# Patent

## Title
Subtilase variants

## Abstract

The present invention relates to novel subtilase variants exhibiting improvements 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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 11/482,424 filed on Jul. 7, 2006 (abandoned) and claims priority or the benefit under 35 U.S.C. 119 of Danish Application no. PA 2005 01007 filed Jul. 8, 2005 and U.S. Provisional Application No. 60/698,254 filed Jul. 11, 2005, the contents of which are fully incorporated herein by reference.


REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
FIELD 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 and catalytic activity. The variants of the invention are suitable for use in e.g., cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions. The present invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention. Further, the present invention relates to cleaning and detergent compositions comprising the variants of the invention.
BACKGROUND OF THE INVENTION
In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, 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 or naturally occurring wild type proteases, e.g., RELASE®, ALCALASE®, SAVINASE®, PRIMASE®, EVERLASE®, ESPERASE®, OVOZYME®, CORONOASE, POLARZYME® and KANNASE® (Novozymes A/S), MAXATASETM, MAXACALTM, MAXAPETM, PROPERASETM, PURAFECTTM, PURAFECT OXPTM, FN2TM, FN3TM, FN4TM and PURAFECT PRIMETM (Genencor International, Inc.), BLAP X and BLAP S (Henkel). Further, a number of protease variants are described in the art. A list of prior art protease variants is given in WO 99/27082.
However, even though a large 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 one or more of the modifications listed in Table 1.







TABLE 1





Modifications in subtilase variants.

















T143K, Y167A, R170S, A194P



Y167A, R170S, A194P, K251R



Y167A, R170S, A194P, S265K



Y167A, R170S, A194P, V244R



S141E, Y167A, R170S, A194P



Y167A, R170S, M175I



Y167A, R170S, A172T



Y167A, R170S, A174V, M175F



Y167A, R170S, A172V, A174V



Y167A, R170S, A172E



Y167A, R170S, M175L



Y167A, R170S, A174T



Y167A, R170S, A174T, M175L



G53C, G61E



A98S, S99D, G100S



S9R, T22A, V68A, S99A, *99Ad



S9R, P14H, R19L, N62D



G61P, *99aS



N43S, N62D



*96aG, P131S, V203A, A228T



N62D, A232C, Q236L, Q245N



*96aA, A98T, R247K



S99D, S101R, S103A, V104I, G160S, A194P, L217D



*61aD



N62D, S106A



V68A, S106M, N184D



S9R, A15T, *97aV, H120N



A15M, A16P, *99aD



*99aE, G160S, S163T, G195S, G211S, K237R, G258A, T260L



G23S, *99aD, A194P, S242T, Q245R



G100S, N173D



Y167A, R170S, A172E



A98T, Q137L, Y167A, R170S, M175L



*98aA, S99D



S99A, *99aD, V203A



N62D, K237R



V11M, N76D, L126F, K251R



S9F, A15L, A16P, T22I, *98aA, S99D, R170H



*96aA, *130aG, P131H



E54D, N62D



*98aA, *98bS, S99G, S101T



S9R, A15T, V68A, I79T, G102S, P131H, Q137H



*100aA, *100bG, *100cS, *100dG



V68A, L111I



*98aA, R170H, Q245R



I35V, N62D, N183D, T224S



*97aG, P131S, V203A, A228T



S9R, R10K, P14Q, T22A, Y167A, R170S



S9R, *22aL, S57A, G61E, *98aA, V139L, N173S



P14T, N18K, Y167A, R170S



S9R, Q12E, P14Q, K27R, Y167A, R170S



N62D, R170L



N62D, R170S, Q245R



Y167A, R170S, A194P, K251R, S265K



P14T, N18K, Y167A, R170S, A194P



N62D, A151G, K237R



N62D, A151G, Q245R



N62D, A151G, K237R, Q245R



S103A, V104I, G159D, A232V, Q236H, Q245R



S9R, A15T, T22A, V139L



S9R, A15T, G61E, A85T, E89Q, P239L, Q245C



S9R, A15T, V68A, H120N, Q245R



N248R



S9R, A15T, *22AI, V139L, N204D, Q245L



N218S



S9R, A15T, V68A, Q245R, N252K



S9R, A15T, V68A, Q245R, H120N



V68A, S106A, H120N



V68A, S106A, N252K



A15T, V68A, S99G, Q245R, N261D



S9R, V68A, S99G, Q245R, N261D



V68A, S99G, Q245R, N261D



S9R, A15T, V68A, S99G, N261D



S9R, A15T, V68A, Q245R, N261D



S9R, A15T, *22aL, V139L, S163G, N204D, Q245L



Q245R, N252H



S9R, *22aL, G61E, *97aA, M119I, Q137H, N173S



V68A, S106A, T213A



S9R, A15T, V68A, H120N, P131S, Q137H, Q245M



S9R, A15T, V68A, I72F, S99G, Q245R, N261D



S9R, A15T, V68A, S99D, Q245R, N261D



S9R, A15T, V68A, S99G, A194P, Q245R, N261D



S9R, A15T, V68A, N76I, S99G, Q245R, N261D



S9R, A15T, V68A, S99G, A228V, Q245R, N261D










The variants listed in Table 1 exhibit protease activity. Each position corresponds to a position of the amino acid sequence of subtilisin BPN′ set forth in FIG. 1 and SEQ ID NO: 1.
In a second aspect the present invention relates to an isolated polynucleotide encoding a subtilase variant of the invention.
In a third aspect the present invention relates to an expression vector comprising the isolated polynucleotide of the invention.
In a fourth aspect the present invention relates to a microbial host cell transformed with the expression vector of the invention.
In a fifth aspect the present invention relates to a method for producing a subtilase variant according to the invention, comprising culturing a host according to the invention under conditions conducive to the expression and secretion of the variant, and recovering the variant.
In a sixth aspect the present invention relates to a cleaning or detergent composition, preferably a laundry or dish wash composition, comprising the variant of the invention. 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, which is herein 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, which is herein 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® which is marketed by Novozymes A/S 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”.

Modification(s) of a subtilase variant. The term “modification(s)” used herein is defined to include chemical modification of a subtilase as well as genetic manipulation of the DNA encoding a subtilase. The modification(s) can be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.

Subtilase variant. In the context of this invention, the term subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host.

Homologous subtilase sequences. The homology between two amino acid sequences is in this context described by the parameter “identity”. In order to determine the degree of identity between two subtilases the GAP routine of the GCG package version 9.1 can be applied (infra) using the same settings. The output from the routine is besides the amino acid alignment the calculation of the “Percent Identity” between the two sequences. Based on this description it is routine for a person skilled in the art to identify suitable homologous subtilases, which can be modified according to the invention.

Isolated polynucleotide. The term “isolated”, when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5′ and 3′ untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985). The term “an isolated polynucleotide” may alternatively be termed “a cloned polynucleotide”.

Isolated protein. When applied to a protein, the term “isolated” indicates that the protein has been removed from its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e., “homologous impurities” (see below)). An isolated protein is more than 10% pure, preferably more than 20% pure, more preferably more than 30% pure, as determined by SDS-PAGE. Further it is preferred to provide the protein in a highly purified form, i.e., more than 40% pure, more than 60% pure, more than 80% pure, more preferably more than 95% pure, and most preferably more than 99% pure, as determined by SDS-PAGE. The term “isolated protein” may alternatively be termed “purified protein”.

Homologous impurities. The term “homologous impurities” means any impurity (e.g., another polypeptide than the subtilase of the invention), which originate from the homologous cell where the subtilase of the invention is originally obtained from.

Obtained from. The term “obtained from” as used herein in connection with a specific microbial source, means that the polynucleotide and/or subtilase produced by the specific source, or by a cell in which a gene from the source has been inserted.

Substrate. The term “substrate” used in connection with a substrate for a protease should be interpreted in its broadest form as comprising a compound containing at least one peptide (amide) bond susceptible to hydrolysis by a subtilisin protease.

Product. The term “product” used in connection with a product derived from a protease enzymatic reaction should, in the context of the present invention, be interpreted to include the products of a hydrolysis reaction involving a subtilase protease. A product may be the substrate in a subsequent hydrolysis reaction.

Wash Performance. In the present context the term “wash performance” is used as an enzyme's ability to remove proteinaceous or organic stains present on the object to be cleaned during e.g., wash or hard surface cleaning. See also the wash performance test in Example 3 herein.




BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 shows an alignment between subtilisin BPN′ (a) and SAVINASE® (b) using the GAP routine mentioned above.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to novel subtilase variants exhibiting alterations relative to the parent subtilase in one or more properties including: Wash performance, thermal stability, storage stability or catalytic activity. Variants which are contemplated as being part of the invention are such variants where, when compared to the wild-type subtilase, one or more amino acid residues have been modified by substitution, deletion or insertion. The variants of the present invention comprise one or more of those modifications listed in Table 1.
The variants listed in Table 1 exhibit protease activity, and each position corresponds to a position of the amino acid sequence of subtilisin BPN′ showed in FIG. 1 and SEQ ID NO: 1.
A subtilase variant of the first aspect of the invention may be a parent or wild-type subtilase identified and isolated from nature. Such a parent wild-type subtilase may be specifically screened for by standard techniques known in the art.
One preferred way of doing this may be by specifically PCR amplify conserved DNA regions of interest from subtilases from numerous different microorganism, preferably different Bacillus strains.
Subtilases are a group of conserved enzymes, in the sense that their DNA and amino acid sequences are homologous. Accordingly it is possible to construct relatively specific primers flanking the polynucleotide sequences of interest.
Using such PCR primers to amplify DNA from a number of different microorganisms, preferably different Bacillus strains, followed by DNA sequencing of said amplified PCR fragments, it will be possible to identify strains which produce subtilase variants of the invention. Having identified the strain and a partial DNA sequence of such a subtilase of interest, it is routine work for a person skilled in the art to complete cloning, expression and purification of such a subtilase. However, it is envisaged that a subtilase variant of the invention is predominantly a variant of a parent subtilase.
A subtilase variant suitable for the uses described herein may be constructed by standard techniques known in the art such as by site-directed/random mutagenesis or by DNA shuffling of different subtilase sequences. See the “Material and Methods” section and Example 1 herein for further details.
As will be acknowledged by the skilled person, the variants described herein may comprise one or more additional modifications, in particular one or more additional 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. 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, S101R, S103A, V104A, V104I, V104N, V104Y, S106A, H120D, H120N, N123S, G159D, Y167A, R1705, R170L, A194P, N204D, V205I, Q206E, L217D, N218S, N218D, M222S, M222A, T224S, A232V, K235L, Q236H, Q245R, N248D, N252K and T274A.
Furthermore variants comprising any of the modifications S101G+V104N, S87N+S101G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V104I, S99D+S101R+S103A+V104I+G160S, S3T+V4I+S99D+S101R+S103A+V104I+G160S+V199M+V205I+L217D, S3T+V4I+S99D+S101R+S103A+V104I+G160S+A194P+V199M+V205I+L217D, S3T+V4I+S99D+S101R+S103A+V104I+G160S+V205I or N76D+V104A, 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, R1705, R170L, A194P, N204D, V205I, Q206E, L217D, N218D, N218S, 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 a modification 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.
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 et al. (eds.) “Current protocols in Molecular Biology”. John Wiley and Sons, 1995; Harwood and Cutting (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 Cell
The DNA sequence encoding the present enzyme introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e., produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. The term “homologous” is intended to include a DNA sequence encoding an enzyme native to the host organism in question. The term “heterologous” is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence 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. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium 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.
Method for Producing a Subtilase Variant
The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
When an expression vector comprising a DNA sequence encoding the enzyme is transformed 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.
Detergent Applications
The enzyme of the invention may be added to and thus become a component of a detergent composition. 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, 68, 76, 87, 97, 101, 104, 106, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, 245, 252 and 274. Preferred commercially used protease enzymes include RELASE®, ALCALASE®, SAVINASE®, PRIMASE®, EVERLASE®, ESPERASE®, OVOZYME®, CORONASE®, POLARZYME® and KANNASE® (Novozymes A/S), MAXATASETM, MAXACALTM, MAXAPEMTM, PROPERASETM, PURAFECTTM, PURAFECT OXPTM, FN2TM, FN3TM, FN4TM and PURAFECT PRIMETM (Genencor International, Inc.), BLAP X and BLAP S (Henkel).
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 407225, EP 260105, 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 used lipase enzymes include LIPOLASE®, LIPOLASE ULTRA® and LIPEX® (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, α-amylases obtained from Bacillus, e.g., a special strain of B. licheniformis, described in more detail in GB 1,296,839. Examples of useful 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 used amylases are DURAMYL®, TERMAMYL®, STAINZYME®, FUNGAMYL® and BAN® (Novozymes A/S), RAPIDASETM, PURASTARTM and PURASTAR OXAMTM (from Genencor International Inc.).
Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases having colour care and whiteness maintenance 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 used cellulases include RENOZYME®, CELLUZYME®, and CAREZYME® (Novozymes A/S), CLAZINASETM, and PURADEX HATM (Genencor Int. Inc.), and KAC-500(B)TM (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 used peroxidases include GUARDZYMETM (Novozymes A/S).

Hemicellulases: Suitable hemicellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable hemicellulases include mannanase, lichenase, xylanase, arabinase, galactanase acetyl xylan esterase, glucorunidase, ferulic acid esterase, coumaric acid esterase and arabinofuranosidase as described in WO 95/35362. Suitable mannanases are described in WO 99/64619.

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 (polyethylene glycol, 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, a gel 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, alkylpoly-glycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, 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, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic 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(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, 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 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.
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.
It is at present contemplated that in the detergent compositions any enzyme, in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liquor, preferably 0.05-5 mg of enzyme protein per liter of wash liquor, in particular 0.1-1 mg of enzyme protein per liter of wash liquor.
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










OTHER APPLICATIONS
The subtilase variants of the present invention may be used in the processing of food, especially in the field of diary products, such as milk, cream and cheese, but also in the processing of meat and vegetables. The subtilase variants of the present invention may also be used in the processing of feed for cattle, poultry, and pigs and especially for pet food. Further, the subtilase variants of the invention may be used for the treatment of hides. The subtilase variants of the invention may also be used in processes for decontaminating instruments, surfaces, and other materials in hospitals, clinics, and meat processing plants, etc. in order to decompose prions or other infectious agents.
Materials and Methods
Method for Producing a Protease 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 transformed 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 RP-II protease 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 variant 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.
Proteolytic Activity
Enzyme activity can be measured using the PNA assay using succinyl-alanine-alanine-proline-glutamicacid-paranitroaniline as a substrate. The principle of the PNA assay is described in the Journal of American Oil Chemists Society, Rothgeb, T. M., Goodlander, B. D., Garrison, P. H., and Smith, L. A., (1988).
Textiles
Standard textile pieces are obtained from EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland or CFT, Center For Testmaterials, Vlaardingen, Netherlands. Especially important are EMPA 116 (cotton textile stained with blood, milk and ink), EMPA 117 (polyester/cotton textile stained with blood, milk and ink), C-03 (cotton textile stained with chocolate milk and soot), C-05 (cotton textile stained with blood, milk and ink) and C-10 (cotton textile stained with milk, oil and pigment).
Wash Conditions













Region













Latin
North

Asia excl.




America
America
Europe
Japan
Japan
















Temperature
20-25° C.
20-32° C.
30-60° C.
15-30° C.
15-20° C.


Washing
14-16 min
12 min
20-40 min
14-20 min
15 min


time


Water
6-12° dH
6° dH
15° dH
14° dH
3° dH


hardness*


Detergent
1.5-4 g/l
1.0-1.5 g/l
4-10 g/l
1.5-2.5 g/l
0.5-0.7 g/l


dosage


Washing pH
As it is
As it is
As it is
As it is
As it is





*° dH: adjusted by adding CaCl2*2H2O, MgCl2*6H2O and NaHCO3 to Milli-Q water.






Detergents

The enzymes of the invention may be tested in the detergent formulations disclosed in WO 97/07202 or in the detergent examples above. Further, tests could be done in detergents formulations purchased from wfk testgewebe GmbH (Germany) or similar supplier, or in commercial detergents.
List of test detergents from wfk testgewebe:

    
    
        IEC 60456 Type A* Base Detergent
        IEC 60456 Type B Base Detergent
        IEC 60456 Type C Detergent
        ECE Reference Detergent with Phosphate (1977)
        ECE Reference Detergent without Phosphate (1998)
        AHAM Standard Detergent
        EU ECOLABEL (detergents) Light Duty Detergent
        EU ECOLABEL (detergents) PVP
    
    


However, also one of the following commercial detergents may be used in the wash assay, e.g., Ariel HDP, P&G, Mexico; Omo Multi Acao HDP, Unilever, Brazil; Breeze HDP, Unilever Thailand; Diao Pai, Nice, China; Tide HDL, P&G, US; Wisk HDL, Unilever, US; TOP HDP, Lion, Japan; Attack HDP, Kao, Japan; Ariel Regular HDP, P&G, Europe; Ariel Compact HDPC, P&G, Europe; Persil Megaperls, Henkel, Germany and Persil, Unilever, UK.
Furthermore, a brand extension or color/compact version for the above specified detergent could be used as well.
If the detergent contains enzymes, the detergent should be in-activated before use in order to eliminate the enzyme activity already present in the detergent. This is done by heating a detergent stock solution to 85° C. in 5 minutes in a micro wave oven. The concentration of the detergent stock solution to be inactivated in the micro wave oven is 4-20 g/l.
Automatic Mechanical Stress Assay
The Automatic Mechanical Stress Assay (AMSA) is described in Example 3 below.
Mini Wash Assay
The milliliter scale wash performance assay is conducted under the following conditions:













Detergent
Latin American HDP


Detergent dose
1.5-4 g/l


pH
As it is


Wash time
14-16 min.


Temperature
20-25° C.


Water hardness
6-12°dH, adjusted by adding CaCl2*2H2O,



MgCl2*6H2O and NaHCO3 to milli-Q water.


Enzyme conc.
5 nM, 10 nM, 30 nM


Test system
125 ml glass beakers. Textile dipped in test



solution.



Continuously lifted up and down into the



detergent solution, 50 times per minute.


Test solution volume
50 ml









After washing the textile piece is flushed in tap water and air-dried and the remission (R) of the test material is measured at 460 nm using a Zeiss MCS 521 VIS spectrophotometer. The measurements are done according to the manufacturer's protocol.
The performance of the new variants is compared to the performance of Savinase by calculating the relative performance:

RP=(Rvariant−RBLANK)/(RSAVINASE−RBLANK)

A variant is considered to exhibit improved wash performance, if it performs better than the reference in at least one detergent composition.
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 oligonucleotides containing the desired mutations.
Briefly, plasmid DNA pSX222 (E. coli/B. subtilis shuttle vector including appropriate selection marker, origins of replication for Bacillus and E. coli, digestion sites, etc. disclosed in WO 96/34946) bearing the subtilisin 309 wild-type or a subtilisin 309 variant gene is used as template in the PCR reaction. In a first PCR an oligonucleotide containing the desired mutation (anti-sense) and a suitable opposite oligonucleotide (sense) is used. The resulting DNA fragment is used as a sense oligonucleotide in a second PCR together with a suitable anti-sense oligonucleotide. The resulting DNA fragment is digested with suitable restriction enzymes and ligated into a suitable E. coli/Bacillus shuttle vector (e.g., pSX222) digested with the same enzymes.
The ligation product is transformed into competent E. coli and plated on a solid agar containing an appropriate selection marker. DNA purified from a single colony is sequenced to confirm the designed mutation. Plasmid DNA is isolated from E. coli cells bearing plasmids containing subtilisin 309 genes with the designed mutation and is transformed into a suitable competent B. subtilis strain, i, B. subtilis DN1885: Disclosed in WO 01/16285 (e.g., as described by Dubnau et al., 1971, J. Mol. Biol. 56:209-221) and plated on a solid agar containing an appropriate selection marker.
Plasmid DNA from single Bacillus colonies showing protease activity is isolated and sequenced to confirm the designed mutation. Bacillus colonies bearing plasmid DNA including subtilisin 309 genes with the desired mutations are fermented in baffled shake flasks in a suitable media.
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 1 herein was purified according to the above procedure and subsequently the enzyme concentration was determined.
Known concentrations of the variants of Table 1 were tested for wash performance in detergent as described below.
Example 3
Wash Performance of Detergent Composition Comprising Subtilase Variants
The enzyme variants of the present application 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 page 23-24. The assay is conducted under the experimental conditions specified below.













Detergent
Latin American type HDP


Detergent dosage
2.2 g/l


Test solution volume
160 micro l


pH
Adjusted to pH 9.5-10.5 with



NaHCO3.


Wash time
14 minutes


Temperature
20° C.


Water hardness
9° dH*


Enzyme concentration in test solution
5 nM, 10 nM and 30 nM


Test material
C-10





*° dH: adjusted by adding CaCl2*2H2O; MgCl2*6H2O (Ratio Ca2+:Mg2+− = 2:1) to milli-Q water.






The Latin American type detergent was composed according to the provisions in Detergent Example 1 herein. After washing the textile pieces are 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 1T8 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 (r), green (g) and blue (b). The intensity value (Int) is calculated by adding the (r), (g) and (b) values together as vectors and then taking the length of the resulting vector:

Int=√{square root over (r2+g2+b2)}.

The wash performance (P) of a variant is defined as the light intensity value of textile surface washed with enzyme variant:

P=Int(v)

The results are presented in Table 2 where the performance is given as relative performance of a new variant versus the performance of Savinase at 10 nM protease concentration:

RP is (PVARIANT−PBLANK)/(PSAVINASE−PBLANK)








TABLE 2







Wash performance test results with subtilase variants


relative to the performance of Savinase.









Relative


Mutations in variant
performance





T143K, Y167A, R170S, A194P
1.8


Y167A, R170S, A194P, K251R
1.6


Y167A, R170S, A194P, S265K
2.0


Y167A, R170S, A194P, V244R
1.9


S141E, Y167A, R170S, A194P
1.1


Y167A, R170S, M175I
1.2


Y167A, R170S, A172T
1.2


Y167A, R170S, A174V, M175F
1.4


Y167A, R170S, A172V, A174V
1.5


Y167A, R170S, A172E
1.3


Y167A, R170S, M175L
1.5


Y167A, R170S, A174T
1.2


Y167A, R170S, A174T, M175L
1.4


G53C, G61E
1.3


A98S, S99D, G100S
1.4


S9R, T22A, V68A, S99A, *99aD
1.7


S9R, P14H, R19L, N62D
1.8


G61P, *99aS
1.7


N43S, N62D
1.7


*96aG, P131S, V203A, A228T
1.8


N62D, A232C, Q236L, Q245N
2.0


*96aA, A98T, R247K
2.0


S99D, S101R, S103A, V104I, G160S, A194P,
1.4


L217D


*61aD
1.3


N62D, S106A
2.2


V68A, S106M, N184D
1.6


S9R, A15T, *97aV, H120N
1.4


A15M, A16P, *99aD
1.6


*99aE, G160S, S163T, G195S, G211S, K237R, G258A,
1.2


T260L


G23S, *99aD, A194P, S242T, Q245R
1.5


G100S, N173D
1.4


Y167A, R170S, A172E
1.1


A98T, Q137L, Y167A, R170S, M175L
1.1


*98aA, S99D
1.8


S99A, *99aD, V203A
1.8


N62D, K237R
2.1


V11M, N76D, L126F, K251R
1.4


S9F, A15L, A16P, T22I, *98aA, S99D, R170H
1.2


*96aA, *130aG, P131H
1.5


E54D, N62D
2.0


*98aA, *98bS, S99G, S101T
1.8


S9R, A15T, V68A, I79T, G102S, P131H, Q137H
1.7


*100aA, *100bG, *100cS, *100dG
1.7


V68A, L111I
1.8


*98aA, R170H, Q245R
1.8


I35V, N62D, N183D, T224S
1.2


*97aG, P131S, V203A, A228T
1.4


S9R, R10K, P14Q, T22A, Y167A, R170S
1.6


S9R, *22aL, S57A, G61E, *98aA, V139L, N173S
1.3


P14T, N18K, Y167A, R170S
2.0


S9R, Q12E, P14Q, K27R, Y167A, R170S
1.7


N62D, R170L
1.7


N62D, R170S, Q245R
1.4


Y167A, R170S, A194P, K251R, S265K
2.0


P14T, N18K, Y167A, R170S, A194P
2.0


N62D, A151G, K237R
2.0


N62D, A151G, Q245R
1.9


N62D, A151G, K237R, Q245R
2.0









Example 4
Wash Performance of Detergent Composition Comprising Subtilase Variants
The milliliter scale wash performance assay was conducted under the following conditions:
Mini Wash Assay













Detergent
Persil, Lever, UK, HDP


Detergent dose
6 g/l


pH
As it is


Wash time
20 min.


Temperature
30° C.


Water hardness
15° dH, adjusted by adding CaCl2*2H2O,



MgCl2*6H2O and NaHCO3 (4:1:7.5) to



milli-Q water.


Enzyme conc.
2.5 nM, 5 nM, 10 nM, 30 nM, 60 nM


Test system
125 ml glass beakers. Textile dipped in test



solution.



Continuously lifted up and down into the



detergent solution, 50 times per minute.



Swatch used: EMPA 116 (2.5 cm × 7 cm)


Test solution volume
50 ml









After washing the textile piece is flushed in tap water and air-dried and the remission (R) of the test material is measured at 460 nm using a Zeiss MCS 521 VIS spectrophotometer. The measurements are done according to the manufacturer's protocol.
The performance of the new variants is compared to the performance of Savinase at 10 nM protease concentration by calculating the relative performance:

RP−(Rvariant−RBLANK)/(RSAVINASE−RBLANK)

A variant is considered to exhibit improved wash performance, if it performs better than the reference in at least one detergent composition.
Scoring: A score=1 is given for variants with an improved wash performance equal to or better than 1.1.














Mutations
Score









S103A, V104I, G159D, A232V, Q236H, Q245R
1



S9R, A15T, T22A, V139L
1



S9R, A15T, G61E, A85T, E89Q, P239L, Q245C
1



S9R, A15T, V68A, H120N, Q245R
1



N248R
1



S9R, A15T, *22aL, V139L, N204D, Q245L
1



N218S
1



S9R, A15T, V68A, Q245R, N252K
1



S9R, A15T, V68A, Q245R, H120N
1



V68A, S106A, H120N
1



V68A, S106A, N252K
1



A15T, V68A, S99G, Q245R, N261D
1



S9R, V68A, S99G, Q245R, N261D
1



V68A, S99G, Q245R, N261D
1



S9R, A15T, V68A, S99G, N261D
1



S9R, A15T, V68A, Q245R, N261D
1



S9R, A15T, *22aL, V139L, S163G, N204D, Q245L
1



Q245R, N252H
1



S9R, *22aL, G61E, *97aA, M119I, Q137H, N173S
1



V68A, S106A, T213A
1



S9R, A15T, V68A, H120N, P131S, Q137H, Q245M
1



S9R, A15T, V68A, I72F, S99G, Q245R, N261D
1



S9R, A15T, V68A, S99D, Q245R, N261D
1



S9R, A15T, V68A, S99G, A194P, Q245R, N261D
1



S9R, A15T, V68A, N76I, S99G, Q245R, N261D
1



S9R, A15T, V68A, S99G, A228V, Q245R, N261D
1

## Claims

1. A variant of a subtilisin 309 comprising the following set of modifications:
S9R, A15T, V68A, S99G, A228V, Q245R, N261D;

wherein the variant has protease activity and each position corresponds to a position of the amino acid sequence of SEQ ID NO: 1.

2. The variant of claim 1, which further comprises one or more of the modifications K27R, *36D, S56P, N62D, N76D, S87N, G97N, S101G, S101R, S103A, V104A, V104I, V104N, V104Y, S106A, H120D, H120N, N123S, G159D, Y167A, R1705, R170L, A194P, N204D, V205I, Q206E, L217D, N218S, N218D, M222S, M222A, T224S, A232V, K235L, Q236H, N248D, N252K, T274A, S101G+V104N, S87N+S101G+V104N, K27R+V104Y+N123S+T274A, and N76D+S103A+V104I.

3. A cleaning or detergent composition, comprising a variant of claim 1 and a surfactant.

4. The composition of claim 3, which additionally comprises a cellulase, a lipase, an amylase, a cutinase, a protease, a hemicellulase, an esterase, a lactase, a glycoamylase, a polygalacturonase, a beta-galactosidase, a ligninase, or a mixture thereof.

5. An isolated DNA sequence encoding a subtilase variant of claim 1.

6. An expression vector comprising the isolated DNA sequence of claim 5.

7. A microbial host cell transformed with the expression vector of claim 6.

8. The microbial host cell of claim 7, which is a bacterium.

9. A method for producing a subtilase variant, comprising
(a) culturing a host cell of claim 7 under conditions conducive to the expression and secretion of the variant, and
(b) recovering the variant.