EP1608744A2 - Subtilases - Google Patents

Subtilases

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Publication number
EP1608744A2
EP1608744A2 EP04722236A EP04722236A EP1608744A2 EP 1608744 A2 EP1608744 A2 EP 1608744A2 EP 04722236 A EP04722236 A EP 04722236A EP 04722236 A EP04722236 A EP 04722236A EP 1608744 A2 EP1608744 A2 EP 1608744A2
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EP
European Patent Office
Prior art keywords
atom
subtilase
variant
parent
amino acid
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EP04722236A
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German (de)
English (en)
Inventor
Allan Svendsen
Stefan Minning
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Novozymes AS
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Novozymes AS
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Priority to EP09152523A priority Critical patent/EP2163616A3/fr
Publication of EP1608744A2 publication Critical patent/EP1608744A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates to JP170 and BPN' like subtilases and to methods of construction such variants with altered properties, such as stability (e.g. thermostability or storage stability), Ca 2+ dependency, pH dependent activity, improved performance in washing and cleaning applications.
  • stability e.g. thermostability or storage stability
  • Ca 2+ dependency e.g. Ca 2+ dependency
  • pH dependent activity e.g. Ca 2+ dependency
  • improved performance in washing and cleaning applications e.g. thermostability or storage stability
  • Enzymes have been used within the detergent industry as part of washing formula- tions for more than 30 years. Proteases are from a commercial perspective the most relevant enzyme in such formulations, but other enzymes including lipases, amylases, cellu- lases or mixtures of enzymes are also often used.
  • proteases w ith altered properties, such as i ncreased activity at low tempera- tures, increased thermostability, increased specific activity at a given pH, altered Ca 2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
  • properties such as i ncreased activity at low tempera- tures, increased thermostability, increased specific activity at a given pH, altered Ca 2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
  • proteases with altered properties include both discovery of naturally occurring proteases, i.e. so called wild-type proteases but also alteration of well-known proteases by e.g. genetic manipulation of the nucleic acid sequence encoding said proteases.
  • Knowledge of the relationship between the three-dimensional structure and the function of a protein has improved the ability to evaluate which areas of a protein to alter to affect a specific characteristic of the protein.
  • subtilases One family of p roteases, which are often u sed i n d etergents, a re the subtilases. This family has previously been further grouped into 6 different sub-groups by Siezen RJ and Leunissen JAM, 1997, Protein Science, 6, 501-523.
  • One of these sub-groups is the Subtilisin family which includes subtilases such as BPN', subtilisin 309 (SAVINASE ® , NOVOZYMES A/S), subtilisin Carlsberg (ALCALASE ® , NOVOZYMES A/S), subtilisin S41 (a subtilase from the psychrophilic Antarctic Bacillus TA41 , Davail S et al.
  • s ubtilisin S 39 (a s ubtilase from the psychrophilic Antarctic Bacillus TA39, Narinx E et al. 1997, Protein Engineering, 10 (11 ), pp. 1271-1279) and TY145 (a subtilase from Bacillus sp. TY145, NCIMB 40339 described in WO 92/17577).
  • subtilases belonging to the Subtilisin subgroup were made based on primary sequence alignments, with only little consideration of three-dimensional structure.
  • modelling of the three-dimensional structure of one subtilase on the basis of the three-dimensional structure of another subtilase may result in an incorrect three-dimensional model structure because of structural differences.
  • subtilisin BPN' that was used by Siezen and Leunis- sen
  • the three-dimensional structure of subtilase TY145 have been elucidated and it was found that there are several differences between this and the three-dimensional structure of BPN' also belonging to the Subtilisin subgroup of subtilases (PCT/DK2004/000066).
  • subtilase JP170 and subtilases similar to JP170 are already known in the art, but the three-dimensional structure has not been disclosed for such subtilases.
  • the JP170 subtilase was described as protease A in WO 88/01293 to Novozymes.
  • Figure 1 shows an alignment of three JP170 type proteases: (a) SD-521 (EP 1 209 233), (b) protease Y a (WO 99/67370), and (c) JP170 (WO 98/56927, mature sequence from Appendix 1 ).
  • Figure 2 shows a superposition of the 3D structures of the proteases JP170 and Savinase ( BLSAVI), with i ndication of calcium b inding s ites.
  • I n the figure J P170 i s i ndicated in light grey with three ion-binding sites, and Savinase in a dark structure with two ion-binding sites.
  • Figure 3 shows a matrix of homology between amino acid sequences of subtilases pertaining to various subtilase subgroups.
  • the sequences are identified by sequence database accession numbers and their derivation. 1 : aam50084; Subtilase derived from Bacillus sp. strain SD-521 2: aaw89547; Subtilase derived from Bacillus sp. JP170 3: q45681 ; Subtilase derived from B. subtilis (BSTA41 )
  • sequences 6 to 11 belong to the "true subtilisins” or I-S1 type
  • sequences 12 to 17 belong to the "highly alkaline” subtilisins or I-S2 type. From Fig. 3 it is clear that these types are quite distinct.
  • Figure 4 shows a three-dimensional alignment of the subtilases: (1 ) Ty145; (2) BPN'; (3) Savinase; and (4) JP170.
  • 3D sequences is meant that the position of homologous residues are chosen by superposition of the 3D structures and subsequently the amino acid sequences are aligned based on these homologous positions.
  • APPENDIX 1 s hows the structural coordinates for the solved crystal structure of JP170.
  • Subtilase JP170 (SEQ ID NO:1) Subtilase Y (SEQ ID NO:2) Subtilase SD-521 (SEQ ID NO:3) Subtilase BPN" (SEQ ID NO:4) Partiel sequence (SEQ ID NO:5) Partiel sequence (SEQ ID NO:6) Subtilase TY145 (SEQ ID NO:7)
  • subtilase JP170 This subtilase has large structural differences to the structures of the subtilisins BPN' and TY145.
  • subtilases having a JP170 type structure and subtilases having a BPN' type structure to obtain variants with improved properties.
  • the variants have altered properties, such as in- creased activity at low temperatures, increased thermostability, increased specific activity at a given pH, altered Ca 2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
  • the object of the present invention is to provide a method for constructing subtilases having altered properties, in particular to provide a method for constructing subtilases having altered properties as described above.
  • the present invention relates to a method for constructing a variant of a parent subtilase, wherein the variant has at least one altered property as compared to said parent subtilase, which method comprises: a) analyzing the three-dimensional structure of the subtilase to identify, on the basis of an evaluation of structural considerations in relation to a JP170 three dimensional structure, at least one amino acid residue or at least one structural region of the subtilase, which is of relevance for altering said property; b) modifying the DNA of the polynucleotide encoding the parent to construct a polynucleotide encoding a variant subtilase, which in comparison to the parent sub- tilase, has been modified by deletion, substitution or insertion of the amino acid residue or structural part identified in i) so as to alter said property; c) expressing the variant subtilase in a suitable host, and d) testing the resulting subtilase variant for said property.
  • the invention relates to a method of producing a subtilase variant, wherein the variant has at least one altered property as compared to a parent subtilase, which method comprises: a) producing a model structure of the parent subtilase on the three-dimensional structure of BPN', TY145 or JP170; or producing an actually determined three- dimensional structure of the parent subtilase, b) comparing the model or actual three-dimensional structure of the parent subtilase to the JP170 structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step b) at least one structural part of the parent subtilase, wherein an alteration in said structural part is predicted to re- suit in an altered property; d) modifying the n ucleic a cid sequence e ncoding the p arent subtilase to p roduce a nucleic acid sequence encoding at
  • modification of the parent subti- lase in certain regions and/or positions is expected to confer a particular effect to the thus produced subtilase variant
  • modification of the parent subtilase in any of such regions may also give rise to any other of the above-mentioned effects.
  • any of the regions and/or positions mentioned as being of particular interest with respect to, e.g., improved thermostability may also give rise to, e.g., higher activity at a lower pH, an altered pH optimum, or increased specific activity, such as increased pepti- dase activity.
  • variants of a subtilase t_he DNA encoding such variants and methods of preparing the variants.
  • Still further aspect s of the present invention relates to the use of the variants for various industrial purposes, in particular as an additive in detergent compositions.
  • Other aspects of the present invent tion will be apparent from the below description as well as from the appended claims.
  • subtilases refer to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
  • Serine proteases or serine peptidases is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate.
  • subtilases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue.
  • Subtilases are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases.
  • the subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
  • the Subtilisin family (EC 3.4.21.62) may be further divided into 3 sub-groups, i.e. I-
  • subtilisins S1 ("true” subtilisins), I-S2 (highly alkaline proteases) and intracellular subtilisins. Definitions or grouping of enzymes may vary or change, however, in the context of the present invention the above division of subtilases into sub-division or sub-groups shall be understood as those described by Siezen et al., Protein Engng. 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
  • parent is in the context of the present invention to be understood as a protein, which is modified to create a protein variant.
  • the parent protein may be a naturally occurring (wild-type) polypeptide or it may be a variant thereof prepared by any suitable means.
  • the parent protein may be a variant of a naturally occurring protein which has been modified by substitution, chemical modification, deletion or truncation of one or more amino acid residues, or by addition or insertion of one or more amino acid residues to the amino acid sequence, of a naturally-occurring polypeptide.
  • parent subtilase refers to a subtilase which is modified to create a subtilase variant.
  • variant is in the context of the present invention to be understood as a protein which has been modified as compared to a parent protein at one or more amino acid residues.
  • modification(s) or “modified” is in the context of the present invention to be understood as to include chemical modification of a protein as well as genetic manipulation of the DNA encoding a protein.
  • the modification(s) may 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.
  • modified protein e.g. "modified subtilase”
  • modified protein e.g. subtilase
  • subtilase is to be understood a s a protein which contains modification(s) compared to a parent p rotein, e.g. subtilase.
  • position is in the context of the present invention to be understood as the number of an amino acid in a peptide or polypeptide when counting from the N- terminal end of said peptide/polypeptide.
  • the position numbers used in the present invention refer to different subtilases depending on which subgroup the subtilase belongs to. As m entioned above the alkaline subtilases KP43, KP1790, KP9860, Y, SD-521 and E1 belong to the JP170 subgroup, based on sequence homology. Due to the exten- sive homology only subtilase Ya and SD-521 are in Fig. 1 aligned with JP170.
  • the JP170 subtilase, Y subtilase and SD-521 subtilase are numbered according to SEQ ID NO:1 , SEQ ID NO:2 and SEQ ID NO:3, respectively.
  • the invention is not limited to variants of these particular subtilases but extends to parent subtilases, especially of the JP170 type, containing amino acid residues at positions which are "equivalent" to the particular identified residues in the JP170 subtilase.
  • a residue (amino acid) position of a JP170 type subtilase is equivalent to a residue (position) of the JP170 subtilase, if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in the JP170 subtilase (i.e., having the same or similar functional capacity to combine, react, or interact chemically).
  • the amino acid sequence of a precursor protease is directly compared to the JP170 subtilase primary sequence by aligning t he a mino a cid s equence of an i solated o r p arent wild type e nzyme with a suitable well-known (standard) enzyme of the same group or class of enzymes to define a frame of reference.
  • a suitable well-known (standard) enzyme of the same group or class of enzymes to define a frame of reference.
  • This type of numbering has been used in numerous patent applications relating to subtilisins of the I-S1 and I-S2 subgroups with subtilisin BPN' as the standard subtilisin. If nothing else is indicated herein, in the present instance the JP170 subtilase has been chosen as standard.
  • the 3D structure based a lignment i n F ig. 1 h as been provided.
  • the amino acid sequence of a precursor JP170 type subtilase may be directly correlated to the JP170 primary sequence.
  • the (3D based) position corresponding to a position in JP170 is found by i) identifying the JP170 type subtilase from the alignment of Fig.
  • the alignment may define a number of deletions and insertions in relation to the sequence of JP170. In the alignment deletions are indicated by asterixes ( * ) in the referenced sequence, and the referenced enzyme will be considered to have a gap at thie position in question.
  • Insertions are indicated by asterixes (*) in the JP170 sequence, and the positions in the referenced enzyme are given as the position number of the last amino acid residue where a corresponding amino acid residue exists in the standard enzyme with a lower case letter appended in alphabetical order, e.g. 82a, 82b, 82c, 82d.
  • the referenced enzyme contains a N- or C-terminal extension in comparison to JP170; an N-terminal extension is given the position number 0a, 0b, etc. in the di rection of the N-terminal; and a C-terminal extension will be given either the position number of the C-terminal amino acid residue of JP170 with a lower case letter appended in alphabetical order, or simply a continued consecutive numbering.
  • JP170 type subtilases are numbered by reference to the positions of the JP170 subtilase (SEQ ID NO: 1 ) as provided in Fig. 1. The position is then indicated as "corresponding to JP170".
  • Subtilases belonging to the BPN' subgroup refers to the positions of Subtilisin Novo
  • Subtilases belonging to the TY145 subgroup refers to the positions of the TY145 subtilase (SEQ ID NO:7), see also PCT/DK2004/000066.
  • subtilases Despite the great homology of the subtilases described above the inventors of the present invention have elucidated the three-dimensional structure of JP170, SEQ ID NO:1 by X-ray crystallography and found that there are several differences between this and the three-dimensional structure of BPN'. The inventors of the present invention have further compared the sequence homology of subtilases belonging to the Subtilisin subgroup. This is shown in Figure 3 of the present invention.
  • JP170 type subtilases become a separate subgroup in addition to the subgroups of BPN' subtilases and TY145 subtilases PCT/DK2004/000066. JP170 type subtilases
  • JP170 subtilase or "JP170 type subtilase” should in the context of the present invention be understood as a subtilase belonging to the Subtilisin group according to Siezen et al. Protein Science 6 (1997) 501-523 and which has at least 58% homology to JP170, SEQ ID NO:1.
  • JP170 type subtilase may have at least 60% homology to SEQ ID NO:1 , such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to JP17O, i.e. to SEQ ID NO:1.
  • the alkaline proteases KP43, KP1790, KP9860, Prote- ase Ya, Protease E-1 and SD-521 are subtilases belonging to the JP170 subgroup of subtilases.
  • a JP170 subtilase suitable for the purpose described herein m ay be a subtilase homologous to the three-dimensional structure of JP170, i.e. it may be homologous to the three-dimensional structure defined by the structure coordinates in Appendix 1.
  • a set of structure coordinates for a protein or a portion thereof is a relative set of points that define a shape in three dimensions, it is possible that an entirely different set of coordinates could define an identical or a similar shape.
  • slight variations in the individual coordinates may have little or no effect on the overall shape. These variations in coordinates may be generated because of mathematical manipulations of the structure coordinates.
  • the structure coordinates of JP170 may be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
  • said variations may be due to differences in the primary amino acid sequence.
  • the standard error may typically be measured as the root mean square deviation of e.g. conserved backbone residues, where the term "root mean square deviation” (RMS) means the square root of the arithmetic mean of the squares of the deviations from the mean.
  • RMS root mean square deviation
  • similarities in 3D structure of different subtilases can be described by the content of homologous structural elements, and/or the similarity in amino acid or DNA sequence. For sequences having no deletions or insertions a RMS for the CA carbon atoms can be calculated.
  • a JP170 type subtilase is further characterised as comprising the following structural characteristics: a) a twisted beta-sheet with 7 strands, b) six alpha helices, c) at least three ion-binding sites, and not comprising the Strong and Weak ion-binding site of the BPN' like subtilases
  • the isolated nucleic acid sequence encoding a JP170 subtilase of the invention hybridizes with a complementary strand of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:1 preferably under low stringency conditions, at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions.
  • Suitable experimental conditions for determining hybridization at low, medium, or high stringency conditions between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5 % SDS and 100 ⁇ g/ml of denatured sonicated salmon sperm DNA (Sambrook et al.
  • the filter is then washed twice for 30 minutes in 2 x SSC, 0.5 % SDS at least * 55°C (low stringency), more preferably at least 60°C (medium stringency), still more pref- erably at least 65°C (medium/high stringency), even more preferably at least 70° C (high stringency), and even more preferably at least 75°C (very high stringency).
  • a BPN' subtilase or BPN' type subtilase is in the context of the present invention to be understood as a subtilase belonging to the Subtilisin group according Siezen et al. Siezen et al. Protein Science 6 (1997) 501-523 and which has at least 61% homology to SEQ ID NO:4.
  • a BPN' subtilase may have at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to BPN', i.e. to SEQ ID NO:4.
  • isolated nucleic acid sequence encoding a BPN' subtilase of the invention hybridizes with a complementary strand of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:4 preferably under low stringency conditions, at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions.
  • a BPN' subtilase suitable for the purpose described herein may be a subtilase homologous to the three-dimensional structure of BPN' as defined by the structure coordinates given in PDB Nos. 1SBT and 1GNS (Protein Data Bank), or one of the several other structures of BPN' that are accessible from the Protein Data Bank. Variations between homologous structures may occur for several reasons as described above.
  • a BPN' subtilase within the context of the present invention is to be understood as any subtilase having the structural characteristics pertaining to the BPN' subtilases as described above, and in addition such subtilases do preferably not have further structural characteristics which are not present in the BPN' subtilases as de- scribed herein.
  • a BPN' type subtilase has two ion- binding sites.
  • a BPN' like subtilase may, in the context of the present invention, belong to branch l-S of the subtilisins i.e. to branch I-S1 , the "true” subtilisins or I-S2, the highly alkaline proteases (Siezen et al., Protein Engng. 4 (1991 ) 719-737).
  • subtilisin 309 subtilisin 309
  • SAVI- NASE ® subtilisin ®
  • subtilisin Carisberg ALCALASE ®
  • NOVOZYMES A/S subtilisin Carisberg
  • the Weak calcium-binding site was shown to be a Na (sodium) binding site when the calcium concentration in the crystallization medium was low.
  • ion- binding sites instead of calcium-binding sites.
  • a TY145 subtilase or TY145 type subtilase is in the context of the present invention to be understood as a subtilase which has at least 63% homology to SEQ ID NO:7.
  • said TY145 subtilase may have at least 65%, such as at least 70%, at least 74%, at least 80%, at least 83%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to TY145, i.e. to SEQ ID NO:7.
  • a TY145 subtilase suitable for the purpose described herein may be a subtilase homologous to the three-dimensional structure of TY145 as defined by the structure coordinates given in PCT/DK2004/000066. Variations between homologous structures may occur for several reasons as described above.
  • a TY145 subtilase within the context of the present invention is to be understood as any subtilase having the structural characteristics pertaining to the TY145 subtilases as described above, and in addition such subtilases do preferably not have further structural characteristics which are not present in the TY145 subtilases as described herein.
  • a TY145 subtilase further comprises the following structural characteristics: a) a twisted beta-sheet with 7 strands, b) six alpha helices, c) at least three ion-binding sites, wherein the Strong ion-binding site of the BPN' type sub- tilases is not present,
  • Examples of s ubtilases of the TY145 type i include the TY145 s ubtilase, the psychrophilic subtilisin protease S41 derived from the Antarctic Bacillus TA41 , herein also called TA41 subtilase (Davail S et al., 1994, J. Biol. Chem., 269, 17448-17453), and the psychrophilic subtilisin protease S39 derived from the Antarctic Bacillus TA39, herein also called TA39 subtilase (Narinx E et al., 1997, Protein Engineering, 10 (11 ), 1271-1279).
  • TA41 subtilase the psychrophilic subtilisin protease S41 derived from the Antarctic Bacillus TA41
  • TA41 subtilase the psychrophilic subtilisin protease S39 derived from the Antarctic Bacillus TA39
  • the JP170 subtilase was used to elucidate the three-dimensional structure forming the basis for the present invention.
  • JP170 was solved in accordance with the principle for x-ray crystallographic methods, for example, as given in X-Ray Structure Determination , Stout, G.K. and Jensen, L.H., John Wiley & Sons, Inc. NY, 1989.
  • the protease coordinates has a chain identification called A, whereas the CI2 inhibitor is called B, the calcium ions are called C, and the water is W.
  • the positions of the mentioned residues refer to the sequence of JP170 as disclosed in SEQ ID NO:1.
  • the JP170 structure consists of two domains, a catalytic domain and a C-terminal domain.
  • the structure of the catalytic domain shows the same overall fold as found in the S8 family of subtilisins.
  • the structure comprises a twisted beta-sheet with 7 strands arranged in the following sequential order S2, S3, S1 , S4, S5, S6, S7.
  • H1 contains residues 9-17
  • H 2 contains residues 68-76
  • H3 contains residues 1 10-1 19
  • H 4 contains residues 139-150
  • H5 contains residues 253-273
  • H6 contains residues 281- 291.
  • the C-terminal domain comprises a strand motif, a so called "beta sandwich” consisting of sheets a and b.
  • the sheet in this domain is combined of strands in an anti- parallel fashion, whereas the strand in the catalytic domain is combined in parallel.
  • the sequential order of the strands can be denoted as: S1a-S1b-S3a-S3b-S4b-S4a-S2b-S2a with the beta sandwich organised as to the two sheets S1a, S3a, S4a, S2a and S1 b, S3b, S4b, S2b.
  • the JP170 subtilases were found to lack the well-known Strong and Weak ion- binding sites of the BPN' subtilases. However, the JP170 subtilases have three ion-binding sites which are not present in the BPN' subtilisin structures. This can be seen in the struc- tural alignment presented in Fig. 2. These three ion-binding sites are hereinafter referred to as Site 1 , which is placed in the catalytic domain, and Site 2 and 3 which are placed in the non-catalytic C-terminal domain.
  • Site 2 - calcium atom named A603 CA
  • Site 3 - calcium atom named A602 CA in the PDB table (Appendix 1).
  • the position of an ion-binding site can be defined by the distance to four specific atoms in the core structure.
  • the distance from the ion-binding site to the c-alpha atoms of the three active site residues has been chosen.
  • the residues Ser, His and Asp in the active site are highly conserved. In JP170 they are Asp30, His68 and Ser254.
  • the fourth distance chosen is the distance to the c-alpha atom of the arnino acid residue coming first after the active site serine residue in the sequence (herein after called "next to Ser"); in the 3D structure of JP170 it is Met255. o
  • the distance between: a) ion-binding site 1 and i) Asp c-alpha atom is 26.70-28.7 ⁇ A, ii) His c-alpha atom is 22.10-24.10A, iii) Ser c-alpha atom is 16.95-18.95A, iv) next to Ser c-alpha atom is 15.30-17.30A, b) ion-binding site 2 and i) Asp c-alpha atom is 33.50-35.5 ⁇ A, ii) His c-alpha atom is 37-39A, iii) Ser c-alpha atom is 29.40-31.40A, iv) next to Ser c-alpha atom is 30.70-32.70A, c) ion-binding site 3 and i) Asp c-alpha atom is 41.50-43.50A, ii) His c-alpha
  • these distances may vary from one subtilase to the other.
  • the present distances are given with a calcium ion in the structure. If a sodium ion was bound instead the distances would be shifted a little bit.
  • the distances can vary ⁇ 0.8 ⁇ A, pref- erably ⁇ 0.7 ⁇ A, ⁇ 0.6 ⁇ A, ⁇ 0.5 ⁇ A, ⁇ 0.4 ⁇ A, or most preferably ⁇ 0.3 ⁇ A.
  • the peptide structure circumscribing ion- binding site 1 up to a distance of 10 A from the metal ion is composed of the amino acid residues placed in positions 183-189, 191-204 and 224-225.
  • the peptide structure circumscribing ion-binding site 2 up to a distance of 10 A from the metal ion is composed of residues 378-393.
  • the peptide structure circumscribing ion-binding site 3 up to a distance of 10 A from the metal ion is composed of residues 348, 350, 352, 363-370, 380-383, 391-400 and 414- 420.
  • the structure of the JP170 like subtilases can be divided into a "core subtilase-like” region, an "intermediate” region and a "nonhomologous” region.
  • the active site can be found in the core subtilase-like region, which is structurally closely related to the BPN' structures.
  • the core subtilase-like region is composed of residues 17-34, 197-209 and 216-232, and contains the alpha-helix H3 and the central alpha- helix H5 in which the active site serine residue is situated in the N-terminal part.
  • the core subtilase-like region has an RMS lower than 1.2.
  • the intermediate region consists of residues 42-46, 150-186, 245-272 and 278- 296.
  • the intermediate region has an RMS bigger than 1.2 and less than 1.8.
  • the relationships between the three-dimensional structure and functionality are potentially difficult to predict in this region of the JP170 like subtilases.
  • the nonhomologous region consists of residues 1-16, 35-41 , 47-149, 187-196, 210-215, 233-244, 273-277 and 297-316.
  • the nonhomologous region has a RMS h igher than 1.8.
  • the relationships between the three-dimensional structure and functionality are very difficult to predict in this region of the JP170 like subtilases.
  • the loops N79-N82 (I75-V81 ) and G208-D218 (G193-D197) are in contact with a ion-binding site in Savinase, but not in JP170.
  • the loop E185-N199 (D181-N 184) is in contact with a ion-binding site in JP170, but not in Savinase.
  • a good example of the difference is the loop S232-K246 in JP170 which has 15 residues compared to the corresponding BPN' type loop G211-T213 (in Savinase), which has only three residues.
  • the loop folds back to the substrate binding site, especially the P' parts of the substrate binding site.
  • the loop is situated close to the substrate as illustrated by the CI2 inhibitor bound in the 3D structure attached (Ap- pendix l ).
  • loop S232-K246 in JP170 can be described in relation to the four specific residues as described above.
  • the distance from the CA atom of residue W240 in the loop to the CA atoms of the active site residues are: Residue H68 D30 S254 M255
  • distances like these can vary ⁇ 0.8 ⁇ A, preferably ⁇ 0.7 ⁇ A, ⁇ 0.6 ⁇ A, ⁇ 0.5 ⁇ A, ⁇ 0.4 ⁇ A, or most preferably ⁇ 0.3 ⁇ A.
  • distances from the residues of JP170 loop S232-K246 to atoms of the CI2 inhibitor can be calculated. These distances are: from CA atom of W240 to CA atom of R62 in CI2 is 7.49A, from CA atom of F239 to CA atom of R62 in CI2 is 8.39A, from CA atom of S238 to CA atom of R62 in CI2 is 8.42A, from CA atom of S237 to CA atom of R62 in CI2 is 9.44A, from CA atom of S238 to CA atom of E60 in CI2 is 9.42A.
  • the distances from JP170 active site residue S254 to atoms of the CI2 inhibitor, as placed in the 3D coordinates of Appendix 1 are: from CA atom of S254 to CA atom of E60 in CI2 is 5.25A, from CA atom of S254 to CA atom of R62 in CI2 is 11.55A, from CA atom of S254 to CA atom of T58 in CI2 is 7.06A, from CA atom of S254 to CA atom of M59 in CI2 is 4.71 A.
  • the distances can vary ⁇ 0.8 ⁇ A, preferably ⁇ 0.7 ⁇ A, ⁇ 0.6 ⁇ A, ⁇ 0.5 ⁇ A, ⁇ 0.4 ⁇ A, or most preferably ⁇ 0.3 ⁇ A.
  • a preferred JP170 like subtilase variant has a deletion in the region S232-K246, and the subsequent insertion of one or more residues to partly or completely remove the loop.
  • Preferred variants comprises the deletion of L233-S245 + insertion of Asn, deletion of L233-D244 + insertion of Gly or deletion of S232-D244 + insertion of Gly. Similar considerations can be made in respect of differences to the TY145 structure.
  • a model structure of a JP170 type subtilase, a BPN' type subtilase or a TY145 type subtilase can be built using the Homology program or a comparable program, e.g., Modeller (both from Molecular Simulations, Inc., San Diego, CA).
  • the principle is to align the amino acid sequence of a protein for which the 3D structure is known with the amino acid sequence of a protein for which a model 3D structure has to be constructed.
  • the structur- ally conserved regions can then be built on the basis of consensus sequences.
  • loop structures can be inserted, or sequences can be deleted with subsequent bonding of the n ecessary residues using, e .g., the program Homology. S ubsequent relaxing and optimization of the structure should be done using either Homology or another molecular simulation program, e.g., CHARMm from Molecular Simulations.
  • the present invention relates to a method for constructing a variant of a parent subtilase, wherein the variant has at least one altered property as compared to said parent subtilase, which method comprises: a) analyzing the three-dimensional structure of the parent subtilase to identify, on the basis of an evaluation of structural considerations in relation to a JP170 three di- mensional structure, at least one amino acid residue or at least one structural region of the subtilase, which is of relevance for altering said property; b) modifying the DNA of the polynucleotide encoding the parent to construct a polynucleotide encoding a variant subtilase, which in comparison to the parent subtilase, has been modified by deletion, substitution or insertion of the amino acid residue or structural part identified in i) so as to alter said property; c) expressing the variant subtilase
  • the invention relates to a method of producing a subtilase variant, wherein the variant has at least one altered property as compared to a parent subtilase, which method comprises: a) producing a model structure of the parent subtilase on the three-dimensional structure of BPN', TY145 or JP170; or producing an actually determined three- dimensional structure of the parent subtilase, b) comparing the model or actual three-dimensional structure of the parent subtilase to the JP170 structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step b) at least one structural part of the parent subtilase, wherein an alteration in said structural part is predicted to re- suit in an altered property; d) modifying the n ucleic a cid sequence e ncoding the parent subtilase to produce a nucleic acid sequence encoding at least one deletion or substitution of one or more
  • the present invention thus generally relates to the use of the JP170 structure as provided herein for the identification of desired modifications in subtilases of any of the three subtilisin types, the BPN' types (I-S1 and I-S2 subgroups), the TY145 types and the JP170 types through modelling the 3-D structure of a parent subtilase to the type it belongs to and subsequent comparison thereof to the JP170 3-D structure, or in instances where the 3-D structure of the parent subtilase is actually known by comparison thereof to the JP170 3-D structure.
  • At least one residue in the parent subtilase is selected for modification by substitution, deletion or insertion in order to provide a subtilase variant with altered properties in comparison to the parent subtilase.
  • the parent subtilase may therefore belong to the sub-group I- S1 , preferably selected from the group consisting of ABSS168, BASBPN, BSSDY, and BLSCAR, or functional variants thereof having retained the characteristic of sub-group I- S1.
  • the parent subtilase belongs to the sub-group I-S2, preferably selected from the g roup consisting of BLS147, BLS309, BAPB92, and BYSYAB, or functional variants thereof having retained the characteristic of sub-group I-S2.
  • subtilase belongs to the TY145 type subgroup, preferably selected from the group comprising TY145, protease S41 also called TA41 protease S39 also called TA39 subtilase, etc.
  • subtilase belongs to the JP170 type subgroup, preferably selected from the group comprising JP170, KP43, KP9860, Protease E-1 , Protease Ya, Protease SD-521 , etc.
  • a further embodiment of the invention relates to a method of producing a JP170 type subtilase variant, wherein the variant has at least one altered property as compared to a parent subtilase, which method comprises: a) producing a model structure of the parent JP170 type subtilase on the three- dimensional structure of JP170; or producing an actually determined three- dimensional structure of the parent subtilase, b) comparing the model or actual three-dimensional structure of the parent JP170 type subtilase to the BPN' or TY145 structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step b) at least one structural part of the parent JP170 type subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent JP170 type subtilase to produce a nucleic acid sequence
  • the invention also comprises the protease variants produced by the above meth- ods.
  • JP170 subtilases As described above the three-dimensional structure of JP170 subtilases as provided in Appendix 1 indicates the presence of three ion-binding sites not present in the BPN' subtilisin structures, thus lacking the Strong and Weak ion-binding site of the BPN' subtilases. Stability of ion-binding sites is important for the functionality of the enzyme. Therefore alterations of the ion-binding sites are likely to result in alterations of the stability of the enzyme.
  • step (c) above identifies amino acid residue positions located at a distance of 1 ⁇ A or less to the ion-binding site of the JP170 type parent, preferably positions located at a distance of 6 A or less.
  • a preferred variant of the present invention has a modification in one or more of the positions located at a distance of 1 ⁇ A to the ion-binding sites of JP170 (SEQ ID NO:1). These positions are:
  • Site 1 183-189 (i.e. positions 183, 184, 185, 186, 187, 188, 189),
  • Site 2 378-393 (i.e. positions 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387,
  • Site 3 348, 350, 352, 363-370 (i.e. positions 363, 364, 365, 366, 367, 368, 369, 370),
  • Preferred variants stabilised in ion-binding site 2 are N390D and N391 D
  • preferred variants stabilised in ion-binding site 3 are G394N,Q,F,Y,S and W392S,N,Q.
  • a variant with improved stability may be obtained by substitution with proline, introduction of a disulfide bond, altering a hydrogen bond contact, altering charge distribution, introduction of a salt bridge, filling in an internal structural cavity with o ne o r m ore a mino acids with b ulkier s ide groups (in e.g. regions which are structurally mobile), substitution of histidine residues with other amino acids, removal of a deamidation site, or by helix capping.
  • the following regions of JP170 have an increased mobility in the crystal structure of the enzyme, and it is presently believed that these regions can be responsible for stability or activity of JP170. Especially thermostabilisation may possibly be obtained by altering the highly mobile regions. Improvements of the enzyme can be obtained by mutation in the below regions and positions. Introducing e.g. larger residues or residues having more atoms in the side chain could increase the stability, or, e.g., introduction of residues having fewer atoms in the side chain could be important for the mobility and thus the activity profile of the enzyme.
  • Two methods extract the highly mobile regions from a 3D structure.
  • One is a molecular dynamics calculation of the isotropic fluctuations by using the program CHARMm from MSI (Molecular Simulations Inc.), and the other is an analysis of the B-factors.
  • the B- factors are listed in Appendix 1 and give a value to the uncertainty of determination of the location of the various atoms of the structure.
  • the uncertainty relates to the mobility of the atoms in the molecules in the crystal lattice. This mobility reflects the thermal motion of the atoms and thus indicates possible sites for thermostabilisation of the enzyme.
  • 131-134 i.e. positions 131 , 132, 133, 134
  • 107-111 i.e. positions 107, 108, 109, 110, 111
  • 337-342 i.e. positions 337, 338, 339, 340, 341 , 342
  • a preferred JP170 subtilase variant of the present invention has been modified in one or more of the above mentioned positions of SEQ ID NO:1.
  • Further preferred variants comprises one or more alterations in the regions 57-60, 66-67, 107-1 1 1 , 236-240, 326-332, 355-360, 372-377, 384-388, 404-41 1.
  • variant W240H.Y and variants modified in the region 355-360 such as variants comprising one or more of the modifications: G355A.S; S356T.N; T357N,Q,D,E,P; T358S; A359S,T,N,Q and S360T.N.
  • Variants modified in the region 355-360 may be produced in accordance with the method for random mutagenesis by use of the DOPE program as described herein. To obtain variants comprising 1-3 modifications in region 355-360 one may introduce the substitutions with the following frequencies: wild-type modified
  • a JP170 variant of the present invention with improved stability, e.g. thermostability, as compared to the parent JP170 subtilase may be obtained by introducing new inter- domain or intra-domain bonds, such as by establishing inter- or intra-domain disulfide bridges.
  • a further aspect of the present invention relates to a method for producing a variant of a parent JP170, wherein step (c) identifies amino acid residue positions in the parent JP170 type subtilase, the modification of which may create at least one disulfide bridge by insertion of or substitution with at least one Cys residue.
  • substitutions are: G21C+A86C, V26C+A265C, G57C+G105C, G74C+A229C, Q111C+N143C, G160C+S170C, A286C+V349C, A27C+A122C, A45C+G78C, V72C+P258C, G78C+A229C, D98C+G104C, Q111C+Y147C, G135C+G167C, R142C+P354C, V144C+A178C, G182C+P217C, A183C+G223C, A195C+Y225C, F271C+P279C, A287C+A430C, A293C
  • a variant with improved stability as compared to the parent subtilase may be obtained by changing the surface charge distribution of the subtilase. For example, when the pH is lowered to about 5 or below histidine residues typically become positively charged and, consequently, unfavorable electrostatic interactions on the protein surface may occur. By engineering the surface charge of the subtilase one may avoid such unfavorable electrostatic interactions that in turn lead to a higher stability of the subtilase.
  • Charged amino acid residues are (a) positively charged: Lys, Arg, His (pH ⁇ 5), Tyr (pH>9) and Cys (pH>10) and (b) negatively charged: Asp and Glu.
  • a further aspect of the present invention relates to method for constructing a variant of a parent subtilase, the method comprising: a) identifying, on the surface of the parent subtilase, preferably a JP170 like or a BPN' like subtilase, at least one amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; b) substituting, on the surface of the parent subtilase, at least one amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His with an uncharged amino acid residue; c) optionally repeating steps a) and b) recursively; d) optionally, making alterations each of which is an insertion, a deletion or a substitution of an amino acid residue at one or more positions other than b); e) preparing the variant resulting from steps a) - d); f) testing the stability of said variant; and g) optionally repeating steps a) - f
  • the above method may be employed. Again, compared to the above method, the only difference being steps a) and b) which, in this case, read: a) identifying, on the surface of the parent subtilase, at least one charged amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; b) substituting, on the surface of the parent subtilase, at least one charged amino acid residue identified in step a) with an amino acid residue having an opposite charge.
  • Lys or His may be substituted with Asp or Glu; Lys may be substituted with Asp or Glu; and His may be substituted with Asp or Glu.
  • the surface accessible area are measured using the DSSP pro- gram (Kabsch and Sander, Biopolymers (1983), 22, 2577-2637). All residues having a surface accessibilty higher than 0 0, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55 or 0.60 are regarded a surface residue.
  • N79, N316, L381 , K246, K9, K313 and K83 are amino acid residues found on the surface of JP170 using the above method.
  • substitutions N79D, N316D and L381 D are of particular interest for stabilisation by introduction of salt bridges
  • substitutions K246R, K9R, K313R and K83R are of particular interest for the stabilisation at high pH.
  • thermostability of a subtilase can be obtained by subjecting the subtilase in question to analysis for secondary structure, identifying residues in the subtilase having dihedral angles ⁇ (phi) and ⁇ (psi) confined to the intervals [-90° ⁇ -40° and -180° ⁇ 180°], preferably the intervals [-90° ⁇ -40° and 120° ⁇ 180°] or [-90° ⁇ -40° and -50° ⁇ 10°] and excluding residues located in regions in which the subtilase is characterized by possessing ⁇ - helical or ⁇ -sheet structure.
  • proline residues are generally not compatible with ⁇ -helical and ⁇ -sheet secondary con- formations.
  • a proline residue is not already at the identified position(s)
  • the naturally occurring amino acid residue is substituted with a proline residue, preferably by site directed mutagenesis applied on a gene encoding the subtilase in question.
  • proline residues can advantageously be intro- quizzed at positions 22, 44, 110, 139, 140, 166, 198, 201 , 203, 231 , 282, 356, 357 and 378.
  • a preferred JP170 variant has one or more of the substitutions: Q22P, E44P, L110P, T139P, D140P, S166P I198P, V201P, Q203P, S231P, S282P, S356P, T357P and K378P.
  • variants comprising one or more of: E44P, Q203P and S356P.
  • JP170 subtilases differ greatly from the BPN' like subtilases in having a long apparently non-catalytic C-terminal.
  • a possible truncation of JP170 is the removal of approx. 115 residues including two ion-binding sites, which can be obtained by deletion of or within the region 311-433, which is the non-catalytic C-terminal.
  • Preferred deletions com- prises the regions 317-433 or 315-433.
  • the new C-terminal will be within the region of 311-325.
  • the deletion can be optimised with additional substitutions, such as one or more of L283N.Q; A290S.N and W306H,Y,K.
  • Preferred truncations comprise: a) deletion of region 317-433 and the substitutions L283N + A290S + W306H, b) deletion of region 315-433 and the substitutions L283N + A290S + W306H.
  • the substrate binding site is identified by the residues in contact with a substrate model, such as the CI2 inhibitor.
  • a substrate model such as the CI2 inhibitor.
  • the 3D structure coordinates of the JP170 subtilase with CI2 bound in the active site are provided in Appendix 1. Without being limited to any theory, it is presently believed that binding between a substrate and an enzyme is supported by favorable interactions found within a sphere 10 A from the substrate molecule. Examples of such favorable bonds are hydrogen bonds, strong electrostatic interaction and/or hydrophobic interactions.
  • residues of the JP170 subtilase are within a distance of 1 ⁇ A from the CI2 inhibitor which is bound to the substrate binding site. These residues are thus believed to be involved in interactions with said substrate: 29-32, (i.e. residues 29, 30, 31 , 32) 64-72, (i.e. residues 64, 65, 66, 67, 68, 69, 70, 71 , 72)
  • 100-110 (i.e. residues 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110) 113-114, 127-136, (i.e. residues 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136) 138-141 , (i.e. residues 138, 139, 140, 141 ) 144, 157, 174,
  • a variant comprises a modification in one or more of the above mentioned positions.
  • a preferred variant is W129L. JP170 with extra ion-binding site
  • the Strong ion-binding site from the BPN' subtilases can be transplanted into JP170 (or other subtilases in JP170 subgroup) by deletion of N79-N82 and subsequent insertion of LNNSIGV (SEQ ID NO:5), followed by the substitution A45D.N and optionally the substitutions E44P,T and/or R47Q.
  • JP170 or other JP170 type subtilases can be removed with guidance from information about the three-dimensional structures of other related subtilases, such as a BPN' type subtilase, such as Savinase or BPN', and a
  • Removal of ion-binding site 1 can be done by deletion of N186-N199 and subse- quent insertion of at least three amino acid residues - or stated differently by the substitution of a region comprising from 3 to 6 amino acid residues for the region comprising 14 amino acid residues in positions 186 to 199, preferably the sequence of the substituting region is SSN (SEQ ID NO:6).
  • SSN SEQ ID NO:6
  • substitutions I7Q and V3Y is further added.
  • the ion-binding site 1 can be removed from a wild-type JP170 subtilase or a JP170 subtilase truncated as described above.
  • JP170 type subti- lases and TY145 type subtilases can be used to remove the Strong and Weak ion-binding sites in BPN' type subtilases, or the ion-binding sites in TY145 type subtilases may be removed on the b asis of structural i nformation a bout the J P170 and BPN' types of s ubtilases.
  • the removal can be done by altering the loops A194-L196 (weak ion-binding site) and L75-L82 (strong ion-binding site) either by a) insertion or deletion of a number of amino acid residues in the loops or b) by deletion of the entire loop or part of the loop and subsequent insertion of a number of residues from a corresponding loop of a JP17J0 or TY145 like subtilase.
  • the ion-binding sites of Savinase can be removed by either i) full or partial deletion of the region A194-L196 (BPN' numbers) and insertion of three or more residues chosen from JP170 positions P209-P217, and ii) full or partial deletion of the region L75-L82 (BPN' numbers) and insertion of at least one residue chosen from TY145 positions H83-Y92 or i) full or partial deletion of the region A194-L196 (BPN 1 numbers) and insertion of three or more residues chosen from JP170 positions P209-P217 and ii) full or partial deletion of the region L75-L82 (BPN' numbers) and insertion of at least one residues chosen from JP170 positions N79-K83.
  • JP170 type subtilase protease it may be advantageous to substitute or delete critical oxidation sites, such as methionines, with other amino acid residues which are not subject to oxidation.
  • the present invention relates to an RP-II pro- tease variant, in which one or more amino acid residues susceptible to oxidation, especially m ethionine residues exposed to the surface of the molecule, is/are d eleted or replaced with another amino acid residue less susceptible to oxidation.
  • the amino acid residue less susceptible to oxidation may for instance be selected from the group consisting of A, E, N, Q, I, L, S and K. Specific such variants comprises at least one of the deletions or substitutions
  • the invention consequently further relates to a subtilase, in which the modificatioins indicated above are either or both residues of any of the Asn-Gly sequence appearing in the amino acid sequence of the parent RP-II protease is/are deleted or substituted with a residue of a different amino acid.
  • the Asn and/or Gly residue may, for instance, be substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y.
  • any of the Asn or Gly residues of the Asn-Gly occupying positions 66-67, 134-135 and/or 375-376 of the SD-521 and Ya protease; and positions 66-67, 134- 135, 301-302 and/or 375-376 of the JP170 protease, may be deleted or substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y. (positions are indicated in relation to the JP170 protease as indicated in Fig. 1 ).
  • JP170 Specific variants of JP170 are: N66f,A,Q,S,P,T,Y ⁇ ; G67f ,A,Q,S,P,T,Y ⁇ ; N134 ⁇ * ,A,Q,S,P,T,Y ⁇ ; G135 ⁇ * ,A,Q,S,P,T,Y ⁇ ; N301 ⁇ * ,A,Q,S,P,T,Y ⁇ ; G302f ,A,Q,S,P,T,Y ⁇ ;
  • SD-521 and Ya proteases are: N66 ⁇ *,A,Q,S,P,T,Y ⁇ ; G67 ⁇ * ,A,Q,S,P,T,Y ⁇ ; N134 ⁇ * ,A,Q,S,P,T,Y ⁇ ; G135f ,A,Q,S,P,T,Y ⁇ ; and N375 ⁇ *,A,Q,S,P,T,Y ⁇ ; G376 ⁇ * ,A,Q,S,P,T,Y ⁇ , and combinations thereof as indicated above.
  • JP170 tyrosine residues may be modified in positions: 20, 54, 1 18, 137, 147, 194, 225, 247, 249, 334, 379, 388, 411 , and 418.
  • the tyrosine residues may be modified in positions: 17, 20, 54, 137, 147, 187, 243, 247, 249, 299, 319, 334, 361 , 379, 386, 388, 411 , and 418.
  • the invention thus relates to the variants: Y17 ⁇ F,W ⁇ , Y20 ⁇ F,W ⁇ ,
  • JP170 type subtilases JP170 type subtilases.
  • the invention relates to JP170 type subtilase variants comprising one or more of the following substitutions: For the SD-521 a nd Ya proteases positions 1 18, 1 29, 240, 306, 350, and 392; and for the JP170 protease positions 129, 240, 306, 350, and 392.
  • the invention relates to a JP170 variant comprising one or more of the following substitutions W129 ⁇ F,T,Q,G ⁇ , W240 ⁇ F,T,Q,G ⁇ , W306 ⁇ F,T,Q,G ⁇ , W350 ⁇ F,T,Q,G ⁇ , and W392 ⁇ F,T,Q,G ⁇ .
  • 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 subtilase variants i.e. the JP170 and BPN' variants of the present invention may be produced by any known method within the art and the present invention also relates to nucleic acid encoding a subtilase variant of the present invention, a DNA construct comprising said nucleic acid and a host cell comprising said nucleic acid sequence.
  • natural occurring proteins may be produced by culturing the organism expressing the protein and subsequently purifying the protein or it may be produced by cloning a nucleic acid, e.g. genomic DNA or cDNA, encoding the protein into an expression vector, introducing said expression vector into a host cell, culturing the host cell and purifying the expressed protein.
  • protein variants may be produced by site-directed mutagenesis of a parent protein, introduction into expression vector, host cell etc.
  • the parent protein may be cloned from a strain producing the polypeptide or from an expression library, i.e. it may be isolated from genomic DNA or prepared from cDNA, or a combination thereof.
  • subtilase or subtilase or subtilase variant of the invention.
  • suitable techniques reference is made to Molecular cloning: A laboratory manual (Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.)); Current protocols in Molecular Biology (John Wiley and Sons, 1995; Harwood, C. R., and Cut- ting, S. M.
  • Random mutagenesis is suitably performed either as localized or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid se- quence shown in question, or within the whole gene.
  • the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions that are to be changed.
  • the doping or spiking may be done so that codons for unwanted amino acids are avoided.
  • the doped or spiked oligonucleotide can be incorporated into the DNA encoding the subtilase enzyme by any published technique, using, e.g., PCR, LCR or any DNA polymerase and ligase as deemed appropriate.
  • the doping is carried out using "constant random doping", in which the percentage of wild-type and modification in each position is predefined.
  • the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues.
  • the doping may be made, e.g., so as to allow for the introduction of 90% wild type and 10% modifications in each position.
  • An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints.
  • the doping scheme may be made by using the DOPE program which, inter alia, ensures that introduction of stop codons is avoided (L.J. Jensen et al. Nucleic Acid Research, 26, 697-702 (1998).
  • PCR-generated mutagenesis When PCR-generated mutagenesis is used, either a chemically treated or non- treated gene encoding a parent subtilase enzyme is subjected to PCR under conditions that increase the misincorporation of nucleotides (Deshler 1992; Leung et al., Technique, 1 , 1989, pp. 11-15).
  • the DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent subtilase.
  • the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenising agent.
  • the DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harbored in the cell. Fi- nally, the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.
  • telomere amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme.
  • the mutated DNA sequence may further comprise a DNA sequence encoding func- tions permitting expression of the mutated DNA sequence.
  • the random mutagenesis may be advantageously localised to a part of the parent subtilase i n q uestion. This may, e .g., be advantageous when certain regions of the e n- zyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.
  • the localised or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art.
  • the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g., by insertion into a suitable vector, and said part m ay be subsequently subjected to m utagenesis by use of any of the m utagenesis methods discussed above.
  • the random mutagenesis may be carried out by the following steps:
  • Suitable dope algorithms for use in step 6 are well known in the art.
  • One such algorithm is described by Tomandl, D. et al., 1997, Journal of Computer-Aided Molecular Design 11 :29-38.
  • Another algorithm is DOPE (Jensen, LJ, Andersen, KV, Svendsen, A, and o Kretzschmar, T (1998) Nucleic Acids Research 26:697-702).
  • a recombinant expression vector comprising a nucleic acid sequence encoding a subtilase variant of the invention may be any vector that may conveniently be subjected to 5 recombinant DNA p rocedures a nd which may bring about the expression of the nucleic acid sequence.
  • the choice of vector will often depend on the host cell into which it is to be intro- prised.
  • a suitable vector include a linear or closed circular plasmid or a virus.
  • the vector may be an autonomously replicating vector, i.e., a vector which exists as an 0 extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini chromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAM ⁇ l .
  • Examples of origin of 5 replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1.
  • the origin of replication may be one having a mutation which makes it function as temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • Vectors which are integrated into the genome of the host cell may contain any nucleic acid sequence enabling integration into the genome, in particular it may contain nucleic acid sequences facilitating integration into the genome by homolo- 5 gous or non-homologous recombination.
  • the vector system may be a single vector, e.g. plasmid or virus, or two or more vectors, e.g. plasmids or virus', which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the vector may in particular be an expression vector in which the DNA sequence encoding the subtilase variant of the invention is operably linked to additional segments or control sequences required for transcription of the DNA.
  • the term, "operably linked" indi- cates 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 encoding the subtilase variant.
  • Additional segments or control sequences include a promoter, a leader, a polyadenylation sequence, a propeptide sequence, a signal sequence and a transcription terminator. At a minimum the control sequences include a pro- moter and transcriptional and translational stop signals.
  • 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 subtilis levansucrase gene (sacB), the Bacillus stearothermophilus malto- genic amylase gene (amyM), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, o
  • promoters for use in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhi- zomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae those phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described in U.S.
  • Patent No. 4,288,627 which is incorporated herein by reference
  • Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes en- coding Aspergillus niger neutral (-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters.
  • Further suitable promoters for use in filamentous fungus host cells are the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093 - 2099) or the tpiA promoter.
  • promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPM (US 4,599,311 ) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters.
  • yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.
  • useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).
  • SV40 Simian Virus 40
  • RSV Rous sarcoma virus
  • BPV bovine papilloma virus
  • Suitable promoters for use in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981 ), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
  • a suitable promoter for use in insect cells is the polyhedrin promoter (US 4,745,051 ; Vasuvedan et al., FEBS Lett. 311 , (1992) 7 - 11), the P10 promoter (J.M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica polyhedro- sis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (US 5,155,037; US 5,162,222), or the baculovirus 39K delayed-early gene promoter (US 5,155,037; US 5,162,222).
  • the polyhedrin promoter US 4,745,051 ; Vasuvedan et al., FEBS Lett. 311 , (1992) 7 - 11
  • the P10 promoter J.M. Vlak et al., J. Gen. Virology 69, 1988, pp
  • the DNA sequence encoding a subtilase variant 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 ampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, neomycin, hygromycin, methotrexate, or resistance to heavy metals, virus or herbicides, or which provides for prototrophy or auxotrophs.
  • bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, resistance.
  • a frequently used mammalian marker is the dihydrofolate reductase gene (DHFR).
  • Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3.
  • a selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate d ecarboxylase), sC ( sulfate a denyltransferase), trpC (anthranilate synthase), and g lufosinate resistance markers, as well as equivalents from other species.
  • amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.
  • selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.
  • 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.
  • More than one copy of a nucleic acid sequence encoding an enzyme of the present invention may be inserted into the host cell to amplify expression of the nucleic acid sequence.
  • Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transformants.
  • the nucleic acid constructs of the present invention may also comprise one or more nucleic acid sequences which encode one or more factors that are advantageous in the expression of the polypeptide, e.g., an activator (e.g., a trans-acting factor), a chaperone, and a processing protease. Any factor that is functional in the host cell of choice may be used in the present invention.
  • an activator e.g., a trans-acting factor
  • a chaperone e.g., a chaperone
  • processing protease e.g., a factor that is functional in the host cell of choice.
  • the nucleic acids encoding one or more of these factors are not necessarily in tandem with the nucleic acid sequence encoding the polypeptide.
  • the DNA sequence encoding a subtilase variant of the present invention may be either homologous or heterologous to the host cell into which it is introduced. 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 se- quence 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 subtilase variants, such as prokaryotes, e.g. bacteria or eukaryotes, such as fungal cells, e.g. yeasts or filamentous fungi, insect cells, plant cells or mammalian cells.
  • prokaryotes e.g. bacteria
  • eukaryotes such as fungal cells, e.g. yeasts or filamentous fungi, insect cells, plant cells or mammalian cells.
  • Examples of bacterial host cells which, on cultivation, are capable of producing the subtilase variants of the invention are gram-positive bacteria such as strains of Bacillus, e.g. 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 or Pseudomonas sp.
  • 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 subtilase variant in bacteria such as E. coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or it 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.
  • gram-positive bacteria such as Bacillus or
  • the enzyme may be retained in the cytoplasm, or it 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.
  • yeast cells include cells of a species of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, Hansehula, or Yarrowia.
  • the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cer- evisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
  • yeast host cells are a Kluyveromyces lactis, Kluyveromyces fragilis, Hansehula polymorpha, Pichia pastoris, Yarrowia lipolytica, Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichia guillermondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US 4,882,279 and US 4,879,231).
  • yeast Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980.
  • yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M.,MHorecker, B.J., and Stopani, A.O.M., editors, 2nd edition, 1987; The Yeasts, Rose, A.H., and Harrison, J.S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981). Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
  • filamentous fungal cells include filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra), in particular it may of the a cell of a species of Acremonium, such as A. chrysogenum, Aspergillus, such as A. awamori, A. foetidus, A. japonicus, A. niger, A. nidulans or A. oryzae, Fusarium, such as F. bactridioides, F. cerealis, F. crookwellense, F. culmorum, F. graminearum, F. graminum, F. heterosporum, F. negundi, F.
  • a species of Acremonium such as A. chrysogenum, Aspergillus, such as A. awamori, A. foetidus, A. japonicus, A. niger, A. nidulans or A.
  • reticulatum F. roseum, F. sambucinum, F. sarcochroum, F. sulphureum, F. trichothecioides or F. oxysporum, Humicola, such as H. insolens or H. lanuginose, Mucor, such as M. miehei, Myceliophthora, such as M. thermo- philum, Neurospora, such as N. crassa, Penicillium, such as P. purpurogenum, Thielavia, such as T. terrestris, Tolypocladium, or Trichoderma, such as T. harzianum, T. koningii, T. longibrachiatum, T. reesei or T. viride, or a teleomorph or synonym thereof.
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023.
  • insect cells include a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214). Culture conditions may suitably be as described in WO 89/01029 or WO 89/01028.Transformation of insect cells and pro- duction of heterologous polypeptides therein may be performed as described in US 4,745,051 ; US 4, 775, 624; US 4,879,236; US 5,155,037; US 5,162,222; EP 397,485).
  • mammalian cells examples include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection. Methods of transfect- ing mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601 - 621 ; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327 - 341 ; Loyter et al., Proc. Natl. Acad. Sci.
  • Mammalian cells may be transfected by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546).
  • the above mentioned host cells transformed or transfected with a vector comprising a nucleic acid sequence encoding an enzyme of the present invention are typically cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth.
  • the medium used to culture the host cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The media may be prepared using procedures known in the art (see, e.g., references o for bacteria and yeast; Bennett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).
  • the enzymes of the present invention are secreted into the nutrient medium, they may be recovered directly from the medium. If they are not secreted, they may be recovered from cell lysates.
  • the enzymes of the present invention may be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the enzyme in question.
  • the enzymes of the invention may be detected using methods known in the art that are specific for these proteins. These detection methods include use of specific antibodies, formation of a product, or disappearance of a substrate. For example, an enzyme assay may be used to determine the activity of the molecule. Procedures for determining various kinds of activity are known in the art.
  • the enzymes of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction
  • IEF isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • extraction see, e.g
  • heterologous host cell An advantage of using a heterologous host cell is that it is possible to make a highly purified enzyme composition, characterized in being free from homologous impurities, which are often present when a protein or peptide is expressed in a homologous host cell.
  • homologous impurities mean any impurity (e.g. other polypeptides than the enzyme of the invention) which originates from the homologous cell where the enzyme of the invention is originally obtained from.
  • the enzyme of the invention may be added to and thus become a component of a detergent composition.
  • the d etergent composition of the i nvention m ay for example be formulated a s 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, especially for automatic dish washing (ADW).
  • 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, especially for automatic dish washing (ADW).
  • ADW automatic dish washing
  • the invention provides a detergent additive comprising the en- zyme 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 galac- tanase, 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 galac- tanase, a
  • Suitable proteases include those of animal, vegetable or microbial origin. Mi- crobial 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 mi- crobial protease or a trypsin-like protease.
  • alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carisberg, 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.
  • useful proteases are the variants described in WO 92/19729, WO
  • Preferred commercially available protease enzymes include AlcalaseTM, Savi- naseTM, PrimaseTM, DuralaseTM, EsperaseTM, and KannaseTM (Novozymes A/S), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect OxPTM, FN2TM, and FN3TM (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 Pseu- domonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. ce- pacia (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 1 05, 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.
  • LipolaseTM and Lipolase UltraTM are preferred commercially available lipase enzymes.
  • Amylases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, ⁇ -amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, de- scribed 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 DuramylTM, TermamylTM, FungamylTM and BANTM (Novozymes A/S), RapidaseTM and PurastarTM (from Genencor International Inc.).
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modi- fied or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits.
  • cellulases examples include cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
  • cellulases include CelluzymeTM, and CarezymeTM (Novozymes A/S), ClazinaseTM, and Puradax HATM (Genencor International Inc.), and KAC- 500(B)TM (Kao Corporation).
  • Peroxidases/Oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include GuardzymeTM (Novozymes A/S).
  • the detergent enzyme(s) may be included i n 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 granu- lates, liquids, in particular stabilized liquids, or slurries.
  • Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 30000; 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.
  • Pro- tected 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, al- pha-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, al- pha-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
  • 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, ethylenediaminetetraacetic a cid, d iethylenetriaminepentaacetic a cid, a Ikyl- o r a Ikenylsuc- cinic 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, ethylenediaminetetraacetic a cid, d iethylenetriaminepentaacetic a cid, a Ikyl- o r a Ikenylsuc- cinic acid, soluble silicates or layered silicates
  • 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), polycarboxylat.es such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the detergent may contain a bleaching system which may comprise a H 2 0 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 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 i ncluding clays, foam boosters, suds s uppressors, a nti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • any enzyme in particular the enzyme of the inven- tion, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per litre of wash liquor, preferably 0.05-5 mg of enzyme protein per litre of wash liquor, in particular 0.1-1 mg of enzyme protein per litre 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.
  • type EMPA 116 cotton textile stained with blood, milk and ink
  • EMPA 117 polyyester/cotton textile stained with blood, milk and ink
  • Other atandard textile pieces are obtained from wfk-Cleaning Technology Research Institute, Chris- tenfeld 10, D-41379 Br ⁇ ggen-Bracht, Germany.
  • type wfk10N cotton textile stained with egg/pigment
  • wfklOeggEG cotton textile stained with egg yolk. Denaturation of wfk10N occurs in an autoclave.
  • 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.
  • a suitable host cell which has been transformed with a DNA sequence encoding the enzyme
  • the resulting enzyme is recovered from the culture.
  • 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.
  • 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.
  • Subtilase JP170 site-directed variants of the invention comprising specific inser- tions/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 JP170. Mutations are introduced by oligo directed mutagenesis to the construction of variants.
  • subtilisin JP170 variants were 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 S. subtilis. Transformation of B. subtilis i s performed as described by Dubnau et al., 1971 , J. M ol. Biol. 56, pp. 209-221.
  • Site-directed mutagenesis in order to introduce mutations in a specific region The overall strategy used to perform site-directed mutagenesis is: Mutagenic primers (oligonucleotides) are synthesized corresponding to the DNA sequence flanking the sites of mutation, separated by the DNA base pairs defining the insertions / deletions / substitutions.
  • 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 sec- ond PCR-reaction, the resulting PCR product is purified and extended in a third PCR- reaction before being digegted 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.
  • the pSX222 expression plasmid comprising a variant of the invention was transformed into a competent B. subtilis strain and fermented as described above.
  • subtilisin variants After fermentation, purification of subtilisin variants is accomplished u sing H ydro- phobic Charge Induction Chromatography (HCIC) and subsequent vacuum filtration.
  • the HCIC uses a cellulose matrix to which 4-Mercapto-Ethyl- Pyridine (4-MEP) is bound. Beads of the cellulose matrix sized 80-100 ⁇ m are mixed with a media containing yeast and the transformed B. subtilis capable of secreting the subtilisin variants and incu- bated at pH 9.5 in Unifilter ® microplates.
  • the pH is now lowered by washing the filter with a n elution b uffer ( pH 5 ) H ereby the e nzymes part from the beads a nd can be retrieved from the buffer.
  • the concentration of the purified subtilisin enzyme variants is assessed by active site titration (AST).
  • the purified enzyme is incubated with the high affinity inhibitor CI-2A at different concentrations to inhibit a varying amount of the active sites.
  • the protease and inhibitor o binds to each other at a 1 :1 ratio and accordingly the enzyme concentration can be directly related to the concentration of inhibitor, at which all protease is inactive.
  • a substrate 0.6 mM Suc-Ala-Ala-Pro-Phe-pNA in Tris/HCI buffer
  • pNA paranitrophenol
  • the performance of the enzyme variant is measured as the brightness of the colour of the textile samples washed with that specific protease. Brightness can also be expressed as the intensity of the light reflected from the textile sample when illuminated with white light. When the textile is stained the intensity of the reflected light is lower, than that of a c lean t extile. T herefore t he i ntensity o f t he r eflected I ight c an be u sed t o m easure wash performance of a shuffled protease.
  • Colour measurements are made with a professional flatbed scanner (PFU DL2400pro, obtainable from: J.M. Thomsen, Dorfgade 2, Dorf, Dronninglund, DK-9330), 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.
  • Detergents for wash performance tests of the subtilases of the invention can be ob- tained 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).
  • a commercial detergent base without enzymes can be purchased directly from the manufacturer.
  • a suitable model detergent can be purchased and used for wash performance tests.
  • the proteases may also be tested in a model detergent composition comprising
  • subtilases according to the invention e xhibits i mproved wash p erformance o n e gg s tains i n c omparison t o t he wiild type JP170 subtilase with SEQ ID NO:1.
  • REMARK Complex of JP170 and CI2A inhibitor REMARK Contents of asymmetric unit subtilisin 2x (433 a. a. x 2) REMARK CI2A inhibitor 2x (a. a. 16 - 83 and 21 - 83) REMARK small peptide (autodigestion product, a. a.
  • REMARK 3 METHOD USED BABINET MODEL WITH MASK REMARK 3 PARAMETERS FOR MASK CALCULATION REMARK 3 VDW PROBE RADIUS 1.40 REMARK 3 ION PROBE RADIUS 0.80 REMARK 3 SHRINKAGE RADIUS 0.80 REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: REMARK 3 HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS REMARK 3 CISPEP 1 GLY A 163 PRO A 164 00 CISPEP 2 ALA A 171 PRO A 172 00 CISPEP 3 PHE A 191 GLY A 192 00 CISPEP 4 ASN A 199 HIS A 200 00 CISPEP 5 GLY A 208 PRO A 209 00 CISPEP 6 LYS A 216 PRO A 217 00 CISPEP 7 ASP A 236 SER A 237 00 CISPEP 8 ASP A 244 SER A 245 00 CISPEP 9
  • HETATM 3 O10 ASN A 1 19.168 22 .251 -2.661 1.00 13 .33 A O
  • ATOM 106 CA ASN A 15 30 .185 10 .847 3. .469 1, .00 20 .71 A C
  • ATOM 180 C GLN A 24 13 .619 2 .022 2, .352 1 .00 13 .19 A C
  • ATOM 182 N ILE A 25 12 .324 2 .066 2, .692 1 .00 13 .28 A N
  • ATOM 220 C ASP A 30 0, .665 13 .113 -5. .327 1, .00 12 .41 A C
  • ATOM 266 CZ ARG A 37 4, .322 5, .434 -20, .575 1, .00 38, .63 A C
  • ATOM 272 CA ASN A 38 7 .254 1. .998 -13, .703 1, .00 20 .37 A C
  • ATOM 308 CA HIS A 43 12, .479 9 .903 -15, .081 1, .00 17, .38 A C
  • ATOM 326 N ALA A 45 15, .665 9 .954 -12, .152 1, .00 16 .39 A N
  • ATOM 373 O ILE A 50 9 .339 -0 .161 -7, .909 1, .00 18 .69 A O
  • ATOM 382 CA ALA A 52 5, .179 0 .338 -6, .052 1, .00 17 .34 A C
  • ATOM 407 CA ALA A 55 -3 .032 5 .260 -8. .926 1 .00 17 .49 A C
  • ATOM 410 O ALA A 55 -5 .174 4 .400 -8. .269 1 .00 19 .60 A O
  • ATOM 429 CZ ARG A 58 -3. ,248 11, .716 -12. ,392 1. ,00 18. .78 A C
  • ATOM 442 CA ASN A 60 -4, .939 3 .618 -14, ,124 1, .00 21 .57 A C
  • ATOM 458 CA ALA A 62 -0, .418 7 .910 -11, .902 1, .00 16 .34 A C
  • ATOM 461 O ALA A 62 1 .583 9 .221 -11, .728 1, .00 15 .61 A O
  • ATOM 472 CB ASP A 64 3 .450 13 .990 -12, .233 1 .00 14 .59 A C
  • ATOM 478 N PRO A 65 1 .065 15 .644 -14 .476 1 .00 16 .18 A N
  • ATOM 479 CA PRO A 65 0 .950 16 .813 -15 .343 1 .00 17 .33 A C
  • ATOM 482 CD PRO A 65 -0 .249 15 .043 -14 .172 1 .00 17 .21 A
  • C ATOM 483 C PRO A 65 1.228 18.102 -14.607 1.00 17.72 A C

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Abstract

La présente invention a trait à des procédés pour la production d'une subtilase mère JP170 et d'une subtilase mère BPN' et des variants de JP170 et de BPN' présentant des propriétés modifiées par rapport à la subtilase mère JP170/BPN'.
EP04722236A 2003-03-21 2004-03-22 Subtilases Ceased EP1608744A2 (fr)

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US7294499B2 (en) 2003-01-30 2007-11-13 Novozymes A/S Subtilases
WO2006032278A1 (fr) * 2004-09-21 2006-03-30 Novozymes A/S Subtilases
US7741095B2 (en) 2004-09-21 2010-06-22 Novozymes A/S Subtilases
US7473544B2 (en) 2004-10-08 2009-01-06 Kao Corporation Alkaline protease
DE102007032111B4 (de) * 2007-07-09 2017-07-20 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel enthaltend diese Proteasen
JP6067409B2 (ja) 2012-04-10 2017-01-25 花王株式会社 アルカリプロテアーゼの溶解性向上方法
EP2832853A1 (fr) * 2013-07-29 2015-02-04 Henkel AG&Co. KGAA Composition détergente comprenant des variantes de protéases
EP3212785A2 (fr) * 2014-10-30 2017-09-06 Novozymes A/S Variants de protéase et polynucléotides les codant
EP3608403A3 (fr) * 2014-12-15 2020-03-25 Henkel AG & Co. KGaA Composition détergente comprenant des variantes de subtilase
US10400230B2 (en) 2014-12-19 2019-09-03 Novozymes A/S Protease variants and polynucleotides encoding same
US11518987B2 (en) 2014-12-19 2022-12-06 Novozymes A/S Protease variants and polynucleotides encoding same
WO2016097357A1 (fr) 2014-12-19 2016-06-23 Novozymes A/S Variants de protéase et polynucléotides les codant
CN107002059A (zh) 2014-12-19 2017-08-01 诺维信公司 蛋白酶变体以及对其进行编码的多核苷酸
CN111615559B (zh) 2018-01-16 2024-01-16 花王株式会社 角质污垢清洁剂及角质污垢分解能力的评价方法
US11760793B2 (en) 2020-12-18 2023-09-19 University Of Tennessee Research Foundation Antibody for skewing sex ratio and methods of use thereof

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DK386586D0 (da) * 1986-08-14 1986-08-14 Novo Industri As Protease, fremgangsmaade til fremstilling deraf og anvendelse deraf
US5567601A (en) * 1993-06-01 1996-10-22 University Of Maryland Subtilisin mutants lacking a primary calcium binding site
EP0724629A1 (fr) * 1993-10-18 1996-08-07 Degussa Aktiengesellschaft Procede de modification controlee d'enzymes, enzymes modifiees et utilisation
DE69637940D1 (de) * 1995-02-03 2009-07-09 Novozymes As Eine methode zum entwurf von alpha-amylase mutanten mit vorbestimmten eigenschaften
US5891701A (en) * 1997-06-12 1999-04-06 Novo Nordisk Biotech Inc. Nucleic acids encoding a polypeptide having protease activity
EP1277762B1 (fr) * 1998-10-30 2008-03-26 Novozymes A/S Variants proteiques faiblement allergenes
EP2298875B1 (fr) * 2000-03-08 2015-08-12 Novozymes A/S Variants possédant des propriétés modifiées
US6803222B2 (en) * 2000-11-22 2004-10-12 Kao Corporation Alkaline proteases
JP2002218989A (ja) * 2000-11-22 2002-08-06 Kao Corp アルカリプロテアーゼ
JP4143274B2 (ja) * 2001-04-12 2008-09-03 花王株式会社 アルカリプロテアーゼ

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WO2004083362A2 (fr) 2004-09-30
JP2012045000A (ja) 2012-03-08
EP2163616A3 (fr) 2010-05-26
CN1791672A (zh) 2006-06-21
EP2163616A2 (fr) 2010-03-17
CN101851615A (zh) 2010-10-06
AR043746A1 (es) 2005-08-10
WO2004083362A3 (fr) 2004-11-04
JP2006521795A (ja) 2006-09-28
CN101851615B (zh) 2014-08-06

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