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US20090181875A1 - Subtilase variants - Google Patents

Subtilase variants Download PDF

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Publication number
US20090181875A1
US20090181875A1 US12/403,859 US40385909A US2009181875A1 US 20090181875 A1 US20090181875 A1 US 20090181875A1 US 40385909 A US40385909 A US 40385909A US 2009181875 A1 US2009181875 A1 US 2009181875A1
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Prior art keywords
g97ga
l96lg
variant
g97gv
subtilase
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US12/403,859
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Henriette Draborg
Peter Kamp Hansen
Mads Eskelund Bjornvad
Mads Norregaard-Madsen
Mikael Mikkelsen
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Novozymes AS
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Novozymes AS
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Priority to US12/403,859 priority Critical patent/US20090181875A1/en
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    • 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
    • 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

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, amylases, 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
  • 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.
  • the present invention relates to a subtilase variant comprising
  • the present invention relates to a subtilase variant comprising at least one of the alterations disclosed in Table I below:
  • each position corresponds to a position of the amino acid sequence of subtilisin BPN′, shown in FIG. 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: G97GA + H120D + P131H + Q137E L111I + Q137E G97GV + Q137H G97GA + Q137H + N218S T22TQ + S101P L96LG + H120N + P131S + Q137H G97GV + H120D + Q137H L96LG + H120N + P131S + Q137H V4A + G97GV + H120D G97GA + H120N + Q137E L111V + Q137E + G211D L111I + H120N + Q137E L21LW + G100S + V139L + Q245V L96LG + P131T + Q137H V68A + Q137D L96LG + H120N + P131S L96LG + H120Q + Q137E G97GA + H120Y + Q137H K94N + H120N + P131H G97GA + Q137D G97GV + H120Q L96LG + P131P131
  • each position corresponds to a position of the amino acid sequence of subtilisin BPN′, shown in FIG. 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.
  • subtilase enzyme variants produced or contemplated according to the invention, 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).
  • 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
  • FIG. 1 subtilisin 309
  • FIG. 1 indicated by asterixes (*).
  • 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′ (BASBPN) sequence.
  • BPN′ sequence see FIG. 1 , SEQ ID NO: 1 or Siezen et al., 1991 , Protein Engng. 4: 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.
  • subtilases A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., 1991 , Protein Engng. 4: 719-737 and Siezen et al., 1997 , Protein Science 6: 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 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., 1999 , Nature Biotechnology 17: 893-896.
  • subtilase may be termed “wild type subtilase”.
  • Organism Bacteria Gram-positive enzyme acronym 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) 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.
  • 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.
  • 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, 1985 , Nature 316: 774-78).
  • 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 80% pure, more preferably more than 95% pure, and most preferably more than 99% pure, as determined by SDS-PAGE.
  • 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 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,
  • 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
  • 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 amplifying 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.
  • 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.
  • 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.
  • 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 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 from the group consisting of BSAPRQ, BLS147 (BSAPRM, BAH101), BLSAVI (BSKSMK, BAALKP, BLSUBL), BYSYAB, BAPB92, TVTHER, and BSAPRS, or functional variants thereof having retained the characteristic of sub-group I-S2.
  • 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 above mentioned subtilase variants in combination with any other modification to the amino acid sequence thereof. Especially combinations 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 Ca 2+ -binding sites stabilizing the enzyme, e.g., position 76, and many other apparent from the prior art.
  • 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 Performance Score of 2, where:
  • 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, N.Y.; 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, 1994 , Nature 370: 389-91). 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 enzyme of the invention may be any vector that may conveniently be subjected to recombinant DNA procedures.
  • 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 P L 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 sequences are 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 may be from another organism, or it may be 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. alkalophilus, B. amyloliquefaciens, B. brevis, B. circulans, B. coagulans, B. lautus, B. lentus, B. licheniformis, B. megaterium, B. stearothermophilus, B. subtilis , or B. 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. alkalophilus, B. amyloliquefaciens, B. brevis, B. circulans, B. coagulans, B. lautus, B. lentus, B. licheniformis, B. megaterium, B. stearothermophilus, B
  • 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 are 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 household hard surface 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 and subtilisin 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.
  • 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), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect OXPTM, FN2TM, FN3TM and FN4TM (Genencor International, Inc.).
  • Suitable lipases include those of bacterial or fungal origin. Chemically modified 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.
  • 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®
  • Amylases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus , e.g., a special strain of B. licheniformis , described in more detail in GB 1,296,839.
  • Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, 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.
  • amylases are Duramyl®, Termamyl®, Fungamyl® and BAN® (Novozymes A/S), RapidaseTM and PurastarTM (from Genencor International Inc.).
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Acremonium, Bacillus, Humicola, Fusarium, Pseudomonas, Thielavia , e.g., the fungal cellulases produced from Fusarium oxysporum, Humicola insolens , and Myceliophthora thermophila disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, and 5,776,757 and WO 89/09259.
  • 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, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.
  • cellulases include Celluzyme®, and Carezyme® (Novozymes A/S), ClazinaseTM, and Puradax HATTM (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 U.S. Pat. Nos. 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, alkyl sulfate (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, alkyl sulfate (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 peracid-forming bleach activator 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.
  • 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% Na 2 OSiO 2 0-10% Na 2 CO 3 0-20% Fillers 0-40% Na 2 SO 4 0-40% Others up to 100% Polymers Enzymes Foam regulators Water Hydrotropes Others
  • 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% NaCl 0-40% Others up to 100% Polymers Enzymes Foam regulators Water Hydrotropes Others
  • 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 liquor, preferably 0.05-50 mg of enzyme protein per liter of wash liquor, in particular 0.1-10 mg of enzyme protein per liter of wash liquor.
  • 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 EMPA17 (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 U.S. Pat. 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 shaking table at 225 rpm in 50 ml tubes containing 15 ml double TY media for 2-3 days.
  • the subtilase variant secreted from the host 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.
  • washing experiments are performed.
  • the enzyme variants are tested using the Automatic Mechanical Stress Assay (AMSA).
  • AMSA Automatic Mechanical Stress Assay
  • 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.
  • 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 pages 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 these transformants is transformed into B. 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: 209-221.
  • 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 In order to purify subtilase variants of the invention, the pSX222 expression plasmid comprising a variant of the invention was transformed into a competent B. 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 micro-m are mixed with a media containing yeast extract and the transformed B. subtilis capable of secreting the subtilisin variants and incubated at pH 9.5 in Unifilter® microplates.
  • 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 Cl-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/HCl buffer
  • pNA paranitrophenol
  • Assay A Commercial detergent base Latin American type Detergent dosage 1.5-2.5 g/l Test solution volume 160 micro l pH 10-10.5 adjusted with NaHCO 3 Wash time 14 min. Temperature 20° C. Water hardness 6-9°dH Enzyme concentration in test solution 5 nM, 10 nM and 30 nM Test material EMPA 117
  • Assay B Commercial detergent base European powder type 1 Detergent dosage 6 g/l Test solution volume 160 micro I pH as it is in detergent (app. 10-10.5) Wash time 20 min. Temperature 30° C. Water hardness 6-9° dH Enzyme concentration in test solution 5 nM, 10 nM and 30 nM Test material EMPA 116
  • 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 is stained the intensity of the reflected light is lower, than that of a clean textile. Therefore the intensity of the reflected light can be used to measure wash performance of an enzyme variant.
  • Color 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).
  • the 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:
  • Int(v) is the light intensity value of textile surface washed with enzyme variant
  • Int(r) is the light intensity value of textile surface washed with the reference enzyme subtilisin 309 (BLSAVI).
  • Assay C Commercial detergent base European powder type 2 Detergent dosage 4 g/l Test solution volume 160 micro l pH as it is in detergent (app. 10-10.5) Wash time 20 min. Temperature 30° C. Water hardness 6-9° dH Enzyme concentration in test solution 5 nM, 10 nM and 30 nM Test material EMPA 116
  • Assay D Commercial detergent base European powder type 1 Detergent dosage 6 g/l Test solution volume 160 micro l Ph as it is in detergent (app. 10-10.5) Wash time 20 min. Temperature 30° C. Water hardness 6-9° dH Enzyme concentration in test solution 5 nM, 10 nM and 30 nM Test material C-10 swatches from Center for Testmaterials, Vlaardingen, NL

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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.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a divisional of U.S. application Ser. No. 10/699,394 filed on Oct. 31, 2003, which claims priority or the benefit under 35 U.S.C. 119 of Danish application nos. PA 200201705 and PA 200201933 filed on Nov. 8, 2002 and Dec. 18, 2002, respectively, and U.S. provisional application Nos. 60/427,156, 60/434,723, and 60/507,537 filed on Nov. 18, 2002, Dec. 19, 2002, and Oct. 1, 2003, respectively, the contents of which are fully incorporated herein by reference.
    • BACKGROUND OF THE INVENTION
  • 1. 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.
  • 2. Description of Related
  • In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, 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,L,V; R10H,K; V11A; Q12D; A13V; P14S,T,D,A,M,V,K,Q,L,H,R,I; A15M,T; A16P; H17R; N18S,H; R19W,K,L,F,G,I; G20*,R,A; L21F,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,R,V,S; S99D,L,A,AD,SD,SM,SG,DA,P,G,N,C,M,V,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; I107T,V,M; A108V,T,S; L111I,V; A114V; N116S,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,*; P131M,F,W,L,A,H,T,*,PA,S,Q,R,E,G,D,C; S132G,T; A133ASA; T134A; Q137H,E,D; A138G,V; 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,I; N204T,D,S; Q206L; Y209C,H; G211D; 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,N,V,W,G,I,S,C,L,A,M; N248P,D,S; K251E,R; N252G,H,D,V,M,S,T,E,Y,S,Q,K,A,L; 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; 18V+R19K+V1391; 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+L111I+Q137E, S9R+A15T+L111I+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+V1391+A194P+N218D+A230V, A108V+A169G+R170A+Y171H, 18V+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.
  • 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,XM,XG,DA
  • X106D,E,T,M,G,A,L,F,I
  • X131M,F,W,L,A,H,T,*,S,Q,R,E,G,XA
  • X170C,S,H
  • X245R,K,E,D,T,F,N,V,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; A1T,V; Q2L; S3T,A,L; V4L,A; I8V,T; S9G,D,R,K,L,V; R10H,K; V11A; Q12D; A13V; P14S,T,D,A,M,V,K,Q,L,H,R,I; A15M,T; A16P; H17R; N18S,H; R19W,K,L,F,G,I; G20*,R,A; L21F,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,R,V,S; S99D,L,A,AD,SD,SM,SG,DA,P,G,N,C,M,V,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; I107T,V,M; A108V,T,S; L111I,V; A114V; N116S,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,*; P131M,F,W,L,A,H,T,*,PA,S,Q,R,E,G,D,C; S132G,T; A133ASA; T134A; Q137H,E,D; A138G,V; 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,I; N204T,D,S; Q206L; Y209C,H; G211D; S212N,L; T213A; Y214C,H; A215D,T; N218D,S; M222L,I,V,A,S; A223G; T224A,S; A228T; A230V; A232S,L,T,P; V2341; Q236A,L,D,T,C,M,F,S; K237R; N238D; P239T,S; S240F; S242T; V244I,M,A; Q245R,K,E,D,T,F,N,V,W,G,I,S,C,L,A,M; N248P,D,S; K251E,R; N252G,H,D,V,M,S,T,E,Y,S,Q,K,A,L; 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.
  • 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:
    G97E + A98S V28I + A98AD + T224S
    G97D + A98D S99AD + M175V + P131F
    V95C + G97W + A98E S99AD + P131L
    V95T + G97A + A98D S9R + S99AD + P131W
    S103Y + V104M + S106D V68A + N116S + V139L + Q245R
    V104T + S106D S3T + A16P + R45C + G100S + A230V
    S3T + A16P + S99SD + S144D + A158T + A230V + I8V + S9R + A15T + R19W + V30I + G61D + S99SD +
    T260R S256N
    S103D + V104T + S106T V30I + S99SD + S256R
    S103D + V104L + S106M G61S + S99SD + V244I
    S103D + V104T + S106G V68A + V139L + S163G + N185S
    S103D + V104S + S106A S99SD + Y263H
    S103H + V104N + S106D V104N + S106T
    S103E + V104I + S106T S99SG + S144D
    S103Q + V104T + S106E V30I + S99SD
    S103E + S106T N18H + S99SD
    S103E + V104R + S106A S9R + T22S + S99SD + K251E
    A108T + L111V A48T + V68A + P131M
    L124I + S125A A15M + S99SM + V139I + V244I
    L124C + P131* P14T + A15M + S99SD
    P129S + S130AT I8V + S99SD + S144D + A228T
    L96LA + A151G + V203A I8V + R19K + V139I
    S99SD + A108V + V139L I35T + N62D
    S99SD + S190A N62D + S265G
    S99SD + V203A Q2L + N62D
    S99SD + V139I N62D + N76D
    S99SD + A108V R45H + G61E + V68A
    S99SD + S106A + A151G N62D + V121A
    V68A + S106A N62D + A215D
    V68A + N185D + V203S N62D + N238D
    V68A + V139L N62D + R145G
    V68A + V139I V4L + N62D + E89G
    V68A + A158V N62D + S188G + K251R
    V68A + V203A S49N + N62D
    V68A + V203S N62NE
    V68A + V203L + S259A V11A + N62DE
    V68A + S106L N62ND + N184S + S256G
    V30I + V68A + V203S N18S + N62D + I107T + A254S
    V51A + V68A + S106T + A168G S57P + N62ND
    V51A + V68A + S106T + A168G N62NE + V234I
    V68A + N76S + V203M + P239T Q137H + R170C + G195E
    V68A + V203L S99A + S101SA
    V68A + L75I + V203Q R10K + P14A + R19K + A98AS + S128N
    V68A + T71A + V139L T22A + R45K + A98AS + S128N
    Y192H + V68A A98AV + S99D + Y167K
    V68A + S106A + A108T S9G + P14K + Y167A + R170S
    V68A + S106T + A108T S9D + P14T + Y167A + R170S
    V68S + A108S S9R + P14M + A98AD
    V68A + N76S + G211D S9R + R19L + A98AD + E271A
    V68A + S106T + A108T S9R + P14S + R19F + A98AD
    A151V + R170C S99DA + P129PSN + P131A
    P14D + A98AS + H120D + G195F + S212N + S99AD + V244M + Q245K + N248D + K251R +
    M222S T255A + S256N
    S49N + V203L + N218D S9R + P14V + R19G + A98AD
    V68A + S106M + N184D S99AD + N248P + T255A + S256G
    P55S + V68L + A158E + G160A *0AQSVPWG + A98AD
    V68A + A158C T22A + S99AD
    V68A + A158L + Y214C K94N + A98T + S99L
    A88V + S99AD + P131F N76D + A174AL + A194P + A230V
    P14T + A16P + I72V + S99SD + V244I + T260A P40L + N218D + A232S + Q236L + Q245E + S259N
    S99AD + P131F A232L + Q236D + Q245E
    R10H + N62D A232T + Q236L + Q245D
    V28I + A98AD + T224S R170H + Q236A + Q245R
    S9K + T22K + S99AD A232L + Q236T + Q245D
    P14S + S99AD + P131W G97GG + P131H + Q137E + V268L
    V68A + I72V + P131F A88V + G97GV + P131H
    S9R + S99AD G97GA + H120Q + S130P + G264E
    S9K + S99AD G97GG + V139L
    V28I + A88V + G100S + P131M G97GG + Q137D
    S103L + V104S + S106G G97GG + H120D + Q137H
    V68A + T224A N185R
    V68A + P131F P131H + Q137E
    A48T + V68A + P131M V104I + H120N + P131H + Q137E
    V68A + I72V + P131F H120Q + Q137E
    G100GE + P131F S9R + A15T + G97GV + H120D
    S99AD + P131F + T260A G100S + H120Q + Q137H
    R19G + A98AS V68A + H120K + Q137E
    G61R + N62D G97GA + H120E
    V68A + S106M + N184D H120D + S128I + Q137D
    P55S + V68L + A158E + G160A G97GG + P131H
    V68A + A158C G97GG + H120N + L126I
    R19W + G61S + S99SD + N204T + Y263H + S9R + A15T + G97GA + H120D + P131H +
    S265R Q137E
    A232T + Q236C S9R + A15T + G97GV + P131T + Q137H
    N62D + A232T + Q236C S9R + A15T + G20* + L21F + N62D + Q245N
    A232P + Q236L + Q245E S9L + A15T + T22TV + V139L + Q245F
    A232S + Q236L + Q245T + K251E S132G + Q245F
    S163C + Q236M + Q245T + S256G S9R + A15T + T22TG + N62D + V139L + Q245V
    N218D + A232L + Q236F + Q245F S9L + A15T + T22TV + V139L + Q245F + L262S
    S163N + A232L + Q236S + Q245E S9R + A15T + T22TL + N62D + Q245W
    A232S + Q236S + Q245E V68A + A158L + Y214C
    V68A + V203L N62D + V150I
    V68S + A158D S3T + P14Q + A15M + R19K + N62D + S144D
    I8V + A15T + R19K + A85T + S99SD + A114V + P14Q + R19W + V51I + G61E + S99SD + V139I +
    V244I + S256N + Y263H T260R
    L111F + 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 + Q236T + S3T + S9R + P14H + A15M + R19L + S99SD +
    Q245R V139I
    S99R + S101SA S9R + A15T + G97GG + H120D + Q137E
    L96LA + A98T + P131AA S9R + A15T + G20A + G97GV + H120D + P131H
    A98E + S99P S163N + A232L + Q236A + Q245G
    V28I + S99AD + P131F 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 + N140K + 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 + N261D V28I + S99A + *99aD + P131F
    G97GV + H120D A85T + G102D + S106T + K237R
    P131A + A133ASA V68A + T71A
    L111F + 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 + P131S + Q137H R45H + Y171C + 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
    S9R + A15T + L96LG + H120D + P131H + R186L A98AE + V203I
    S9R + A15T + G97GA + H120D + Q137D V51A + V68A + S163G + V203A
    N62D + N252T N62D + Q245W + N252H
    V4A + S9R + A15T + G97GV + H120D N62D + Q245W + N252A
    S9R + A15T + G97GV + H120D + Q137H G20R + N62D + V244I + Q245W + N252E
    S9R + A15T + L96LG + H120N + P131H + Q137E N204D + Q245S
    S9R + A15T + L96LG + H120D + P131S + Q137E N62D + Q245W + N252E
    S9R + A15T + H120N + P131T + N218D N62D + Q245R + N252V
    S9R + A15T + L21LP + T22TV + M119I + N218D + S9R + A15T + S24P + G61E + A85T + P239S +
    Q245I Q245A
    S9R + A15T + L96LG + H120D + G160D G102S + M222S + Q245L + N252D
    V68A + S106A + G118D + Q245R + T255S + A15M + V30I + N62D + S99N + L111I + V244A +
    L257G + T274L S265N
    S9R + A15T + G61E + A85T + P239L + Q245C S9R + A15T + T22TG + N62D + V139L + Q245S
    S9R + A15T + P131H + S144P S3T + Q12D + R19W + V30I + S106G + I107M
    S9R + A15T + G97GA + Q137E V68A + A88T + V139L
    S9R + A15T + G97GA + H120Q + P131H + Q137E V51I + L111I + G118D + Q245R
    S9R + A15T + L21LW + G100S + V139L + Q245V V68A + V203L
    S9R + A15T + G97GA + Q137H + N218S A1T + V68A + N116D + G118D
    S9R + A15T + L96LG + H120N + P131S + Q137H V68A + G118D + Q245R
    S9R + A15T + G97GA + H120N + Q137E N62D + V139I + N183D + N185S + V203I +
    Q245R + L262S
    S9R + A15T + L96LG + P131T + Q137H N62D + I72V
    S9R + A15T + L96LG + H120N + P131S N62D + V81A + Q245R
    S9R + A15T + V68A + Q137D T22A + V68A + S106T + G118D
    S9R + A15T + G97GA + H120Y + Q137H V68A + L111I + V203I
    S9R + A15T + G97GA + Q137D G61E + V68A + A169G
    S9R + A15T + K94N + H120N + P131H V68A + L111V
    S9R + A15T + L96LG + P131H + Q137D V68A + G118D + V203A + K251R
    S9R + A15T + F50S + H120D + P131H V68A + G118D
    S9R + A15T + G97GA + H120N + Q137D + N248D A1V + V51A + V68A + V203I
    S9R + A15T + L96LG + P131Q + Q137D V68A + V139L + A223G
    S9R + A15T + T22G + V139L + Q245L N62D + Y214H + K237R
    V139L + Q245R V68A + S106A + G118D + Q245R
    S9R + A15T + Q245F S9R + A15T + T22A + N62D
    S9R + A15T + Q245S A98Q + S99D
    S9R + A15T + G97GV + H120Q S9R + P14I + R19K + A98AD
    S9R + A15T + G97GA + Q137E + L262V S9R + A15M + A16P + T22S + S99AD
    S9R + A15T + G127E + P131R + Q137H S99AD + T255R + S256N
    S9R + A13V + A15T + I35V + N6D + Q245F S9R + A15T + T22TQ + S101P
    S9R + A15T + Q245V S9R + A15T + H120R + Q137D + N173S
    V139L + Q245F G97E
    S9R + A15T + T22A + V139L + Q245E Q245W
    S9R + A15T + T22L + V139L + Q245V + A254S S9R + A15T + L96LG + Q137E + Y209H
    S9R + R19L + A98AD S9R + A15T + L111V + Q137E + G211D
    P14R + A98AD S9R + A15T + L111I + Q137E
    S9R + A15T + Q245L S9R + A15T + L111I + H120N + Q137E
    S9R + A15T + G61E + A85T + P239S + Q245V S9R + A15T + L96LG + H120Q + Q137E
    S9R + A15T + G61E + A85T + Q206L + Q245R S9R + A15T + T260M
    P239T + Q245R S9R + A15T
    S9R + A15T + N62NG + Q245T Q245I
    S9R + A15T + G61GP + Q245L S9R + A15T + H120G + Q137E + N218D
    S9R + A15T + G61E + A85T + Q137H + Y209C + I8V + P14L + R19L + V30I + I35V + S57P + P129S +
    Q245G Q137D + S144D + S256N
    S9R + A15T + G61E + A85T + P239S + Q245C Q245F
    V68I + A98AD S9R + A15T + N218D
    V68A + N269K G63E + N76D + A194P + A230V
    N62D + Q245A + N252G + S265G S9R + A15T + T224A
    N218D + Q245G + N252H G100S
    S9R + A15T + G102S + M175T + Q245R + N252D S9R + A15T + D60DG
    S9R + A15T + N62D + Q245W + N252V A138V + V139I + A194P + N218D + A230V
    S9R + A15T + N62D + Q245R + N252M A108V + A169G + R170A + Y171H
    S9R + A15T + N62D + Q245W + N252S S9R + A15T + S130P
    S99SD + N204S + Q245R A133D + T134S + Q137A
    N62D + Q245R Q137D
    N62D + A151G A98AH
    V68A + S106T V51D
    S99A + S99SD + V203L Q12E + P14L + A15T
    A98AD + A215T G63E + N76D + A194P + A230V
    N62D + Q245G + N252T Q12E + P14L + A15T
    A152P + Q245R + N252T G97GS
    S163N + T213A + Q245R Q245W + N252Y
    S106L + Q245R + N252E A169G + R170H
    S9V + P14R + R19F + A98AD Q12E + P14L + A15T
    S9R + A15T + L111I + Q137E P14R + A98AD
    S9R + A15T + G97GA + Q137E G100S
    S9R + A15T + L96LG + Q137E + Y209H A169G + R170H
    S9R + A15T + L96LG + H120N + P131S A98AD + A169G
    S9R + A15T + G97GV + H120Q A138V + V139I + A194P + N218D + A230V
    S9R + A15T + L96LG + H120Q + Q137E S99A + S99SD + V203L
    S9R + A15T + G97GV + P131S V68A + S106T
    S9R + A15T + K94N + H120N + P131H A98AD + A215T
    S9R + A15T + N76S + L111V + P131H + Q137D A108V + A169G + R170A + Y171H
    S9R + A15T + F50S + H120D + P131H S3L + N62D + S163A + S190A
    S9R + A15T + L96LG + S130* S9R + P14I + R19K + A98AD + T274S
    S9R + A15T + T22TL + N62D + I107V + V139L + S9R + A15T + G61E + A85T + N218D + P239S +
    Q245W Q245L
    S9R + A15T + G97GA + H120D + Q137H + S9R + A15T + S24P + G61E + A85T + P239S +
    M222V Q245A
    S9R + A15T + G97GA + H120N + Q137D + N248D S99SD + P131F
    S9R + A15T + L21LW + G100S + V139L + Q245V N62D + P131F + A172V
    S9R + A15T + G20* + L21F + N62D + Q245N N62D + P131F
    S9R + A15T + L21LC + V139L + R186H + Q245M V68A + A88T + V139L
    S132G + Q245 V68A + G118D + V203A
    S9R + A15T + T22TG + N62D + V139L + Q245G P40L + V68A + A108T + A138V + V203I
    S9R + A15T + L96LG + P131Q + Q137D I8T + A98AD + T274R
    S9R + A15T + T22TQ + S101P A98AE + V203I
    S9R + A15T + T22TG + N62D + V139L + Q245V V51A + V68A + S163G + V203A
    S9R + A15T + T22TL + N62D + Q245W A1V + V51A + V68A + V203I
    S9R + A15T + T22TW + N204D + Q245I V68A + G100S
    S9R + A15T + T22TG + N62D + V139L + Q245S V68A + V203L
    S9R + A15T + S130P A1T + V68A + N116D + G118D
    Q245W N62D + A169G + V203I + Q245R
    S9R + A15T + L21LP + T22TY + V139L + G160D + G23S + S99SD + A194P + S242T + Q245R +
    Q245L T274R
    S9R + A15T + G61E + A85T + P239L + Q245C S99SD + N204S + Q245R
    S9R + A15T + L21LP + T22TV + M119I + N218D + N62D + V139I + N183D + N185S + V203I +
    Q245I Q245R + L262S
    S9R + A15T + V68A + Q245R V68A + S106A + G118D + Q245R
    S9R + A15T + T22A + V139L + Q245E V51I + L111I + G118D + Q245R
    V139L + Q245R N62D + Q245R
    S9R + A15T + Q245F N62D + I72V
    S9R + A15T + Q245S S9R + R19L + A98AD
    S9R + A15T + T260M S9G + P14R + R19I + A98AD
    S9R + A15T S9R + A15T + T22L + V139L + Q245V + A254S
    S9R + A15T + L21LG + T22TV + V139L + N204D + S99G + S128N + N183D + A232L + Q236T +
    Q245N Q245R
    V139L + Q245F S9R + A15T + Q245L
    S9R + A15T + T22G + V139L + Q245L S9R + A15T + N62NG + Q245T
    S9R + A15T + Q245V S9R + A15T + N62ND + V139L + Q245E
    Q245F S9R + A15T + N62ND + V139L + N261D
    S9R + Q245C Y167I + R170L + Q245E
    S9R + A15T + N218D Y167I + R170L + Q245R
    S9R + A13V + A15T + I35V + N62D + Q245F Y167I + R170L + Q245M
    S9R + A15T + T224A Y167I + R170L
    S163N + A232L + Q236A + Q245G S99SE + Q245R
    S9R + A15T + A16P + G97GA + P131S + Q137D + S9R + A15T + G61E + A85T + Q137H + Y209C +
    N204S Q245G
    N218D + A232L + Q236F + Q245F S9R + A15T + G61E + A85T + P239S + Q245C
    S163N + A232L + Q236S + Q245E G102S + M222S + Q245L + N252D
    G97GA + H120E N62D + Q245A + N252G + S265G
    G97GG + P131H N62D + Q245G + N252T
    S9R + A15T + G97GA + H120D + P131H + Q137E S9R + A15T + N62D + Q245W + N252V
    S9R + A15T + G97GV + Q137H S9R + A15T + N62D + Q245R + N252M
    S9R + A15T + G97GV + H120N S9R + A15T + N62D + Q245W + N252S
    S9R + A15T + G97GG + P131S + Q137H S163N + T213A + Q245R
    S9R + A15T + G97GG + H120N + Q137D S106L + Q245R + N252E
    S9R + A15T + H120Q + P131C + Q137H Q245W + N252Y
    S9R + A15T + G97GV + H120D + Q137H Q245W + N252V
    S163C + Q236M + Q245T + S256G G20R + A48T + R170C + Q245W + N252Q
    S9R + A15T + G97GG + H120D + P131H + N62D + N252T
    Q137H
    S9R + A15T + G97GV + H120E + Q137H N218D + Q245W + N252E
    S9R + A15T + G97GV + P131T + Q137H G20R + R170C + Q245R + N252V
    S9R + A15T + G97GV +0 H120Q + Y263F N62D + Q245W + N252H
    S9R + A15T + G97GV + S106A + P131H N62D + Q245W + N252A
    S9R + A15T + G97GG + L111I + P131T + Q137H G20R + N62D + V244I + Q245W + N252E
    S9R + A15T + G97GV + P131H + Q137H N204D + Q245S
    S9R + A15T + G20A + G97GV + H120D + P131H N62D + Q245W + N252E
    S9R + A15T + G97GA + H120D + P131S + Q137E N62D + Q245R + N252V
    S9G + A15T + G97GA + Q137H A98L + S99C + Q245R
    S9R + A15T + H120R + Q137D + N173S N62D + A98R + Q245R
    S9R + A15T + L96LG + H120N + P131H + Q137E S9R + A15T + V68A + S99G + Q245R + N261D
    S9R + A15T + L96LG + H120D + P131S + Q137E S9R + A15T + G20* + L21F + N62D + Q245R
    S9R + A15T + H120N + P131T + N218D S9R + A15T + G20* + L21F + N62E + Q245R
    S9R + A15T + G97GA + H120D + Q137D V68I + A98AD
    S9R + A15T + L96LG + H120D + P131H + R186L S9R + A15T + H120D + Q137D
    S9R + A15T + G97GA + R186C S9R + A15T + N77S + L96LG + H120D + P131Q
    V4A + S9R + A15T + G97GV + H120D S9R + A15T + G97GA + H120N + Q137E
    S9R + A15T + L96LG + H120D + G160D S9R + A15T + G97GA + Q137E + L262V
    S9R + A15T + G97GA + H120N + S212L S9R + A15T + P131H + S144P
    S9R + A15T + G97GA + Q137H + N218S S9R + A15T + G127E + P131R + Q137H
    M222S + Q245G + N252G S9R + A15T + V68A + S99G + Q245R + N261D
    V68I + V203L N62D + P131F + A172V
    V51A + S163T N62D + P131F
    S106A + A138G S99SD + Q245R
    V139I + A151G S9R + A13T + S99A + S99SD + P131F
    A98R + G100C + Q245R S9R + A15T + N62S + H120N + P131T + N218D
    S9R + A15T + S99G + G100S + H120N + P131S + S9R + A15T + S99C + H120N + P131S + Q137H +
    Q137H M222S
    A15T + N185D + M222S + Q245R + N252V A98G + S99C + Q245R
    S9R + A15T + T22TL + G61E + L96LG + Q137D + A98T + S99G + G100S + S240F + Q245R
    Q245R
    S9R + T22TL + G61E + G97GG + M119I + P131T S9R + A15T + H120N + P131T + N218D + N269T
    Y209H + M222S + Q245G + N252L M222S + Q245M + N252E
    S9R + A15T + G61E + H120S + Q137D + V139L + S9R + A15T + L96LG + H120N + P131S + Q137H +
    N218D M222S
    S9R + A15T + N62D + H120N + P131T S9R + A15T + G61E + A98S + S99M + Q245R
    S9R + A15T + V68A + N218D + Q245R A98G + G100S + Q245R + N261D
    S9R + A15T + V68A + H120N + N218D + Q245R S9R + A15T + V68A + A98L + Q245R
    S9R + A15T + V68A + A174V + Q245R S9R + A15T + V68A + A98G + S99V + Q245R
    S9R + A15T + G46D + V68A + N218D + Q245R S9R + A15T + V68A + A98M + Q245R + N248D
    G97D + A98N + S128G + S130T + P131D + T134A S9R + A15T + G61E + V68A + A98S + S99G +
    Q245R
    S9R + A15T + V68A + A98M + S99G + Q245R + S9R + A15T + A88V + A98R + S99G + G100C +
    T274A H120N + P131S + Q137H
    S9R + A15T + V68A + A98L + S99G + Q245R A98V + S99C + Q245R
    S9R + A15T + A98 + S99C + H120N + P131S + S9R + A15T + G20* + L21F + G61E + *61aP +
    Q137H Q245R
    S9R + A15T + T38S + A98R + S99C + G100S + S9R + A15T + V68A + A98G + S99I + K237R +
    H120N + P131S + Q137H Q245R
    S9R + A15T + A98C + G100S + H120N + P131S + S9R + A15T + V68A + H120N + P131S + Q137H +
    Q137H Q245R
    S9R + A15T + A98S + G100S + H120N + P131S + S9R + A15T + V68A + H120D + P131S + Q137H +
    Q137H Q245R
    S9R + A15T + G20* + L21F + N62D + Q245R A98S + S99G + G100S + Q245R
    S9R + A15T + G20* + L21F + N62D + Q245R + S9R + A15T + A98S + S99G + G100S + H120N +
    S259G P131S + Q137H
    A98S + G100S + Q245R A98T + S99G + G100S + Q245R
    S9R + A15T + G20* + L21F + *61aA + V68A + S9R + A15T + G20* + L21F + P52T + N62D + Q245R
    Q245R
    S9R + A15T + G20* + L21F + N62E + Q245R A98L + S99C + Q245R
    V68A + S105G + S106A V68A + S106A + T213A
    S9R + A15T + Y167I + R170L S9R + A15T + V68A
    V68A + S106A + N252M + Y263C V68A + S106A + Q245W
    V68A + S106A + Q245R + N252D V68A + S106A + Q245W + N252K
    V68A + S106A + A174V + Q245R + N252D S9R + A15T + V68A + Q245R + N252S
    S9R + A15T + V28I + V68A + Q245R + N252A S9R + A15T + V68A + A194T + Q245R + N252E
    S9R + A15T + G20* + L21F + *63aG + Q245R + S9R + A15T + G20* + L21F + *62aS + N218D +
    N272V Q245R
    S9R + A15T + G20* + L21F + *61aS + V68A + S9R + A15T + V68A + H120N + P131S + Q137H +
    G160D + Q245R Q245M

    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 BPN′, shown in FIG. 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:
    G97GA + H120D + P131H + Q137E L111I + Q137E
    G97GV + Q137H G97GA + Q137H + N218S
    T22TQ + S101P L96LG + H120N + P131S + Q137H
    G97GV + H120D + Q137H L96LG + H120N + P131S + Q137H
    V4A + G97GV + H120D G97GA + H120N + Q137E
    L111V + Q137E + G211D L111I + H120N + Q137E
    L21LW + G100S + V139L + Q245V L96LG + P131T + Q137H
    V68A + Q137D L96LG + H120N + P131S
    L96LG + H120Q + Q137E G97GA + H120Y + Q137H
    K94N + H120N + P131H G97GA + Q137D
    G97GV + H120Q L96LG + P131H + Q137D
    G97GA + Q137E + L262V F50S + H120D + P131H
    D60DG G97GA + H120N + Q137D + N248D
    T22TL + N62D + Q245W L96LG + P131Q + Q137D
    T22TW + N204D + Q245I T22G + V139L + Q245L
    T22TG + N62D + V139L + Q245V Q245F
    G97GG + P131S + Q137H Q245S
    G20* + L21F + N62D + Q245N T260M
    T22TG + N62D + V139L + Q245G H120G + Q137E + N218D
    T22TL + N62D + I107V + V139L + Q245W G127E + P131R + Q137H
    G97GG + H120D + Q137E S130P
    H120R + Q137D + N173S Q245V
    V68A + Q245R N218D
    G97GA + H120N + S212L T22A + V139L + Q245E
    G97GA + H120N + S212L T22L + V139L + Q245V + A254S
    L96LG + H120D + P131H + R186L T224A
    G97GA + H120D + Q137D Q245L
    S9R + A15T + A16P + G97GA + P131S + Q137D + G61E + A85T + P239S + Q245V
    N204S
    L21LP + T22TV + M119I + N218D + Q245I G61E + A85T + Q206L+ Q245R
    L96LG + H120N + P131H + Q137E N62NG + Q245T
    L96LG + H120D + P131S + Q137E G61GP + Q245L
    H120N + P131T + N218D G61E + A85T + Q137H + Y209C + Q245G
    L96LG + H120D + G160D G61E + A85T + P239S + Q245C
    T22TG + N62D + V139L + Q245S G102S + M175T + Q245R + N252D
    G61E + A85T + P239L + Q245C N62D + Q245W + N252V
    G61E + A85T + P239L + Q245C N62D + Q245R + N252M
    P131H + S144P N62D + Q245W + N252S
    G97GA + Q137E S24P + G61E + A85T + P239S + Q245A
    L96LG + Q137E + Y209H T22A + N62D
    G97aA + H120Q + P131H + Q137E
    L111I + Q137E G61E + A85T + N218D + P239S + Q245L
    G97GA + Q137E S24P + G61E + A85T + P239S + Q245A
    L96LG + Q137E + Y209H T22L + V139L + Q245V + A254S
    L96LG + H120N + P131S T224A
    G97GV + H120Q Q245L
    L96LG + H120Q + Q137E N62NG + Q245T
    G97GV + P131S N62ND + V139L + Q245E
    K94N + H120N + P131H N62ND + V139L + N261D
    N76S + L111V + P131H + Q137D G61E + A85T + Q137H + Y209C + Q245G
    F50S + H120D + P131H G61E + A85T + P239S + Q245C
    L96LG + S130* N62D + Q245W + N252V
    L96LG + P131Q + Q137D N62D + Q245R + N252M
    G97GA + H120D + Q137H + M222V N62D + Q245W + N252S
    G97GA + H120N + Q137D + N248D V68A + S99G + Q245R + N261D
    L21LW + G100S + V139L + Q245V G20* + L21F + N62D + Q245R
    G20* + L21F + N62D + Q245N G20* + L21F + N62E + Q245R
    L21LC + V139L + R186H + Q245M H120D + Q137D
    T22TG + N62D + V139L + Q245G N77S + L96LG + H120D + P131Q
    T22TL + N62D + I107V + V139L + Q245W G97GA + H120N + Q137E
    T22TQ + S101P G97GA + Q137E + L262V
    T22TG + N62D + V139L + Q245V P131H + S144P
    T22TL + N62D + Q245W G127E + P131R + Q137H
    T22TW + N204D + Q245I G97GG + H120D + P131H + Q137H
    T22TG + N62D + V139L + Q245S G97GV + H120E + Q137H
    L21LP + T22TY + V139L + G160D + Q245L G97GV + P131T + Q137H
    S130P G97GV + H120Q + Y263F
    G61E + A85T + P239L + Q245C G97GV + S106A + P131H
    L21LP + T22TV + M119I + N218D + Q245I G97GG + L111I + P131T + Q137H
    V68A + Q245R G97GV + P131H + Q137H
    T22A + V139L + Q245E G20A + G97GV + H120D + P131H
    Q245F G97GA + H120D + P131S + Q137E
    Q245S G97GA + Q137H
    T260M H120R + Q137D + N173S
    S9R + A15T L96LG + H120N + P131H + Q137E
    L21LG + T22TV + V139L + N204D + Q245N L96LG + H120D + P131S + Q137E
    T22G + V139L + Q245L H120N + P131T + N218D
    Q245V G97GA + H120D + Q137D
    N218D G97GG + P131S + Q137H
    A13V + I35V + N62D + Q245F G97GG + H120N + Q137D
    G97GA + H120D + P131H + Q137E H120Q + P131C + Q137H
    G97GV + Q137H G97GV + H120D + Q137H
    G97GV + H120N A16P + G97GA + P131S + Q137D + N204S
    L96LG + H120D + P131H + R186L L96LG + H120D + G160D
    G97GA + R186C G97GA + H120N + S212L
    V4A + G97GV + H120D G97GA + Q137H + N218S
    G20* + L21F + *63aG + Q245R + N272V V68A + H120N + P131S + Q137H + Q245M
    G20* + L21F + *61aA + V68A + Q245R V28I + V68A + Q245R + N252A
    V68A + A194T + Q245R + N252E V68A + Q245R + N252S
    G20* + L21F + *62aS + N218D + Q245R G20* + L21F + *61aS + V68A + G160D + Q245R

    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 BPN′, shown in FIG. 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.
    • DEFINITIONS
  • 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 invention, 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., 1991, Protein Engng. 4: 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., 1991, Protein Engng. 4: 719-737 and Siezen et al., 1997, Protein Science 6: 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., 1999, Nature Biotechnology 17: 893-896.
  • 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., 1991, Protein Engng. 4: 719-737 and Siezen et al., 1997, Protein Science 6: 501-523.
  • TABLE III
    Organism
    Bacteria: Gram-positive enzyme acronym
    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 Variants
  • 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 Polynucleotides
  • 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, 1985, Nature 316: 774-78). The term “an isolated polynucleotide” may alternatively be termed “a cloned polynucleotide”.
    • Isolated Proteins
  • 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,L,V; R10H,K; V11A; Q12D; A13V; P14S,T,D,A,M,V,K,Q,L,H,R,I; A15M,T; A16P; H17R; N18S,H; R19W,K,L,F,G,I; G20*,R,A; L21F,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,R,V,S; S99D,L,A,AD,SD,SM,SG,DA,P,G,N,C,M,V,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; I107T,V,M; A108V,T,S; L111I,V; A114V; N116S,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,*; P131M,F,W,L,A,H,T,*,PA,S,Q,R,E,G,D,C; S132G,T; A133ASA; T134A; Q137H,E,D; A138G,V; 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,I; N204T,D,S; Q206L; Y209C,H; G211D; 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,N,V,W,G,I,S,C,L,A,M; N248P,D,S; K251E,R; N252G,H,D,V,M,S,T,E,Y,S,Q,K,A,L; 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+A111G+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+L111I+Q137E, S9R+A15T+L111I+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+V1391+A194P+N218D+A230V, A108V+A169G+R170A+Y171H, 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 amplifying 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, BAH101), 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 above mentioned subtilase variants in combination with any other modification to the amino acid sequence thereof. Especially combinations 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 K27R+V104Y+N123S+T274A, N76D+S103A+V104I, N76D+V104A, S87N+S101G+V104N, S99D+S101R+S103A+V104I+G160S, S3T+V4I+S99D+S101R+S103A+V104I+G160S+A194P+V199M+V205I+L217D, S3T+V4I+S99D+S101R+S103A+V104I+G160S+V199M+V205I+L217D, S3T+V4I+S99D+S101R+S103A+V104I+G160S+V205I, S101G+V104N, or other combinations 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, M222A, M222S, 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:
    • S101G+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, 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, N.Y.; 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, 1994, Nature 370: 389-91). 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 enzyme of the invention may be any vector that may conveniently be subjected to recombinant DNA procedures.
  • 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 sequences are 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 Cells
  • 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 may be from another organism, or it may be 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. alkalophilus, B. amyloliquefaciens, B. brevis, B. circulans, B. coagulans, B. lautus, B. lentus, B. licheniformis, B. megaterium, B. stearothermophilus, B. subtilis, or B. 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 are 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.
    • Methods 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 household hard surface 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 and subtilisin 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: Suitable lipases include those of bacterial or fungal origin. Chemically modified 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 B. 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).
  • Amylases: Suitable amylases (alpha and/or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of B. licheniformis, described in more detail in GB 1,296,839.
  • Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, 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 amylases 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 Acremonium, Bacillus, Humicola, Fusarium, Pseudomonas, Thielavia, e.g., the fungal cellulases produced from Fusarium oxysporum, Humicola insolens, and Myceliophthora thermophila disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, and 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, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 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 HAT™ (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 U.S. Pat. Nos. 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, alkyl sulfate (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 peracid-forming bleach activator 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%
    NaCl 0-40%
    Others up to 100%
    Polymers
    Enzymes
    Foam regulators
    Water
    Hydrotropes
    Others
  • 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 liquor, preferably 0.05-50 mg of enzyme protein per liter of wash liquor, in particular 0.1-10 mg of enzyme protein per liter of wash liquor.
  • 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 EMPA17 (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 U.S. Pat. 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 Spring Harbor, N.Y.; 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).
    • 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 shaking table at 225 rpm in 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 subtilase variant secreted from the host 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 assess the wash performance of selected subtilase variants in detergent compositions, washing experiments are performed. The enzyme variants are 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 pages 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 these transformants is transformed into B. 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: 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 B. subtilis strain and fermented as described above.
    • Example 2 Purification and Assessment of Enzyme 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 micro-m are mixed with a media containing yeast extract and the transformed B. 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 Cl-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/HCl buffer) is added after the incubation with inhibitor and during the following 4 minutes the development of the degradation product pNA (paranitrophenol) is measured periodically at 405 nm on an Elisa Reader.
  • Each of the variants of the invention listed in Table I 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 assess 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 pages 23-24.
  • Two assays were conducted under the experimental conditions specified below:
  • Assay A
    Commercial detergent base Latin American type
    Detergent dosage 1.5-2.5 g/l
    Test solution volume 160 micro l
    pH 10-10.5 adjusted with NaHCO3
    Wash time 14 min.
    Temperature 20° C.
    Water hardness 6-9°dH
    Enzyme concentration in test solution 5 nM, 10 nM and 30 nM
    Test material EMPA 117
  • 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 CaCl2 and MgCl2 (Ca2+:Mg2+=4:1) to the test system. After washing the textile pieces were flushed in tap water and air-dried.
  • Assay B
    Commercial detergent base European powder type 1
    Detergent dosage 6 g/l
    Test solution volume 160 micro I
    pH as it is in detergent (app. 10-10.5)
    Wash time 20 min.
    Temperature 30° C.
    Water hardness 6-9° dH
    Enzyme concentration in test solution 5 nM, 10 nM and 30 nM
    Test material EMPA 116
  • 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 CaCl2*2H2O; MgCl2*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 is stained the intensity of the reflected light is lower, than that of a clean textile. Therefore the intensity of the reflected light can be used to measure wash performance of an enzyme variant.
  • Color 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). The intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:

  • Int=√{square root over (r2 +g 2 +b 2)}
  • The wash performance (P) of the variants was calculated in accordance with the below formula:

  • P=Int(v)−Int(r)
  • where
  • Int(v) is the light intensity value of textile surface washed with enzyme variant and
  • Int(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.
  • TABLE IV
    Wash performance test results, Assay A.
    Mutations Score Mutations Score
    G97E + A98S 2 V28I + A98AD + T224S 2
    G97D + A98D 2 S99AD + M175V + P131F 1
    V95C + G97W + A98E 2 S99AD + P131L 2
    V95T + G97A + A98D 2 S9R + S99AD + P131W 1
    S103Y + V104M + S106D 1 V68A + N116S + V139L + Q245R 2
    V104T + S106D 2 S3T + A16P + R45C + G100S + A230V 2
    S3T + A16P + S99SD + S144D + 2 I8V + S9R + A15T + R19W + V30I + G61D + 2
    A158T + A230V + T260R S99SD + S256N
    S103D + V104T + S106T 1 V30I + S99SD + S256R 2
    S103D + V104L + S106M 2 G61S + S99SD + V244I 2
    S103D + V104T + S106G 2 V68A + V139L + S163G + N185S 2
    S103D + V104S + S106A 2 S99SD + Y263H 2
    S103H + V104N + S106D 2 V104N + S106T 2
    S103E + V104I + S106T 1 S99SG + S144D 1
    S103Q + V104T + S106E 2 V30I + S99SD 1
    S103E + S106T 2 N18H + S99SD 2
    S103E + V104R + S106A 2 S9R + T22S + S99SD + K251E 1
    A108T + L111V 2 A48T + V68A + P131M 2
    L124I + S125A 1 A15M + S99SM + V139I + V244I 2
    L124C + P131* 2 P14T + A15M + S99SD 2
    P129S + S130AT 2 I8V + S99SD + S144D + A228T 2
    L96LA + A151G + V203A 1 I8V + R19K + V139I 2
    S99SD + A108V + V139L 2 I35T + N62D 2
    S99SD + S190A 2 N62D + S265G 2
    S99SD + V203A 2 Q2L + N62D 2
    S99SD + V139I 1 N62D + N76D 2
    S99SD + A108V 2 R45H + G61E + V68A 2
    S99SD + S106A + A151G 2 N62D + V121A 2
    V68A + S106A 2 N62D + A215D 2
    V68A + N185D + V203S 2 N62D + N238D 2
    V68A + V139L 2 N62D + R145G 2
    V68A + V139I 2 V4L + N62D + E89G 2
    V68A + A158V 2 N62D + S188G + K251R 2
    V68A + V203A 2 S49N + N62D 2
    V68A + V203S 2 N62NE 2
    V68A + V203L + S259A 2 V11A + N62DE 2
    V68A + S106L 2 N62ND + N184S + S256G 2
    V30I + V68A + V203S 2 N18S + N62D + I107T + A254S 2
    V51A + V68A + S106T + A168G 1 S57P + N62ND 2
    V51A + V68A + S106T + A168G 1 N62NE + V234I 2
    V68A + N76S + V203M + P239T 2 Q137H + R170C + G195E 1
    V68A + V203L 2 S99A + S101SA 2
    V68A + L75I + V203Q 2 R10K + P14A + R19K + A98AS + S128N 2
    V68A + T71A + V139L 2 T22A + R45K + A98AS + S128N 2
    Y192H + V68A 2 A98AV + S99D + Y167K 2
    V68A + S106A + A108T 2 S9G + P14K + Y167A + R170S 2
    V68A + S106T + A108T 2 S9D + P14T + Y167A + R170S 2
    V68S + A108S 2 S9R + P14M + A98AD 1
    V68A + N76S + G211D 2 S9R + R19L + A98AD + E271A 2
    V68A + S106T + A108T 1 S9R + P14S + R19F + A98AD 2
    A151V + R170C 2 S99DA + P129PSN + P131A 2
    P14D + A98AS + H120D + G195F + 1 S99AD + V244M + Q245K + N248D + K251R + 2
    S212N + M222S T255A + S256N
    S49N + V203L + N218D 2 S9R + P14V + R19G + A98AD 2
    V68A + S106M + N184D 2 S99AD + N248P + T255A + S256G 2
    P55S + V68L + A158E + G160A 2 *0AQSVPWG + A98AD 2
    V68A + A158C 2 T22A + S99AD 2
    V68A + A158L + Y214C 2 K94N + A98T + S99L 2
    A88V + S99AD + P131F 2 N76D + A174AL + A194P + A230V 1
    P14T + A16P + I72V + S99SD + 2 P40L + N218D + A232S + Q236L + Q245E + 2
    V244I + T260A S259N
    S99AD + P131F 2 A232L + Q236D + Q245E 1
    R10H + N62D 2 A232T + Q236L + Q245D 2
    V28I + A98AD + T224S 2 R170H + Q236A + Q245R 2
    S9K + T22K + S99AD 2 A232L + Q236T + Q245D 2
    P14S + S99AD + P131W 2 G97GG + P131H + Q137E + V268L 2
    V68A + I72V + P131F 2 A88V + G97GV + P131H 2
    S9R + S99AD 1 G97GA + H120Q + S130P + G264E 2
    S9K + S99AD 2 G97GG + V139L 2
    V28I + A88V + G100S + P131M 2 G97GG + Q137D 1
    S103L + V104S + S106G 2 G97GG + H120D + Q137H 2
    V68A + T224A 2 N185R 2
    V68A + P131F 2 P131H + Q137E 1
    A48T + V68A + P131M 1 V104I + H120N + P131H + Q137E 2
    V68A + I72V + P131F 2 H120Q + Q137E 1
    G100GE + P131F 2 S9R + A15T + G97GV + H120D 1
    S99AD + P131F + T260A 1 G100S + H120Q + Q137H 2
    R19G + A98AS 2 V68A + H120K + Q137E 2
    G61R + N62D 1 G97GA + H120E 2
    V68A + S106M + N184D 2 H120D + S128I + Q137D 2
    P55S + V68L + A158E + G160A 2 G97GG + P131H 2
    V68A + A158C 2 G97GG + H120N + L126I 2
    R19W + G61S + S99SD + N204T + 2 S9R + A15T + G97GA + H120D + P131H + 2
    Y263H + S265R Q137E
    A232T + Q236C 2 S9R + A15T + G97GV + P131T + Q137H 1
    N62D + A232T + Q236C 2 S9R + A15T + G20* + L21F + N62D + Q245N 2
    A232P + Q236L + Q245E 2 S9L + A15T + T22TV + V139L + Q245F 2
    A232S + Q236L + Q245T + K251E 2 S132G + Q245F 1
    S163C + Q236M + Q245T + S256G 2 S9R + A15T + T22TG + N62D + V139L + Q245V 1
    N218D + A232L + Q236F + Q245F 2 S9L + A15T + T22TV + V139L + Q245F + L262S 2
    S163N + A232L + Q236S + Q245E 2 S9R + A15T + T22TL + N62D + Q245W 2
    A232S + Q236S + Q245E 2 V68A + A158L + Y214C 2
    V68A + V203L 2 N62D + V150I 2
    V68S + A158D 2 S3T + P14Q + A15M + R19K + N62D + S144D 2
    I8V + A15T + R19K + A85T + S99SD + 2 P14Q + R19W + V51I + G61E + S99SD + 2
    A114V + V244I + S256N + Y263H2 V139I + T260R
    L111F + Y263H 2 S3T + P14L + H17R + S99SD + V139I + S144D 2
    P52V + S78T + S99SD 2 S3A + V30I + S99SD + S106G + N248S 2
    A15M + S99SD + V268I 2 I8V + A15T + S99SD 2
    S99G + S128N + N183D + A232L + 1 S3T + S9R + P14H + A15M + R19L + S99SD + 2
    Q236T + Q245R 1 V139I
    S99R + S101SA 1 S9R + A15T + G97GG + H120D + Q137E 2
    L96LA + A98T + P131AA 2 S9R + A15T + G20A + G97GV + H120D + P131H 2
    A98E + S99P 2 S163N + A232L + Q236A + Q245G 2
    V28I + S99AD + P131F 2 N173D + A232L + Q236A + Q245N 2
    S9R + A15T + G97GV + Q137H 1 P55S + V68A + S106M + A108T + P129T 2
    V81A + P131T + A133S + Q137E 1 K27R + V68L + G118D + A158E 1
    N43D + V68A + S106F + N238D 2 A98E + S99A + S101SK 2
    V68A + V203F 2 V68A + N140D + T143A + S144N 2
    V68A + S106E 2 N62D + N140K + T143A + S144D 2
    V68A + S106I 2 S9F + P14T + R19L + A98AD 2
    V68A + A158M + R170C 1 S9V + P14R + R19F + A98AD 2
    V68A + P129T + N218D 2 S99A + S99SD + G258K + L262Q 2
    V68S + P129E 2 S87C + S99SA + S99D + P131A 2
    V68S + P129D 2 S99A + S99SD + G258K + L262Q 2
    V68L + P129E + N261D 2 V28I + S99A + *99aD + P131F 1
    G97GV + H120D 2 A85T + G102D + S106T + K237R 2
    P131A + A133ASA 2 V68A + T71A 2
    L111F + Y263H 2 G61GS 2
    V11A + G61GE + V227A + S240F 2 G100L 2
    A133E + S144K + N218D 2 A133D 2
    S128A + P129S + S130SP 2 V68A 2
    G61GE 2 N123D 2
  • TABLE V
    Wash performance test results, Assay B.
    Mutations Score
    S9R + A15T + T22TW + N204D + Q245I 2
    S9R + A15T + G97GG + P131S + Q137H 2
    S9R + A15T + T22TG + N62D + V139L + Q245G 2
    S9R + A15T + T22TL + N62D + I107V + V139L + Q245W 2
    S9G + A15T + G97GA + Q137H 1
    S9R + A15T + V68A + Q245R 2
    S9R + A15T + G97GA + H120N + S212L 2
    S9R + A15T + L96LG + H120D + P131H + R186L 2
    S9R + A15T + G97GA + H120D + Q137D 2
    S9R + A15T + A16P + G97GA + P131S + Q137D + N204S 2
    S9R + A15T + L21LP + T22TV + M119I + N218D + Q245I 2
    S9R + A15T + G97GV + H120D + Q137H 1
    S9R + A15T + L96LG + H120N + P131H + Q137E 2
    S9R + A15T + L96LG + H120D + P131S + Q137E 2
    S9R + A15T + H120N + P131T + N218D 2
    V4A + S9R + A15T + G97GV + H120D 1
    S9R + A15T + L96LG + H120D + G160D 2
    S9R + A15T + T22TG + N62D + V139L + Q245S 2
    S9R + A15T + G61E + A85T + P239L + Q245C 2
    S9R + A15T + P131H + S144P 2
    S9R + A15T + G97GA + Q137E 2
    S9R + A15T + G97GA + H120Q + P131H + Q137E 2
    S9R + A15T + L21LW + G100S + V139L + Q245V 1
    S9R + A15T + G97GA + Q137H + N218S 2
    S9R + A15T + L96LG + H120N + P131S + Q137H 2
    S9R + A15T + G97GA + H120N + Q137E 2
    S9R + A15T + L96LG + P131T + Q137H 2
    S9R + A15T + L96LG + H120N + P131S 2
    S9R + A15T + V68A + Q137D 1
    S9R + A15T + G97GA + H120Y + Q137H 2
    S9R + A15T + G97GA + Q137D 2
    S9R + A15T + K94N + H120N + P131H 1
    S9R + A15T + L96LG + P131H + Q137D 2
    S9R + A15T + F50S + H120D + P131H 2
    S9R + A15T + G97GA + H120N + Q137D + N248D 2
    S9R + A15T + L96LG + P131Q + Q137D 2
    S9R + A15T + T22G + V139L + Q245L 2
    V139L + Q245R 2
    S9R + A15T + Q245F 2
    S9R + A15T + Q245S 2
    S9R + A15T + G97GV + H120Q 1
    S9R + A15T + G97GA + Q137E + L262V 1
    S9R + A15T + G127E + P131R + Q137H 2
    S9R + A13V + A15T + I35V + N62D + Q245F 2
    S9R + A15T + Q245V 2
    V139L + Q245F 2
    S9R + A15T + T22A + V139L + Q245E 2
    S9R + A15T + T22L + V139L + Q245V + A254S 2
    S9R + R19L + A98AD 2
    P14R + A98AD 2
    S9R + A15T + Q245L 2
    S9R + A15T + G61E + A85T + P239S + Q245V 2
    S9R + A15T + G61E + A85T + Q206L + Q245R 2
    P239T + Q245R 2
    S9R + A15T + N62NG + Q245T 2
    S9R + A15T + G61GP + Q245L 2
    S9R + A15T + G61E + A85T + Q137H + Y209C + Q245G 2
    S9R + A15T + G61E + A85T + P239S + Q245C 2
    V68I + A98AD 2
    V68A + N269K 1
    N62D + Q245A + N252G + S265G 2
    N218D + Q245G + N252H 2
    S9R + A15T + G102S + M175T + Q245R + N252D 2
    S9R + A15T + N62D + Q245W + N252V 2
    S9R + A15T + N62D + Q245R + N252M 2
    S9R + A15T + N62D + Q245W + N252S 2
    S99SD + N204S + Q245R 1
    N62D + Q245R 2
    N62D + A151G 1
    V68A + S106T 2
    S99A + S99SD + V203L 2
    A98AD + A215T 2
    N62D + Q245G + N252T 2
    A152P + Q245R + N252T 2
    S163N + T213A + Q245R 2
    S106L + Q245R + N252E 2
    Q245W + N252Y 2
    Q245W + N252V 2
    R45H + Y171C + Q245W + N252S 2
    G20R + A48T + R170C + Q245W + N252Q 2
    N62D + N252T 2
    N218D + Q245W + N252E 2
    G20R + R170C + Q245R + N252V 2
    S9R + P14I + R19K + A98AD + T274S 2
    A98AE + V203I 2
    V51A + V68A + S163G + V203A 2
    N62D + Q245W + N252H 2
    N62D + Q245W + N252A 2
    G20R + N62D + V244I + Q245W + N252E 2
    N204D + Q245S 2
    N62D + Q245W + N252E 2
    N62D + Q245R + N252V 2
    S9R + A15T + S24P + G61E + A85T + P239S + Q245A 2
    G102S + M222S + Q245L + N252D 2
    A15M + V30I + N62D + S99N + L111I + V244A + S265N 2
    V68A + S106A + G118D + Q245R + T255S + L257G + T274L 2
    S3T + Q12D + R19W + V30I + S106G + I107M 2
    V68A + A88T + V139L 2
    V51I + L111I + G118D + Q245R 2
    V68A + V203L 1
    A1T + V68A + N116D + G118D 1
    V68A + G118D + Q245R 2
    N62D + V139I + N183D + N185S + V203I + Q245R + L262S 2
    N62D + I72V 1
    N62D + V81A + Q245R 1
    T22A + V68A + S106T + G118D 1
    V68A + L111I + V203I 1
    G61E + V68A + A169G 1
    V68A + L111V 1
    V68A + G118D + V203A + K251R 1
    V68A + G118D 1
    A1V + V51A + V68A + V203I 2
    V68A + V139L + A223G 1
    N62D + Y214H + K237R 1
    V68A + S106A + G118D + Q245R 2
    S9R + A15T + T22A + N62D 2
    A98Q + S99D 1
    S9R + P14I + R19K + A98AD 2
    S9R + A15M + A16P + T22S + S99AD 1
    S99AD + T255R + S256N 2
    S9R + A15T + T22TQ + S101P 1
    S9R + A15T + H120R + Q137D + N173S 2
    G97E 1
    Q245W 2
    S9R + A15T + L96LG + Q137E + Y209H 2
    S9R + A15T + L111V + Q137E + G211D 1
    S9R + A15T + L111I + Q137E 2
    S9R + A15T + L111I + H120N + Q137E 2
    S9R + A15T + L96LG + H120Q + Q137E 1
    S9R + A15T + T260M 2
    S9R + A15T 2
    Q245I 2
    S9R + A15T + H120G + Q137E + N218D 2
    S9R + A15T + S130P 2
    Q245F 2
    S9R + A15T + N218D 2
    G63E + N76D + A194P + A230V 2
    S9R + A15T + T224A 2
    G100S 2
    S9R + A15T + D60DG 1
    A138V + V139I + A194P + N218D + A230V 2
    A108V + A169G + R170A + Y171H 1
    I8V + P14L + R19L + V30I + I35V + 2
    S57P + P129S + Q137D + S144D + S256N
    A133D + T134S + Q137A 1
    Q137D 2
    A98AH 1
    V51D 2
    Q12E + P14L + A15T 2
    G63E + N76D + A194P + A230V 2
    Q12E + P14L + A15T 2
    G97GS 1
    M222S + Q245G + N252G 1
    V68I + V203L 2
    V51A + S163T 2
    S106A + A138G 2
    V139I + A151G 2
    S9R + A15T + S99C + H120N + P131S + Q137H + M222S 2
    S9R + A15T + S99G + G100S + H120N + P131S + Q137H 2
    A15T + N185D + M222S + Q245R + N252V 2
    S9R + A15T + T22TL + G61E + L96LG + Q137D + Q245R 2
    S9R + T22TL + G61E + G97GG + M119I + P131T 2
    Y209H + M222S + Q245G + N252L 2
    M222S + Q245M + N252E 2
    S9R + A15T + N62D + H120N + P131T 2
    S9R + A15T + V68A + N218D + Q245R 2
    S9R + A15T + V68A + H120N + N218D + Q245R 1
    S9R + A15T + V68A + A174V + Q245R 2
    S9R + A15T + G46D + V68A + N218D + Q245R 2
    G97D + A98N + S128G + S130T + P131D + T134A 2
    S9R + A15T + V68A + A98M + Q245R + N248D 2
    S9R + A15T + V68A + A98L + S99G + Q245R 2
    S9R + A15T + A98G + S99C + H120N + P131S + Q137H 2
    S9R + A15T + T38S + A98R + S99C + 2
    G100S + H120N + P131S + Q137H
    A98V + S99C + Q245R 1
    S9R + A15T + A98S + G100S + H120N + P131S + Q137H 1
    S9R + A15T + G20* + L21F + N62D + Q245R 2
    S9R + A15T + G20* + L21F + N62D + Q245R + S259G 2
    A98S + G100S + Q245R 2
    A98L + S99C + Q245R 2
    S9R + A15T + G20* + L21F + N62E + Q245R 2
    S9R + A15T + G20* + L21F + P52T + N62D + Q245R 2
    S9R + A15T + V68A + S99G + Q245R + N261D 2
    N62D + P131F + A172V 2
    N62D + P131F 2
    S99SD + Q245R 2
    S9R + A13T + S99A + S99SD + P131F 2
    S9R + A15T + N62S + H120N + P131T + N218D 2
    A98R + G100C + Q245R 2
    A98G + S99C + Q245R 2
    A98T + S99G + G100S + S240F + Q245R 2
    S9R + A15T + H120N + P131T + N218D + N269T 2
    S9R + A15T + G61E + H120S + Q137D + V139L + N218D 2
    S9R + A15T + L96LG + H120N + P131S + Q137H + M222S 1
    S9R + A15T + G61E + A98S + S99M + Q245R 2
    A98G + G100S + Q245R + N261D 2
    S9R + A15T + V68A + A98L + Q245R 1
    S9R + A15T + V68A + A98G + S99V + Q245R 1
    S9R + A15T + V68A + A98M + S99G + Q245R + T274A 2
    S9R + A15T + G61E + V68A + A98S + S99G + Q245R 2
    S9R + A15T + A88V + A98R + S99G + 1
    G100C + H120N + P131S + Q137H
    S9R + A15T + A98C + G100S + H120N + P131S + Q137H 1
    S9R + A15T + G20* + L21F + G61E + *61aP + Q245R 1
    S9R + A15T + V68A + A98G + S99I + K237R + Q245R
    S9R + A15T + V68A + H120N + P131S + Q137H + Q245R 2
    A98S + S99G + G100S + Q245R 2
    S9R + A15T + V68A + H120D + P131S + Q137H + Q245R 2
    A98T + S99G + G100S + Q245R 2
    S9R + A15T + A98S + S99G + G100S + 2
    H120N + P131S + Q137H
    V68A + S106A + Q245R + N252D 2
    V68A + S106A + Q245W 2
    V68A + S106A + N252M + Y263C 1
    V68A + S106A + Q245W + N252K 0
    V68A + S106A + A174V + Q245R + N252D 1
    S9R + A15T + V68A + Q245R + N252S 2
    S9R + A15T + V68A 2
    S9R + A15T + G20* + L21F + *61aS + V68A + G160D + Q245R 2
    S9R + A15T + Y167I + R170L 2
    S9R + A15T + G20* + L21F + *63aG + Q245R + N272V 2
    S9R + A15T + G20* + L21F + *61aA + V68A + Q245R 2
    S9R + A15T + V68A + A194T + Q245R + N252E 2
    S9R + A15T + G20* + L21F + *62aS + N218D + Q245R 2
    V68A + S106A + T213A 2
    S9R + A15T + V28I + V68A + Q245R + N252A 2
    V68A + S105G + S106A 2
    S9R + A15T + V68A + H120N + P131S + Q137H + Q245M 2
  • Assay C
    Commercial detergent base European powder type 2
    Detergent dosage 4 g/l
    Test solution volume 160 micro l
    pH as it is in detergent (app. 10-10.5)
    Wash time 20 min.
    Temperature 30° C.
    Water hardness 6-9° dH
    Enzyme concentration in test solution 5 nM, 10 nM and 30 nM
    Test material EMPA 116
  • The European powder type detergent was composed according to the provisions in Detergent Example 2. Water hardness was adjusted to 15° dH by addition of CaCl2*2H2O; MgCl2*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.
    Mutations Score
    Q12E + P14L + A15T 2
    P14R + A98AD 2
    G100S 2
    A169G + R170H 1
    A98AD + A169G 1
    A138V + V139I + A194P + N218D + A230V 2
    S99A + S99SD + V203L 1
    V68A + S106T 1
    A98AD + A215T 2
    A108V + A169G + R170A + Y171H 1
    S3L + N62D + S163A + S190A 2
    S9R + P14I + R19K + A98AD + T274S 2
    S9R + A15T + G61E + A85T + N218D + P239S + Q245L 2
    S9R + A15T + S24P + G61E + A85T + P239S + Q245A 2
    S99SD + P131F 1
    N62D + P131F + A172V 1
    N62D + P131F 1
    V68A + A88T + V139L 2
    V68A + G118D + V203A 2
    P40L + V68A + A108T + A138V + V203I 2
    I8T + A98AD + T274R 2
    A98AE + V203I 2
    V51A + V68A + S163G + V203A 2
    A1V + V51A + V68A + V203I 2
    V68A + G100S 1
    V68A + V203L 1
    A1T + V68A + N116D + G118D 1
    N62D + A169G + V203I + Q245R 1
    G23S + S99SD + A194P + S242T + Q245R + T274R 1
    S99SD + N204S + Q245R 2
    N62D + Q245R 2
    V68A + S106A + G118D + Q245R 2
    V51I + L111I + G118D + Q245R 2
    N62D + V139I + N183D + N185S + V203I + Q245R + L262S 2
    N62D + I72V 1
    S9R + R19L + A98AD 2
    S9G + P14R + R19I + A98AD 1
    S9R + A15T + T22L + V139L + Q245V + A254S 2
    S9R + A15T + T224A 2
    S9R + A15T + Q245L 2
    S9R + A15T + N62NG + Q245T 1
    S9R + A15T + N62ND + V139L + Q245E 1
    S9R + A15T + N62ND + V139L + N261D 2
    Y167I + R170L + Q245E 1
    Y167I + R170L + Q245R 2
    Y167I + R170L + Q245M 1
    Y167I + R170L 1
    S99SE + Q245R 2
    S9R + A15T + G61E + A85T + Q137H + Y209C + Q245G 2
    S9R + A15T + G61E + A85T + P239S + Q245C 1
    G102S + M222S + Q245L + N252D 1
    N62D + Q245A + N252G + S265G 1
    N62D + Q245G + N252T 1
    S9R + A15T + N62D + Q245W + N252V 2
    S9R + A15T + N62D + Q245R + N252M 2
    S9R + A15T + N62D + Q245W + N252S 1
    S163N + T213A + Q245R 2
    S106L + Q245R + N252E 2
    Q245W + N252Y 2
    Q245W + N252V 1
    G20R + A48T + R170C + Q245W + N252Q 2
    N62D + N252T 2
    N218D + Q245W + N252E 2
    G20R + R170C + Q245R + N252V 2
    N62D + Q245W + N252H 2
    N62D + Q245W + N252A 2
    G20R + N62D + V244I + Q245W + N252E 2
    N204D + Q245S 1
    N62D + Q245W + N252E 2
    N62D + Q245R + N252V 2
    A98L + S99C + Q245R 2
    N62D + A98R + Q245R 2
    S9R + A15T + V68A + S99G + Q245R + N261D 2
    S9R + A15T + G20* + L21F + N62D + Q245R 2
    S9R + A15T + G20* + L21F + N62E + Q245R 2
    V68I + A98AD 2
  • Assay D
    Commercial detergent base European powder type 1
    Detergent dosage 6 g/l
    Test solution volume 160 micro l
    Ph as it is in detergent (app. 10-10.5)
    Wash time 20 min.
    Temperature 30° C.
    Water hardness 6-9° dH
    Enzyme concentration in test solution 5 nM, 10 nM and 30 nM
    Test material C-10 swatches from Center for
    Testmaterials, Vlaardingen, NL
  • 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 CaCl2*2H2O; MgCl2*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.
    Mutations Score
    G97GS 1
    S9V + P14R + R19F + A98AD 1
    S9R + A15T + L111I + Q137E 1
    S9R + A15T + G97GA + Q137E 2
    S9R + A15T + L96LG + Q137E + Y209H 1
    S9R + A15T + L96LG + H120N + P131S 2
    S9R + A15T + G97GV + H120Q 2
    S9R + A15T + L96LG + H120Q + Q137E 2
    S9R + A15T + G97GV + P131S 2
    S9R + A15T + K94N + H120N + P131H 1
    S9R + A15T + N76S + L111V + P131H + Q137D 1
    S9R + A15T + F50S + H120D + P131H 2
    S9R + A15T + L96LG + S130* 2
    S9R + A15T + L96LG + P131Q + Q137D 2
    S9R + A15T + G97GA + H120D + Q137H + M222V 1
    S9R + A15T + G97GA + H120N + Q137D + N248D 2
    S9R + A15T + L21LW + G100S + V139L + Q245V 1
    S9R + A15T + G20*+ L21F + N62D + Q245N 2
    S9R + A15T + L21LC + V139L + R186H + Q245M 1
    S132G + Q245F 1
    S9R + A15T + T22TG + N62D + V139L + Q245G 2
    S9R + A15T + T22TL + N62D + I107V + V139L + Q245W 2
    S9R + A15T + T22TQ + S101P 2
    S9R + A15T + T22TG + N62D + V139L + Q245V 1
    S9R + A15T + T22TL + N62D + Q245W 2
    S9R + A15T + T22TW + N204D + Q245I 2
    S9R + A15T + T22TG + N62D + V139L + Q245S 2
    S9R + A15T + L21LP + T22TY + V139L + G160D + Q245L 1
    Q245W 2
    S9R + A15T + S130P 2
    S9R + A15T + G61E + A85T + P239L + Q245C 2
    S9R + A15T + L21LP + T22TV + M119I + N218D + Q245I 2
    S9R + A15T + V68A + Q245R 2
    S9R + A15T + T22A + V139L + Q245E 2
    V139L + Q245R 2
    S9R + A15T + Q245F 2
    S9R + A15T + Q245S 2
    S9R + A15T + T260M 2
    S9R + A15T 2
    S9R + A15T + L21LG + T22TV + V139L + N204D + Q245N 1
    V139L + Q245F 2
    S9R + A15T + T22G + V139L + Q245L 2
    S9R + A15T + Q245V 1
    Q245F 2
    S9R + Q245C 2
    S9R + A15T + N218D 1
    S9R + A13V + A15T + I35V + N62D + Q245F 2
    S99G + S128N + N183D + A232L + Q236T + Q245R 2
    S163N + A232L + Q236A + Q245G 2
    S163C + Q236M + Q245T + S256G 1
    N218D + A232L + Q236F + Q245F 1
    S163N + A232L + Q236S + Q245E 2
    G97GA + H120E 1
    G97GG + P131H 2
    S9R + A15T + G97GA + H120D + P131H + Q137E 1
    S9R + A15T + G97GV + Q137H 2
    S9R + A15T + G97GV + H120N 2
    S9R + A15T + G97GG + P131S + Q137H 2
    S9R + A15T + G97GG + H120N + Q137D 2
    S9R + A15T + H120Q + P131C + Q137H 2
    S9R + A15T + G97GV + H120D + Q137H 2
    S9R + A15T + A16P + G97GA + P131S + Q137D + N204S 2
    S9R + A15T + G97GG + H120D + P131H + Q137H 1
    S9R + A15T + G97GV + H120E + Q137H 2
    S9R + A15T + G97GV + P131T + Q137H 1
    S9R + A15T + G97GV + H120Q + Y263F 2
    S9R + A15T + G97GV + S106A + P131H 1
    S9R + A15T + G97GG + L111I + P131T + Q137H 1
    S9R + A15T + G97GV + P131H + Q137H 2
    S9R + A15T + G20A + G97GV + H120D + P131H 1
    S9R + A15T + G97GA + H120D + P131S + Q137E 1
    S9G + A15T + G97GA + Q137H 1
    S9R + A15T + H120R + Q137D + N173S 1
    S9R + A15T + L96LG + H120N + P131H + Q137E 2
    S9R + A15T + L96LG + H120D + P131S + Q137E 2
    S9R + A15T + H120N + P131T + N218D 2
    S9R + A15T + G97GA + H120D + Q137D 2
    S9R + A15T + L96LG + H120D + P131H + R186L 2
    S9R + A15T + G97GA + R186C 2
    V4A + S9R + A15T + G97GV + H120D 1
    S9R + A15T + L96LG + H120D + G160D 2
    S9R + A15T + G97GA + H120N + S212L 2
    S9R + A15T + G97GA + Q137H + N218S 2
    S9R + A15T + H120D + Q137D 2
    S9R + A15T + N77S + L96LG + H120D + P131Q 1
    S9R + A15T + G97GA + H120N + Q137E 1
    S9R + A15T + G97GA + Q137E + L262V 2
    S9R + A15T + P131H + S144P 2
    S9R + A15T + G127E + P131R + Q137H 2

Claims (20)

1-15. (canceled)
16. A variant of a parent subtilase comprising substitutions at positions 9 and 15, wherein the variant has protease activity and each position corresponds to a position of the amino acid sequence of SEQ ID NO: 1.
17. The variant of claim 16, wherein the substitutions are S9R+A15T.
18. The variant of claim 16, further comprising a substitution at position 68.
19. The variant of claim 18, wherein the substitution at position 68 is V68A.
20. The variant of claim 17, further comprising a substitution at position 68.
21. The variant of claim 20, wherein the substitution at position 68 is V68A.
22. The variant of claim 16, comprising a set of substitutions selected from the group consisting of:
S3T + S9R + P14H + A15M + R19L + S99SD + V139I V4A + S9R + A15T + G97GV + H120D S9L + A15T + T22TV + V139L + Q245F S9L + A15T + T22TV + V139L + Q245F + L262S S9R + A13V + A15T + I35V + N62D + Q245F S9R + A15M + A16P + T22S + S99AD S9R + A15T S9R + A15T + A16P + G97GA + P131S + Q137D + N204S S9R + A15T + G20A + G97GV + H120D + P131H S9R + A15T + G20* + L21F + P52T + N62D + Q245R S9R + A15T + G20* + L21F + G61E + *61aP + Q245R S9R + A15T + G20* + L21F + *61aA + V68A + Q245R S9R + A15T + G20* + L21F + *61aS + V68A + G160D + Q245R S9R + A15T + G20* + L21F + N62D + Q245N S9R + A15T + G20* + L21F + N62D + Q245R S9R + A15T + G20* + L21F + N62D + Q245R + S259G S9R + A15T + G20* + L21F + N62E + Q245R S9R + A15T + G20* + L21F + *62aS + N218D + Q245R S9R + A15T + G20* + L21F + *63aG + Q245R + N272V S9R + A15T + L21LC + V139L + R186H + Q245M S9R + A15T + L21LG + T22TV + V139L + N204D + Q245N S9R + A15T + L21LP + T22TV + M119I + N218D + Q245I S9R + A15T + L21LP + T22TY + V139L + G160D + Q245L S9R + A15T + L21LW + G100S + V139L + Q245V S9R + A15T + T22A + N62D S9R + A15T + T22A + V139L + Q245E S9R + A15T + T22G + V139L + Q245L S9R + A15T + T22L + V139L + Q245V + A254S S9R + A15T + T22TG + N62D + V139L + Q245G S9R + A15T + T22TG + N62D + V139L + Q245S S9R + A15T + T22TG + N62D + V139L + Q245V S9R + A15T + T22TL + G61E + L96LG + Q137D + Q245R S9R + A15T + T22TL + N62D + I107V + V139L + Q245W S9R + A15T + T22TL + N62D + Q245W S9R + A15T + T22TQ + S101P S9R + A15T + T22TW + N204D + Q245I S9R + A15T + S24P + G61E + A85T + P239S + Q245A S9R + A15T + V28I + V68A + Q245R + N252A S9R + A15T + T38S + A98R + S99C + G100S + H120N + P131S + Q137H S9R + A15T + G46D + V68A + N218D + Q245R S9R + A15T + F50S + H120D + P131H S9R + A15T + D60DG S9R + A15T + G61E + V68A + A98S + S99G + Q245R S9R + A15T + G61E + A85T + Q137H + Y209C + Q245G S9R + A15T + G61E + A85T + N218D + P239S + Q245L S9R + A15T + G61E + A85T + P239L + Q245C S9R + A15T + G61E + A85T + P239S + Q245C S9R + A15T + G61E + A85T + P239S + Q245V S9R + A15T + G61E + A98S + S99M + Q245R S9R + A15T + G61E + H120S + Q137D + V139L + N218D S9R + A15T + G61GP + Q245L S9R + A15T + N62D + H120N + P131T S9R + A15T + N62D + Q245R + N252M S9R + A15T + N62D + Q245W + N252S S9R + A15T + N62D + Q245W + N252V S9R + A15T + N62ND + V139L + Q245E S9R + A15T + N62ND + V139L + N261D S9R + A15T + N62NG + Q245T S9R + A15T + N62S + H120N + P131T + N218D S9R + A15T + V68A S9R + A15T + V68A + A98G + S99I + K237R + Q245R S9R + A15T + V68A + A98G + S99V + Q245R S9R + A15T + V68A + A98L + S99G + Q245R S9R + A15T + V68A + A98L + Q245R S9R + A15T + V68A + A98M + S99G + Q245R + T274A S9R + A15T + V68A + A98M + Q245R + N248D S9R + A15T + V68A + S99G + Q245R + N261D S9R + A15T + V68A + H120D + P131S + Q137H + Q245R S9R + A15T + V68A + H120N + P131S + Q137H + Q245M S9R + A15T + V68A + H120N + P131S + Q137H + Q245R S9R + A15T + V68A + H120N + N218D + Q245R S9R + A15T + V68A + Q137D S9R + A15T + V68A + A174V + Q245R S9R + A15T + V68A + A194T + Q245R + N252E S9R + A15T + V68A + N218D + Q245R S9R + A15T + V68A + Q245R S9R + A15T + V68A + Q245R + N252S S9R + A15T + N76S + L111V + P131H + Q137D S9R + A15T + N77S + L96LG + H120D + P131Q S9R + A15T + A88V + A98R + S99G + G100C + H120N + P131S + Q137H S9R + A15T + K94N + H120N + P131H S9R + A15T + L96LG + H120D + P131H + R186L S9R + A15T + L96LG + H120D + P131S + Q137E S9R + A15T + L96LG + H120D + G160D S9R + A15T + L96LG + H120N + P131H + Q137E S9R + A15T + L96LG + H120N + P131S S9R + A15T + L96LG + H120N + P131S + Q137H S9R + A15T + L96LG + H120N + P131S + Q137H + M222S S9R + A15T + L96LG + H120Q + Q137E S9R + A15T + L96LG + S130* S9R + A15T + L96LG + P131H + Q137D S9R + A15T + L96LG + P131Q + Q137D S9R + A15T + L96LG + P131T + Q137H S9R + A15T + L96LG + Q137E + Y209H S9R + A15T + G97GA + H120D + P131H + Q137E S9R + A15T + G97GA + H120D + P131S + Q137E S9R + A15T + G97GA + H120D + Q137D S9R + A15T + G97GA + H120D + Q137H + M222V S9R + A15T + G97GA + H120N + Q137D + N248D S9R + A15T + G97GA + H120N + Q137E S9R + A15T + G97GA + H120N + S212L S9R + A15T + G97GA + H120Q + P131H + Q137E S9R + A15T + G97GA + H120Y + Q137H S9R + A15T + G97GA + Q137D S9R + A15T + G97GA + Q137E S9R + A15T + G97GA + Q137E + L262V S9G + A15T + G97GA + Q137H S9R + A15T + G97GA + Q137H + N218S S9R + A15T + G97GA + R186C S9R + A15T + G97GG + L111I + P131T + Q137H S9R + A15T + G97GG + H120D + P131H + Q137H S9R + A15T + G97GG + H120D + Q137E S9R + A15T + G97GG + H120N + Q137D S9R + A15T + G97GG + P131S + Q137H S9R + A15T + G97GV + S106A + P131H S9R + A15T + G97GV + H120D + Q137H S9R + A15T + G97GV + H120E + Q137H S9R + A15T + G97GV + H120N S9R + A15T + G97GV + H120Q S9R + A15T + G97GV + H120Q + Y263F S9R + A15T + G97GV + P131H + Q137H S9R + A15T + G97GV + P131S S9R + A15T + G97GV + P131T + Q137H S9R + A15T + G97GV + Q137H S9R + A15T + A98C + G100S + H120N + P131S + Q137H S9R + A15T + A98G + S99C + H120N + P131S + Q137H S9R + A15T + A98S + S99G + G100S + H120N + P131S + Q137H S9R + A15T + A98S + G100S + H120N + P131S + Q137H S9R + A13T + S99A + S99SD + P131F S9R + A15T + S99C + H120N + P131S + Q137H + M222S S9R + A15T + S99G + G100S + H120N + P131S + Q137H S9R + A15T + G102S + M175T + Q245R + N252D S9R + A15T + L111I + H120N + Q137E S9R + A15T + L111I + Q137E S9R + A15T + L111V + Q137E + G211D S9R + A15T + H120D + Q137D S9R + A15T + H120G + Q137E + N218D S9R + A15T + H120N + P131T + N218D S9R + A15T + H120N + P131T + N218D + N269T S9R + A15T + H120Q + P131C + Q137H S9R + A15T + H120R + Q137D + N173S S9R + A15T + G127E + P131R + Q137H S9R + A15T + S130P S9R + A15T + P131H + S144P S9R + A15T + Y167I + R170L S9R + A15T + N218D S9R + A15T + T224A S9R + A15T + Q245F S9R + A15T + Q245L S9R + A15T + Q245S S9R + A15T + Q245V and S9R + A15T + T260M.
23. The variant of claim 16, wherein the parent subtilase belongs to the sub-group I-S1.
24. The variant of claim 16, wherein the parent subtilase belongs to the sub-group I-S2.
25. The variant of claim 16, wherein the parent subtilase is subtilisin 309.
26. The variant of claim 16, further comprising one 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+S101R+S103A+V1041+G160S, S3T+V4I+S99D+S100R+S103A+V104I+G160S+V199M+V205I+L217D, S3T+V4I+S99D+S100R+S103A+V104I+G160S+A194P+V199M+V205I+L217D, S3T+V4I+S99D+S100R+S103A+V104I+G160S+V2051 and N76D+V104A.
27. The variant of claim 16, further comprising the following substitutions: S101G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K.
28. A cleaning or detergent composition, comprising a variant of claim 16 and a surfactant.
29. A composition of claim 28, which additionally comprises one or more of an amylase, cellulase, cutinase, esterase, beta-galactosidase, glycoamylase, hemicellulase, lactase, ligninase, lipase, polygalacturonase, and protease.
30. An isolated DNA sequence encoding a protease variant of claim 16.
31. An expression vector comprising the isolated DNA sequence of claim 30.
32. A microbial host cell transformed with the expression vector of claim 31.
33. A microbial host cell of claim 32, which is a Bacillus.
34. A method for producing a protease variant, comprising
(a) culturing a host of claim 32 under conditions conducive to the expression and secretion of the variant, and
(b) recovering the protease variant.
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