases
A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. (1997).
One subgroup of the subtilases, I-S1 or "true" subtilisins, comprises the "classical" subtilisins, such as subtilisin 168 (BSS168), subtilisin BPN", subtilisin Carlsberg (ALCALASE®, NOVOZYMES A/S), and subtilisin DY (BSSDY).
A further subgroup of the subtilases, I-S2 or high alkaline subtilisins, is recognized by Siezen et al. (supra). Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprises enzymes such as subtilisin PB92 (BAALKP) (MAXACAL®, Genencor International Inc.), subtilisin 309 (SAVINASE®, NOVOZYMES A/S), subtilisin 147 (BLS147) (ESPERASE®, NOVOZYMES A S), and alkaline elastase YaB (BSEYAB).
"SAVINASE®"
SAVINASE® is marketed by NOVOZYMES A/S. It is subtilisin 309 from B. Lentus and differs from BAALKP only in one position (N87S). SAVINASE® has the amino acid sequence designated b) in Fig. 1 and in SEQ ID NO:2.
Parent subtilase
The term "parent subtilase" describes a subtilase defined according to Siezen et al. (1991 and 1997). For further details see description of "Subtilases" above. A parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modifications have been made while retaining the characteristic of a subtilase. Furthermore, a parent subtilase may be a subtilase which has been prepared by the DNA shuffling technique, such as described by J.E. Ness et al., Nature Biotechnology, 17, 893-896 (1999). Alternatively the term "parent subtilase" may be termed "wild type subtilase".
For reference a table of the acronyms for various subtilases mentioned herein is provided, for further acronyms, see Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
Table III
Organism enzyme acronym
Bacteria: Gram-positive
Bacillus subtilis 168 subtilisin I168,apr BSS168
Bacillus amyloliquefaciens subtilisin BPN' (NOVO) BASBPN
Bacillus subtilis DY subtilisin DY BSSDY
Bacillus licheniformis subtilisin Carlsberg BLSCAR
Bacillus lentus subtilisin 309 BLSAVI
Bacillus lentus subtilisin 147 BLS147
Bacillus alcalophilus PB92 subtilisin PB92 BAPB92
Bacillus YaB alkaline elastase YaB BYSYAB
Thermoactinomyces vulgaris thermitase TVTHER
Modification(s) of a subtilase variant The term "modification(s)" used herein is defined to include chemical modification of a subtilase as well as genetic manipulation of the DNA encoding a subtilase. The modification(s) can be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.
Subtilase variant
In the context of this invention, the term subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host. Homologous subtilase sequences
The homology between two amino acid sequences is in this context described by the parameter "identity".
In order to determine the degree of identity between two subtilases the GAP routine of the
GCG package version 9.1 can be applied (infra) using the same settings. The output from the routine is besides the amino acid alignment the calculation of the "Percent Identity" between the two sequences.
Based on this description it is routine for a person skilled in the art to identify suitable homologous subtilases, which can be modified according to the invention.
Isolated polynucleotide
The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3" untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985). The term "an isolated polynucleotide" may alternatively be termed "a cloned polynucleotide".
Isolated protein
When applied to a protein, the term "isolated" indicates that the protein has been removed from its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. "homologous impurities" (see below)).
An isolated protein is more than 10% pure, preferably more than 20% pure, more preferably more than 30% pure, as determined by SDS-PAGE. Further it is preferred to provide the protein in a highly purified form, i.e., more than 40% pure, more than 60% pure, more than
80% pure, more preferably more than 95% pure, and most preferably more than 99% pure, as determined by SDS-PAGE.
The term "isolated protein" may alternatively be termed "purified protein".
Homologous impurities
The term "homologous impurities" means any impurity (e.g. another polypeptide than the subtilase of the invention), which originate from the homologous cell where the subtilase of the invention is originally obtained from.
Obtained from
The term "obtained from" as used herein in connection with a specific microbial source, means that the polynucleotide and/or subtilase produced by the specific source, or by a cell in which a gene from the source has been inserted.
Substrate
The term "substrate" used in connection with a substrate for a protease should be interpreted in its broadest form as comprising a compound containing at least one peptide (amide) bond susceptible to hydrolysis by a subtilisin protease.
Product
The term "product" used in connection with a product derived from a protease enzymatic reaction should, in the context of the present invention, be interpreted to include the products of a hydrolysis reaction involving a subtilase protease. A product may be the substrate in a subsequent hydrolysis reaction.
Wash Performance In the present context the term "wash performance" is used as an enzyme's ability to remove proteinaceous or organic stains present on the object to be cleaned during e.g. wash or hard surface cleaning. See also the wash performance test in Example 3 herein.
BRIEF DESCRIPTION OF THE DRAWING Fig. 1 shows an alignment between subtilisin BPN' (a) and Savinase®(b) using the GAP routine mentioned above.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to novel subtilase variants exhibiting alterations relative to the parent subtilase in one or more properties including: Wash performance, thermal stability, storage stability or catalytic activity.
Variants which are contemplated as being part of the invention are such variants where, when compared to the wild-type subtilase, one or more amino acid residues has been substituted, deleted or inserted, said variants comprising at least
a) an insertion, substitution or deletion of one of the amino acid residues K,H,R,E,D,Q,N,C,V,L,I,P,M,F,W,Y,G,A,S,T in one or more of the positions
62, 68, 97, 98, 99, 106, 131 , 170, 245, 252
in combination with at least one of the following modifications *0AQSVPWG; A1T.V; Q2L; S3T,A,L; V4L,A; I8V.T; S9G,D,R,K,LN; R10H.K; V11A; Q12D;
A13V; P14S,T,D,A,MN,K,Q,L,H,R,I; A15M,T; A16P; H17R; Ν18S.H; R19W,K,L,F,G,I;
G20*,R,A; L21 F,LP,LW,LA,LG; T22S,A,K,TV,TG,TL,TW,TV,G,L,TY; G23S; S24P; K27R,
V28I; V30I; I35T,V;T38S; P40L; N43D; R45H.K; G46D; A48T; S49N; F50S; V51A.I.D; P52V.A;
P55S.A; S57P; G61E,D,S,R,GP; N62D,ND,NE,DE,NG,E,S; V68A,S,L,I; T71A; I72V; L75I; N76S.D; N77S; S78T; V81A; A85T; S87C; A88V.T; E89G; K94N; V95C,T; L96LA,LG;
G97E,D,W,A,GG,GA,GV,N,GS; A98S,D,E,T,AS,AD,AV,AE,AH,Q,N,M,L,G,RN,S;
S99D,L,A,AD,SD,SM,SG,DA,P,G,Ν,C,MN,I; G100S,GE,C; S101SA, SK; G102D.S;
S103D,E,Y,L,Q,H,T; V104T,S,R,I,N,M,L,D; S106D,E,T,M,G,A,L,F,I; I107TN.M; A108V,T,S;
L1111N; A114V; Ν116S,D; G118D; M119L,I,V,A,S; H120N,D,Q,K,E,Y,S; V121 A; L124C; L126I; G127E; S128N,I,G,C; P129PSN,T,E,D,S,N,A; S130P,T,C,*;
P131M,F,W,L,A,H,T,*,PA,S,Q,R,E,G,D,C; S132G.T; A133ASA; T134A; Q137H,E,D; A138GN;
V139L.I; Ν140D, K; T143A; S144D, N,P; R145G; V150I; A151V.G; A152P; A158TN,C,E,L,D,
M; G160A.D; S163G,C,Ν,A; Y167K,A,I; A168G; A169G; R170C,S,H,L; Y171C; A172V;
N173D; A174V; M175L,IN,A,S,T; Ν183D; N184D.S; N185S.D; R186L,C,H; S188G; S190A; Y192H; G195F.E; V203S,A,L,Q,M, F,l; N204T,D,S; Q206L; Y209C.H; G211 D; S212N.L;
T213A; Y214C.H; A215D ; N218D,S; M222L,I,V,A,S; A223G; T224A,S; A228T; A230V;
A232S,L,T,P; V234I; Q236A,L,D,T,C,M,F,S; K237R; N238D; P239T.S; S240F; S242T;
V244I,M,A; Q245R,K,E,D,T,F,NN,W,G,I,S,C,L,A,M; Ν248P,D,S; K251E.R;
N252G,H,DN,M,S,T,EN,S,Q,K,A,L; A254S; T255A.S; S256Ν,R,G; L257G; G258K, S259A,N,G; T260A.R; N261D; L262S, Q,V; Y263H.F; G264E; S265G,R,N; V268L.I; N269T;
N296K; E271A; T274S,L,A,R or
b) one of the following combination variants
A108T+L111V; L124I+S125A; P129S+S130AT; L96LA+A151G+V203A; S49N+V203L+N218D; S3T+A16P+R45C+G100S+A230V; I8V+R19K+V139I;
N76D+A174AL+A194P+A230V; N185R; N62NE; H120Q+Q137E, G61GE, G61GS, G100L, A133D, V68A, N123D, L111F+Y263H, V11A+G61GE+V227A+S240F, A133E+S144K+N218D, S128A+P129S+S130SP, S9R+A15T+T22TQ+S101P, S9R+A15T+H120R+Q137D+N173S, G97E, Q245W, S9R+A15T+L96LG+Q137E+Y209H, S9R+A15T+L111V+Q137E+G211 D, S9R+A15T+L111 I+Q137E, S9R+A15T+L111 I+H120N+Q137E,
S9R+A15T+L96LG+H120Q+Q137E, S9R+A15T+T260M, S9R+A15T, Q245I, S9R+A15T+H120G+Q137E+N218D, S9R+A15T+S130P, Q245F, S9R+A15T+N218D, G63E+N76D+A194P+A230V, S9R+A15T+T224A, G100S, S9R+A15T+D60DG, A138V+V139I+A194P+N218D+A230V, A108V+A169G+R170A+Y171 H, I8V+P14L+R19L+V30I+I35V+S57P+P129S+Q137D+S144D+S256N, A133D+T134S+Q137A, Q137D, A98AH, V51D, Q12E+P14L+A15T, G63E+N76D+A194P+A230V, Q12E+P14L+A15T, G97GS or
c) one or more modifications in position 68, wherein said modification(s) comprise(s): deletion, insertion and/or substitution of an amino acid residue selected from the group consisting of K,H,R,E,D,Q,N,C,V,L,I,P,M,F,W,Y,G,A,S and T.
Further, variants of the present invention comprises at least one or more of the alterations indicated in Table I and II, wherein
(a) the variants of Table I and II has protease activity, and (b) each position corresponds to a position of the amino acid sequence of subtilisin BPN' (SEQ ID NO:1).
A subtilase variant of the first aspect of the invention may be a parent or wild-type subtilase identified and isolated from nature. Such a parent wild-type subtilase may be specifically screened for by standard techniques known in the art.
One preferred way of doing this may be by specifically PCR amplify conserved DNA regions of interest from subtilases from numerous different microorganism, preferably different Bacillus strains.
Subtilases are a group of conserved enzymes, in the sense that their DNA and amino acid sequences are homologous. Accordingly it is possible to construct relatively specific primers flanking the polynucleotide sequences of interest.
Using such PCR primers to amplify DNA from a number of different microorganisms, preferably different Bacillus strains, followed by DNA sequencing of said amplified PCR fragments, it will be possible to identify strains which produce subtilase variants of the invention. Having identified the strain and a partial DNA sequence of such a subtilase of interest, it is routine work for a person skilled in the art to complete cloning, expression and purification of such a subtilase. However, it is envisaged that a subtilase variant of the invention is predominantly a variant of a parent subtilase. A subtilase variant suitable for the uses described herein may be constructed by standard techniques known in the art such as by site-directed/random mutagenesis or by DNA shuffling of different subtilase sequences. See the "Material and Methods" section and Example 1 herein (vide infra) for further details.
As will be acknowledged by the skilled person, the variants described herein may comprise one or more further modifications, in particular one or more further substitutions or insertions. Moreover, the variants described herein may encompass mutation at more than just one position. For example the variant according to the invention may contain mutations at one position, two positions, three positions or more than three positions, such as four to eight positions.
It is preferred that the parent subtilase belongs to the subgroups I-S1 or I-S2, especially subgroup I-S2, both for enzymes from nature or from the artificial creation of diversity, and for designing and producing variants from a parent subtilase.
In relation to variants from subgroup I-S1 , it is preferred to select a parent subtilase from the group consisting of BSS168 (BSSAS, BSAPRJ, BSAPRN, BMSAMP), BASBPN, BSSDY, BLSCAR (BLKERA, BLSCA1 , BLSCA2, BLSCA3), BSSPRC, and BSSPRD, or functional variants thereof having retained the characteristic of sub-group I-S1.
In relation to variants from subgroup I-S2 it is preferred to select a parent subtilase from the group consisting of BSAPRQ, BLS147 (BSAPRM, BAH 101 ), BLSAVI (BSKSMK, BAALKP, BLSUBL), BYSYAB, BAPB92, TVTHER, and BSAPRS, or functional variants thereof having retained the characteristic of sub-group I-S2. In particular, the parent subtilase is BLSAVI (Savinase®, NOVOZYMES A/S), and a preferred subtilase variant of the invention is accordingly a variant of Savinase®.
The present invention also encompasses any of the a bove m entioned s ubtilase variants i n combination with any other modification to the amino acid sequence thereof. Especially com- binations with other modifications known in the art to provide improved properties to the enzyme are envisaged. The art describes a number of subtilase variants with different improved properties and a number of those are mentioned in the "Background of the invention" section herein (vide supra). Those references are disclosed here as references to identify a subtilase variant, which advantageously can be combined with a subtilase variant described herein. Such combinations comprise the positions: 222 (improves oxidation stability), 218 (improves thermal stability), substitutions in the Ca2+-binding sites stabilizing the enzyme, e.g. position 76, and many other apparent from the prior art. In further embodiments a subtilase variant described herein may advantageously be combined with one or more modification(s) in any of the positions:
27, 36, 56, 76, 87, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 120, 123, 159, 167, 170, 206, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274.
Specifically, the following BLSAVI, BLSUBL, BSKSMK, and BAALKP modifications are considered appropriate for combination:
K27R, *36D, S56P, N62D, V68A, N76D, S87N, G97N, S99SE, S101G, S103A, V104A, V104I, V104N, V104Y, S106A, H120D, H120N, N123S, G159D, Y167A, R170S, R170L, A194P, N204D, V205I, Q206E, L217D, N218S, N218D, M222S, M222A, T224S, A232V, K235L, Q236H, Q245R, N248D, N252K and T274A. Furthermore variants comprising any of the modifications S101G+V104N, S87N+S101G+ V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V104I, S99D+S101 R+S103A+V104I+ G160S, S3T+V4I+S99D+S101 R+S103A+V104I+G160S+V199M+V205I+L217D, S3T+V4I+ S99D+S101 R+S103A+V104I+G160S+A194P+V199M+V205I+L217D, S3T+V4I+S99D+
S101R+S103A+V104I+G160S+V205I or N76D+V104A, or other combinations of the modifica- tions K27R, *36D, S56P, N62D, V68A, N76D, S87N, G97N, S99SE, S101 G, S103A, V104A, V104I, V104N, V104Y, S106A, H120D, H120N, N123S, G159D, Y167A, R170S, R170L, A194P, N204D, V205I, Q206E, L217D, N218S, N218D, M222S, M222A, T224S, A232V, K235L, Q236H, Q245R, N248D, N252K and T274A in combination with any one or more of the modification(s) mentioned above exhibit improved properties.
A particular interesting variant is a variant, which, in addition to modifications according to the invention, contains the following substitutions:
S101 G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K.
Moreover, subtilase variants of the main aspect(s) of the invention are preferably combined with one or more modification(s) in any of the positions 129, 131 and 194, preferably as 129K, 131H and 194P modifications, and most preferably as P129K, P131H and A194P modifications. Any of those modification(s) are expected to provide a higher expression level of the subtilase variant in the production thereof.
The wash performance of a selected variant of the invention may be tested in the wash performance test disclosed in Example 3 herein. The wash performance test may be employed to assess the ability of a variant, when incorporated in a standard or commercial detergent composition, to remove proteinaceous stains from a standard textile as compared to a reference system, namely the parent subtilase or a similar subtilase exhibiting an even better wash performance (incorporated in the same detergent system and tested under identical conditions). The enzyme variants of the present application were tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA test the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined rapidly. Using this test, the wash performance of a selected variant can be initially investigated, the rationale being that if a selected variant does not show a significant improvement in the test compared to the parent subtilase, it is normally not necessary to carry out further test experiments.
Therefore, variants which are particularly interesting for the purposes described herein, are such variants which, when tested in a commercial detergent composition such as a US type detergent, an Asian type, a European type or a Latin American type detergent as described in the wash performance test (Example 3), shows an improved wash performance as compared to the parent subtilase tested under identical conditions.
The improvement in the wash performance may be quantified by calculating the so-called intensity value (Int) defined in Example 3, herein.
In a very interesting embodiment of the invention, the variant of the invention, when tested in the wash performance test has a Performance Score (S) of at least 1 , preferably a
Performance Score of 2, where:
S (2) = variant performs better than the reference at all three enzyme concentrations (5, 10 and 30 nM),
S (1) = variant performs better than the reference at one or two concentrations.
Evidently, it is preferred that the variant of the invention fulfils the above criteria on at least the stated lowest level, more preferably at the stated highest level.
PRODUCING A SUBTILASE VARIANT
Many methods for cloning a subtilase and for introducing substitutions, deletions or insertions into genes (e.g. subtilase genes) are well known in the art.
In general standard procedures for cloning of genes and introducing mutations (random and/or site directed) into said genes may be used in order to obtain a subtilase variant of the invention. For further description of suitable techniques reference is made to Example 1 herein (vide infra) and (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990), and WO 96/34946.
Further, a subtilase variant may be constructed by standard techniques for artificial creation of diversity, such as by DNA shuffling of different subtilase genes (WO 95/22625; Stemmer WPC, Nature 370:389-91 (1994)). DNA shuffling of e.g. the gene encoding Savinase® with one or more partial subtilase sequences identified in nature, will after subsequent screening for improved wash performance variants, provide subtilase variants suitable for the purposes described herein.
EXPRESSION VECTORS
A recombinant expression vector comprising a DNA construct encoding the e nzyme o f t he invention may be any vector that may conveniently be subjected to recombinant D NA p rocedures.
The choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector that exists as an extra- chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
Alternatively, the vector may be one that on introduction into a host cell is integrated into the host cell genome in part or in its entirety and replicated together with the chromosome(s) into which it has been integrated.
The vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the enzyme.
The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha- amylase gene, the Bacillus amyloliquefaciens alpha-amylase gene, the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, or the phage Lambda PR or PL promoters or the E. coli lac, trp or tac promoters. The DNA sequence encoding the enzyme of the invention may also, if necessary, be operably connected to a suitable terminator.
The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, or the like, or resistance to heavy metals or herbicides.
To direct an enzyme of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame. Secretory signal s equences a re commonly positioned 5' to the DNA sequence encoding the enzyme. The secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.
The procedures used to ligate the DNA sequences coding for the present enzyme, the promoter and optionally the terminator and/or secretory signal sequence, respectively, or to assemble these sequences by suitable PCR amplification schemes, and to insert them into suitable vectors containing the information necessary for replication or integration, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.cit).
HOST CELL
The DNA sequence encoding the present enzyme introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. The term "homologous" is intended to include a DNA sequence encoding an enzyme native to the host organism in question. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence m ay b e f rom a nother o rganism, o r it may b e a synthetic sequence. The host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell that is capable of producing the present enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells including plants.
Examples of bacterial host cells which, on cultivation, are capable of producing the enzyme of the invention are gram-positive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium or β. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gram-negative bacteria such as Escherichia coli.
The transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
When expressing the enzyme in bacteria such as E. coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells a re lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme.
When expressing the enzyme in gram-positive bacteria such as Bacillus or Streptomyces strains, the enzyme may be retained in the cytoplasm, or may be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme may be recovered from the medium as described below.
METHOD FOR PRODUCING A SUBTILASE VARIANT The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
When an expression vector comprising a DNA sequence encoding the enzyme is trans-formed into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme of the invention. Thereby it is possible to make a highly purified subtilase composition, characterized in being free from homologous impurities.
The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed subtilase may conveniently be secreted into the culture medium and may be recovered there-from by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
CLEANING AND DETERGENT COMPOSITIONS
The enzyme of the invention may be added to and thus become a component of a detergent composition. In general, cleaning and detergent compositions are well described in the art and reference is made to WO 96/34946; WO 97/07202; WO 95/30011 for further description of suitable cleaning and detergent compositions.
The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general h ousehold h ard s urface cleaning operations, or be formulated for hand or machine dishwashing operations.
In a specific aspect, the invention provides a detergent additive comprising the enzyme of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 a nd s ubtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583. Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274. Preferred commercially available protease enzymes include Durazym®, Relase®, Alcalase®, Savinase®, Primase®, Duralase®, Esperase®, Ovozyme® and Kannase® (Novozymes A S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, FN3™ and FN4™ (Genencor International, Inc.).
Lipases: S uitable I ipases i nclude t hose o f b acterial o r f ungal o rigin. C hemically m odified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1 ,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131 , 253-360), B. stearothermophilus (JP 64/744992) or β. pumilus (WO 91/16422). Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541 , EP 407 225, EP 260 105, WO 95/35381 , WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
Preferred commercially available lipase enzymes include Lipex®, Lipolase® and Lipolase Ultra® (Novozymes A/S).
Amyiases: Suitable amyiases (α and/or β) include those of bacterial or fungal origin.
Chemically modified or protein engineered mutants are included. Amyiases include, for example, α-amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in GB 1 ,296,839.
Examples of useful amyiases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181 , 188, 190, 197, 202, 208,
209, 243, 264, 304, 305, 391 , 408, and 444.
Commercially available amyiases are Duramyl®, Termamyl®, Fungamyl® and BAN®
(Novozymes A/S), Rapidase™ and Purastar™ (from Genencor International Inc.).
Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299. Commercially available cellulases include Celluzyme®, and Carezyme® (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).
Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include Guardzyme® (Novozymes A/S).
The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216. The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non-aqueous.
The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight.
When included therein the detergent will usually contain from about 1 % to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, a lkyl s ulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanol- amide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine ("glucamides").
The detergent may contain 0-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylene- diaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenyl-succinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
The detergent may comprise one or more polymers. Examples are carboxymethyl-cellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinyl-pyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as poly-acrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
The detergent may contain a bleaching system which may comprise a H2O2 source such as perborate or percarbonate which may be combined with a p eracid-forming b leach a ctivator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.
The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
Variations in local and regional conditions, such as water hardness and wash temperature calls for regional detergent compositions. Detergent Examples 1 and 2 provide ranges for the composition of a typical Latin American detergent and a typical European powder detergent respectively.
Detergent Example 1. Typical Latin American detergent composition.
Group Subname Content
Surfactants 0-30%
Sulphonates 0-30%
Sulphates 0-5%
Soaps 0-5%
Non-ionics 0-5%
Cationics 0-5%
FAGA 0-5%
Bleach 0-20%
SPT / SPM 0-15%
NOBS, TAED 0-5%
Builders 0-60%
Phosphates 0-30%
Zeolite 0-5%
Na2OSiO2 0-10%
Na2CO3 0-20%
Fillers 0-40%
Na2SO4 0-40%
Others up to 100%
Polymers Enzymes
Foam regulators
Water
Hydrotropes
Others
Detergent Example 2. Typical European powder detergent composition.
Group Subname Content
Surfactants 0-30%
Sulphonates 0-20%
Sulphates 0-15%
Soaps 0-10%
Non-ionics 0-10%
Cationics 0-10%
Other 0-10%
Bleach 0-30%
SPT / SPM 0-30%
NOBS+ TAED 0-10%
Builders 0-60%
Phosphates 0-40%
Zeolite 0-40%
Na2OSiO2 0-20%
Na2CO3 0-20%
Fillers 0-40%
Na2SO4 0-40%
NaCI 0-40%
Others up to 100%
Polymers Enzymes
Foam regulators
Figure imgf000039_0001
The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
It is at present contemplated that in the detergent compositions any single enzyme, in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-200 mg of enzyme protein per liter of wash liqour, preferably 0.05-50 mg of enzyme protein per liter of wash liqour, in particular 0.1-10 mg of enzyme protein per liter of wash liqour. The enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.
MATERIALS AND METHODS TEXTILES:
Standard textile pieces are obtained from EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland. Especially type EMPA116 (cotton textile stained with blood, milk and ink) and EMPA117 (polyester/cotton textile stained with blood, milk and ink).
STRAINS AND PLASMIDS:
Bacillus lentus strain 309 is deposited with the NCIB and accorded the accession number NCIB 10309, and described in US Patent No. 3,723,250 incorporated by reference herein. The parent subtilase 309 or Savinase® can be obtained from Strain 309. The expression host organism is Bacillus subtilis.
The plasmid pSX222 is used as E. coli - B. subtilis shuttle vector and B. subtilis expression vector (as described in WO 96/34946).
GENERAL MOLECULAR BIOLOGY METHODS:
Unless otherwise mentioned the DNA manipulations and transformations are performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold S pring H arbor, N Y; Ausubel, F . M . et a l. (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990).
ENZYMES FOR DNA MANIPULATIONS
Unless otherwise mentioned all enzymes for DNA manipulations, such as e.g. restriction endonucleases, ligases etc., are obtained from New England Biolabs, Inc. Enzymes for DNA manipulations are used according to the specifications of the suppliers.
FERMENTATION:
Fermentations for the production of subtilase enzymes are performed at pH 7.3 and 37°C on a rotary s haking table at 225 rpm. i n 50 ml tubes containing 15 ml double TY media for 2-3 days.
For a description of TY media, see page 1.1.3, Media Preparation and Bacteriological Tools in "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.).
PURIFICATION
The s ubtilase variant s ecreted from the h ost cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
WASH PERFORMANCE TEST In order to asses the wash performance of selected subtilase variants in detergent compositions, washing experiments are performed. The enzyme variants of the present application is tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA test the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined. The AMSA plate has a number of slots for test solutions and a lid firmly squeezing the textile swatch to be washed against all the slot openings. During the washing time, the plate, test solutions, textile and lid are vigorously shaken to bring the test solution in contact with the textile and apply mechanical stress. For further description see WO 02/42740 especially the paragraph "Special method embodiments" at page 23-24.
DETERGENTS
Detergents for wash performance tests of the subtilase enzymes of the invention can be obtained by purchasing fully formulated commercial detergents at the market and subsequently inactivate the enzymatic components by heat treatment (5 minutes at 85°C in aqueous solution). Moreover a commercial detergent base without enzymes can be purchased directly from the manufacturer. Further a suitable model detergent can be composed according to the provisions at page 19-24 herein and used for wash performance tests.
EXAMPLE 1
CONSTRUCTION AND EXPRESSION OF ENZYME VARIANTS:
SITE-DIRECTED MUTAGENESIS:
Subtilisin 309 (Savinase®) site-directed variants of the invention comprising specific insertions/deletions/substitutions are made by traditional cloning of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) produced by PCR with oligos containing the desired mutations.
The template plasmid DNA may be pSX222, or an analogue of this containing a variant of subtilisin 309. Mutations are introduced by oligo directed mutagenesis to the construction of variants.
The subtilisin 309 variants are transformed into E. coli. DNA purified from an over night culture of t hese t ransformants is transformed i nto β. subtilis by restriction endonuclease digestion, purification of DNA fragments, ligation, transformation of B. subtilis. Transformation of B. subtilis is performed as described by Dubnau et al., 1971 , J. Mol. Biol. 56, pp. 209-221. SITE-DIRECTED MUTAGENESIS IN ORDER TO INTRODUCE MUTATIONS IN A SPECIFIC REGION:
The overall strategy used to perform site-directed mutagenesis is:
Mutagenic primers (oligonucleotides) are synthesized corresponding to the DNA sequence flanking the sites of mutation, separated by the DNA base pairs defining the insertions / deletions / substitutions.
Subsequently, the resulting mutagenic primers are used in a PCR reaction with the modified plasmid pSX222. The resulting PCR fragment is purified and extended in a second PCR- reaction, the resulting PCR product is purified and extended in a third PCR-reaction before being digested by endonucleases and cloned into the E. coli - B. subtilis shuttle vector pSX222. The PCR reactions are performed under normal conditions. The plasmid DNA is transformed into E. coli by well-known techniques and one E. coli colony is sequenced to confirm the mutation designed.
Each of the variants listed in Table I at page 2 herein can be constructed as described above. In order to purify subtilase variants of the invention, the pSX222 expression plasmid comprising a variant of the invention was transformed into a competent β. subtilis strain and fermented as described above.
EXAMPLE 2
PURIFICATION AND ASSESMENT OF ENZYM CONCENTRATION
After fermentation purification of subtilisin variants is accomplished using Hydrophobic Charge Induction Chromatography (HCIC) and subsequent vacuum filtration.
To capture the enzyme, the HCIC uses a cellulose matrix to which 4-Mercapto-Ethyl-Pyridine (4-MEP) is bound.
Beads of the cellulose matrix sized 80-100 μm are mixed with a media containing yeast extract and the transformed β. subtilis capable of secreting the subtilisin variants and incubated at pH
9.5 in Unifilter® microplates. As 4-MEP is hydrophobic at pH > 7 and the subtilisin variants are hydrophobic at pH 9.5 a hydrophobic association is made between the secreted enzyme and the 4-MEP on the beads.
After incubation the media and cell debris is removed by vacuum filtration while the beads and enzyme are kept on the filter.
To elute the enzyme from the beads the pH is now lowered by washing the filter with an elution buffer (pH 5). Hereby the enzymes part from the beads and can be retrieved from the buffer.
The concentration of the purified subtilisin enzyme variants is assessed by active site titration (AST). The purified enzyme is incubated with the high affinity inhibitor CI-2A at different concentrations to inhibit a varying amount of the active sites. The protease and inhibitor binds to each other at a 1 :1 ratio and accordingly the enzyme concentration can be directly related to the concentration of inhibitor, at which all protease is inactive. To measure the residual protease activity, a substrate (0.6 mM Suc-Ala-Ala-Pro-Phe-pNA in Tris/HCI buffer) is added after the incubation with inhibitor and during the following 4 minutes the development of the degradation p roduct p NA ( paranitrophenol) i s m easured p eriodically at 405 nm on an Elisa Reader.
Each of the variants of the invention listed in Table I at page 2 herein was purified according to the above procedure and subsequently the enzyme concentration was determined.
Known concentrations of the variants of Table I were tested for wash performance in detergents as described below. EXAMPLE 3
WASH PERFORMANCE OF SAVINASE VARIANTS
In order to asses the wash performance of selected subtilase variants in a commercial detergent base composition, washing experiments was performed. The enzyme variants of the present application were tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA test the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined. The AMSA plate has a number of slots for test solutions and a lid firmly squeezing the textile swatch to be washed against all the slot openings. During the washing time, the plate, test solutions, textile and lid are vigorously shaken to bring the test solution in contact with the textile and apply mechanical stress. For further description see WO 02/42740 especially the paragraph "Special method embodiments" at page 23-24.
Two assays were conducted under the experimental conditions specified below: Assay A
Figure imgf000043_0001
The Latin American type detergent was composed according to the provisions in Detergent Example 1 at page 24 herein. Water hardness was adjusted to 6-9°dH by addition of CaCI2 and MgCI2 (Ca2+:Mg2+ = 4:1 ) to the test system. After washing the textile pieces were flushed in tap water and air-dried.
Assay B
Figure imgf000043_0002
Figure imgf000044_0002
The European powder type detergent was composed according to the provisions in Detergent Example 2 at page 24 herein. Water hardness was adjusted to 15°dH by addition of CaCI2 *2H2O; MgCI2*6H2O; NaHCO3 (Ca2+:Mg2+:HCO3" = 4:1 :10) to the test system. After washing the textile pieces were flushed in tap water and air-dried.
The performance of the enzyme variant is measured as the brightness of the colour of the textile samples washed with that specific enzyme variant. Brightness can also be expressed as the intensity of the light reflected from the textile sample when luminated with white light. When the textile i s stained the i ntensity o f t he reflected l ight i s l ower, than that of a clean textile. Therefore the intensity of the reflected light can be used to measure wash performance of an enzyme variant.
Colour measurements are made with a professional flatbed scanner (PFU DL2400pro), which is used to capture an image of the washed textile samples. The scans are made with a resolution of 200 dpi and with an output colour dept of 24 bits. In order to get accurate results, the scanner is frequently calibrated with a Kodak reflective IT8 target.
To extract a value for the light intensity from the scanned images, a special designed software application is used (Novozymes Color Vector Analyzer). The program retrieves the 24 bit pixel values from the image and converts them into values for red, green and blue (RGB). T he intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:
Figure imgf000044_0001
The wash performance (P) of the variants was calculated in accordance with the below formula:
P = lnt(v) - lnt(r) where lnt(v) is the light intensity value of textile surface washed with enzyme variant and lnt(r) is the light intensity value of textile surface washed with the reference enzyme subtilisin 309 (BLSAVI).
The results presented in Table IV and V below are Performance Scores (S) summing up the performances (P) of the tested enzyme variants as:
S (2) which indicates that the variant performs better than the reference at all three concentrations (5, 10 and 30 nM) and
S (1) which indicates that the variant performs better than the reference at one or two concentrations.
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Table V, Wash performance test results, Assay B.
Figure imgf000048_0002
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Assay C
Figure imgf000057_0001
The European powder type detergent was composed according to the provisions in Detergent Example 2 at page 24 herein. Water hardness was adjusted to 15°dH by addition of CaCI2 *2H2O; MgCI2*6H2O; NaHCO3 (Ca2+:Mg2+:HCO3" = 4:1 :10) to the test system. After washing the textile pieces were flushed in tap water and air-dried.
Table VI, Wash performance test results, Assay C.
Figure imgf000057_0002
Figure imgf000058_0001
Figure imgf000059_0001
Assay D
Commercial detergent base European powder type 1
Detergent dosage 6 g/l
Figure imgf000060_0001
The European powder type detergent was composed according to the provisions in Detergent Example 2 at page 24 herein. Water hardness was adjusted to 15°dH by addition of CaCI2*2H2O; MgCI2*6H2O; NaHCO3 (Ca2+:Mg2+:HCO3" = 4:1:10) to the test system. After washing the textile pieces were flushed in tap water and air-dried.
Table VII, Wash performance test results, Assay D.
Figure imgf000060_0002
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001

Claims

1. A protease variant comprising:
a) an insertion, substitution or deletion of one of the amino acid residues K,H,R,E,D,Q,N,CN,L,I,P,M,F,WN,G,A,S,T in one or more of the positions 62, 68, 97, 98, 99, 106, 131 , 170, 245, 252
in combination with at least one of the following modifications *0AQSVPWG; A1TN; Q2L; S3T,A,L; V4L.A; I8V,T; S9G,D,R,K,LN; R10H.K; V11A; Q12D;
A13V; P14S,T,D,A,MN,K,Q,L,H,R,I; A15M.T; A16P; H17R; Ν18S.H; R19W,K,L,F,G,I;
G20*,R,A; L21 F,LP,LW,LA,LG; T22S,A,K,TV,TG,TL,TW,TV,G,L,TY; G23S; S24P; K27R,
V28I; V30I; I35T,V;T38S; P40L; N43D; R45H.K; G46D; A48T; S49N; F50S; V51A,I,D; P52V,A;
P55S,A; S57P; G61 E,D,S,R,GP; N62D,ND,NE,DE,NG,E,S; V68A,S,L,I; T71A; I72V; L75I; N76S.D; N77S; S78T; V81A; A85T; S87C; A88V.T; E89G; K94N; V95C,T; L96LA,LG;
G97E,D,W,A,GG,GA,GV,N,GS; A98S,D,E,T,AS,AD,AV,AE,AH,Q,N,M,L,G,RN,S;
S99D,L,A,AD,SD,SM,SG,DA,P,G,Ν,C,MN,I; G100S,GE,C; S101SA, SK; G102D,S;
S103D,E,Y,L,Q,H,T; V104T,S,R,I,N,M,L,D; S106D,E,T,M,G,A,L,F,I; I107TN,M; A108V,T,S;
L1111N; A114V; Ν116S.D; G118D; M119L,I,V,A,S; H120N,D,Q,K,E,Y,S; V121A; L124C; L126I; G127E; S128N,I,G,C; P129PSN,T,E,D,S,N,A; S130P,T,C,*;
P131 M,F,W,L,A,H,T,*,PA,S,Q,R,E,G,D,C; S132G.T; A133ASA; T134A; Q137H,E,D; A138GN;
V139L.I; Ν140D, K; T143A; S144D, N,P; R145G; V150I; A151V.G; A152P; A158TN,C,E,L,D,
M; G160A.D; S163G,C,Ν,A; Y167K,A,I; A168G; A169G; R170C,S,H,L; Y171C; A172V;
N173D; A174V; M175L,IN,A,S,T; Ν183D; N184D.S; N185S.D; R186L,C,H; S188G; S190A; Y192H; G195F.E; V203S,A,L,Q,M, F,l; N204T,D,S; Q206L; Y209C.H; G211 D; S212N.L;
T213A; Y214C,H; A215D.T; N218D.S; M222L,IN,A,S; A223G; T224A.S; A228T; A230V;
A232S,L,T,P; V234I; Q236A,L,D,T,C,M,F,S; K237R; Ν238D; P239T,S; S240F; S242T;
V244I,M,A; Q245R,K,E,D,T,F,NN,W,G,I,S,C,L,A,M; Ν248P,D,S; K251 E,R;
N252G,H,DN,M,S,T,EN,S,Q,K,A,L; A254S; T255A.S; S256Ν,R,G; L257G; G258K, S259A,N,G; T260A.R; N261D; L262S, Q,V; Y263H,F; G264E; S265G,R,N; V268LJ; N269T;
N296K; E271A; T274S,L,A,R, or
b) one of the following combination variants
A108T+L111V; L124I+S125A; P129S+S130AT; L96LA+A151G+V203A; S49N+V203L+N218D; S3T+A16P+R45C+G100S+A230V; I8V+R19K+V139I;
N76D+A174AL+A194P+A230V; N185R; N62NE; H120Q+Q137E, G61GE, G61GS, G100L, A133D, V68A, N123D, L111 F+Y263H, V11A+G61GE+V227A+S240F, A133E+S144K+N218D, S128A+P129S+S130SP, S9R+A15T+T22TQ+S101 P, S9R+A15T+H120R+Q137D+N173S, G97E, Q245W, S9R+A15T+L96LG+Q137E+Y209H, S9R+A15T+L111V+Q137E+G211 D, S9R+A15T+L1111+Q137E, S9R+A15T+L1111+H120N+Q137E, S9R+A15T+L96LG+H120Q+Q137E, S9R+A15T+T260M, S9R+A15T, Q245I, S9R+A15T+H120G+Q137E+N218D, S9R+A15T+S130P, Q245F, S9R+A15T+N218D, G63E+N76D+A194P+A230V, S9R+A15T+T224A, G100S, S9R+A15T+D60DG, A138V+V139I+A194P+N218D+A230V, A108V+A169G+R170A+Y171 H, I8V+P14L+R19L+V30I+I35V+S57P+P129S+Q137D+S144D+S256N, A133D+T134S+Q137A, Q137D, A98AH, V51 D, Q12E+P14L+A15T, G63E+N76D+A194P+A230V, Q12E+P14L+A15T, G97GS, V51A+S163T; V139I+A151 G; S9R+A15T+L96LG+S130*; A169G+R170H or
c) one or more modifications in position 68, wherein said modification(s) comprise(s): deletion, insertion and/or substitution of an amino acid residue selected from the group consisting of K,H,R,E,D,Q,N,C,V,L,I,P,M,F,W,Y,G,A,S and T.
2. A protease variant according to claim 1 comprising the combination of one or more of the modifications
X62D,E,S,XD,XE,XG,DE
X68A,S,L,I X97E,D,W,A,N,XG,XA,XV,XS
X98S,D,E,T,N,M,L,G,RN,XS,XD,XV
X99D,L,A,P,G,Ν,C,MN,I,AD,XD,XM,XG,DA
X106D,E,T,M,G,A,L,F,I
X131 M,F,W,L,A,H,T,*,S,Q,R,E,G,D,C,XA X170C,S,H,L
X245R,K,E,D,T,F,ΝN,W,G,I,S,C,L,A,M
X252G,H,D,V,M,S,T,E,Y,S,Q,K,A,L
with at least one of the following modifications *0AQSVPWG; A1TN; Q2L; S3T,A,L; V4L,A; I8V,T; S9G,D,R,K,LN; R10H.K; V11A; Q12D;
A13V; P14S,T,D,A,MN,K,Q,L,H,R,I; A15M.T; A16P; H17R; Ν18S,H; R19W,K,L,F,G,I;
G20*,R,A; L21 F,LP,LW,LA,LG; T22S,A,K,TV,TG,TL,TW,TV,G,L,TY; G23S; S24P; K27R,
V28I; V30I; I35T,V;T38S; P40L; N43D; R45H.K; G46D; A48T; S49N; F50S; V51A,I,D; P52V.A;
P55S.A; S57P; G61 E,D,S,R,GP; N62D,ND,NE,DE,NG,E,S; V68A,S,L,I; T71A; I72V; L75I; N76S,D; N77S; S78T; V81A; A85T; S87C; A88V.T; E89G; K94N; V95C,T; L96LA,LG;
G97E,D,W,A,GG,GA,GV,N,GS; A98S,D,E,T,AS,AD,AV,AE,AH,Q,N,M,L,G,RN,S;
S99D,L,A,AD,SD,SM,SG,DA,P,G,Ν,C,MN,I; G100S,GE,C; S101SA, SK; G102D.S; S103D,E,Y,L,Q,H,T; V104T,S,R,I,N,M,L,D; S106D,E,T,M,G,A,L,F,I; I107TN.M; A108V,T,S; L111 IN; A114V; Ν116S.D; G118D; M119L,IN,A,S; H120Ν,D,Q,K,EN,S; V121A; L124C; L126I; G127E; S128N,I,G,C; P129PSN,T,E,D,S,N,A; S130P,T,C,*;
P131 M,F,W,L,A,H,T,*,PA,S,Q,R,E,G,D,C; S132G.T; A133ASA; T134A; Q137H,E,D; A138GN; V139L.I; N140D, K; T143A; S144D, N,P; R145G; V150I; A151V.G; A152P; A158T,V,C,E,L,D, M; G160A,D; S163G,C,N,A; Y167K,A,I; A168G; A169G; R170C,S,H,L; Y171C; A172V; N173D; A174V; M175L,I,V,A,S,T; N183D; N184D.S; N185S.D; R186L,C,H; S188G; S190A; Y192H; G195F.E; V203S,A,L,Q,M, F,l; N204T,D,S; Q206L; Y209C.H; G211 D; S212N.L; T213A; Y214C.H; A215D.T; N218D.S; M222L,I,V,A,S; A223G; T224A.S; A228T; A230V; A232S,L,T,P; V234I; Q236A,L,D,T,C,M,F,S; K237R; N238D; P239T,S; S240F; S242T; V244I,M,A; Q245R,K,E,D,T,F,NN,W,G,I,S,C,L,A,M; Ν248P,D,S; K251E.R; N252G,H,DN,M,S,T,EN,S,Q,K,A,L; A254S; T255A,S; S256Ν,R,G; L257G; G258K, S259A,N,G; T260A.R; N261D; L262S, Q,V; Y263H.F; G264E; S265G,R,N; V268L.I; N269T; N296K; E271A; T274S,L,A,R.
3. A subtilase variant according to claim 1 or 2 comprising one or more of the following alterations:
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
V68A+V203L N62D+V150I
V68S+A158D S3T+P14Q+A15M+R19K+N62D+S144D
I8V+A15T+R19K+A85T+S99SD+ P14Q+R19W+V511+G61 E+S99SD+ A114V+V244I+S256N+Y263H V139I+T260R
L111 F+Y263H S3T+P14L+H17R+S99SD+V139I+S144D
P52V+S78T+S99SD S3A+V30I+S99SD+S106G+N248S
A15M+S99SD+V268I I8V+A15T+S99SD
S99G+S128N+N183D+A232L+ S3T+S9R+P14H+A15M+R19L+S99SD+ Q236T+Q245R V139I
S99R+S101SA S9R+A15T+G97GG+H120D+Q137E
L96LA+A98T+P131AA S9R+A15T+G20A+G97GV+H120D+P131 H
A98E+S99P S163N+A232L+Q236A+Q245G
V28I+S99AD+P131 F N 173D+A232L+Q236A+Q245N
S9R+A15T+G97GV+Q137H P55S+V68A+S106M+A108T+P129T
V81A+P131T+A133S+Q137E K27R+V68L+G118D+A158E
N43D+V68A+S106F+N238D A98E+S99A+S101SK
V68A+V203F V68A+N 140D+T143A+S144N
V68A+S106E N62D+N 140K+T143A+S144D
V68A+S106I S9F+P14T+R19L+A98AD
V68A+A158M+R170C S9V+P14R+R19F+A98AD
V68A+P129T+N218D S99A+S99SD+G258K+L262Q
V68S+P129E S87C+S99SA+S99D+P131 A
V68S+P129D S99A+S99SD+G258K+L262Q
V68L+P129E+N261 D V28l+S99A+*99aD+P131 F
G97GV+H120D A85T+G102D+S106T+K237R
P131A+A133ASA V68A+T71A
L111 F+Y263H G61GS
V11A+G61GE+V227A+S240F G100L
A133E+S144K+N218D A133D
S128A+P129S+S130SP V68A
G61GE N123D
S9R+A15T+T22TW+N204D+Q245I Q245W+N252V
S9R+A15T+G97GG+P131 S+Q137H R45H+Y171 C+Q245W+N252S
S9R+A15T+T22TG+N62D+V139L+Q245G G20R+A48T+R170C+Q245W+N252Q
S9R+A15T+T22TL+N62D+I107V+V139L+ S9R+A15T+A16P+G97GA+P131 S+Q137D+
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
wherein
(a) the variant has protease activity, and
(b) each position corresponds to a position of the amino acid sequence of subtilisin B PN', shown in SEQ ID NO:1.
4. The variant according to any of claims 1-3, wherein the parent subtilase belongs to the subgroup I-S1.
5. The variant according to any of claims 1-3, wherein the parent subtilase belongs to the subgroup I-S2, and wherein the parent preferably is BLSAVI (Savinase®),
6. An isolated DNA sequence encoding a subtilase variant as defined in any of claims 1-5.
7. An expression vector comprising the isolated DNA sequence of claim 6.
8. A microbial host cell transformed with the expression vector of claim 7.
9. A microbial host cell according to claim 8, which is a bacterium, preferably a Bacillus, espe- daily a β. lentus.
10. A microbial host cell according to claim 9, which is a fungus or yeast, preferably a filamentous fungus, especially an Aspergillus.
11. A method for producing a subtilase variant as defined in any of claims 1-5, wherein a host as defined in any of claims 8-10 is cultured under conditions conducive to the expression and secretion of the variant, and the variant is recovered.
12. A cleaning or detergent composition, preferably a laundry or dish wash composition, com- prising the variant as defined in any of claims 1-5.
13. A composition according to claim 12, which additionally comprises a cellulase, a lipase, an amylase, a cutinase, a protease, a hemicellulase, an esterase, a lactase, a glycoamylase, a polygalacturonase, a beta-galactosidase, a ligninase or a mixture thereof.
14. Use of a variant as defined in any of claims 1-5 in a laundry and/or a dish wash detergent.
15. A s ubtilase variant a ccording to claim 3 , wherein s aid variant f urther comprises o ne or more of the modifications K27R, *36D, S56P, N62D, V68A, N76D, S87N, G97N, S99SE, S101G, S103A, V104A, V104I, V104N, V104Y, S106A, H120D, H120N, N123S, G159D, Y167A, R170S, R170L, A194P, N204D, V205I, Q206E, L217D, N218S, N218D, M222S, M222A, T224S, A232V, K235L, Q236H, Q245R, N248D, N252K, T274A, S101G+V104N, S87N+S101G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V104I, S99D+S101 R+ S103A+V104I+G160S, S3T+V4I+S99D+S101 R+S103A+V104I+G160S+V199M+V205I+ L217D, S3T+V4I+S99D+S101 R+S103A+V104I+G160S+A194P+V199M+V205I+L217D,
S3T+V4I+S99D+ S101 R+S103A+V104I+G160S+V205I and N76D+V104A.
16. A subtilase variant according to claim 3 comprising the following substitutions: S101G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K.
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