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  • 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

• subtilase or "(a) TY145 like subtilase” should in the context of the pre- sent 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 63% homology to TY145, SEQ ID NO:1. In the context of the present invention a TY145 subtilase has three ion-binding sites.
• 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.
• position numbers used in the present invention refer to different subtilases depending on which subgroup the subtilase belongs to.
• subtilases belonging to the TY145 subgroup i.e. subtilases obtained from TY145, TA39, TA41 and Bacillus sphaericus are numbered individually according to each of SEQ ID NO:1 ,2,3 and 4.
• 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 preferably 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 is in the context of the present invention to be understood as a subtilase which has at least 61% homology to SEQ ID NO:5.
• said BPN' subtilase may have at least 70%, such as 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:5.
• subtilase consists of 6-8 helices, 11 strands of which 7 are central in a twisted beta-sheet. Two ion-binding sites are mentioned, one of which is the so called "Weak" calcium-binding site. It was later discovered that for some structures (subtilisin DY PDB no. 1 BH6, 1998), this calcium-binding site was shown to be a Na (sodium) binding site when the calcium concentration in the crystallization medium was low. Thus, in the following we refer to ion-binding sites instead of calcium-binding sites.
• the structure of TY145 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 structural coordinates for the solved crystal structure of TY145 are given in standard PDB format (Protein Data Bank, Brookhaven National Laboratory, Brookhaven, CT) as set forth in Appendix 1. It is to be understood that Appendix 1 forms part of the present application.
• the present invention comprises a method of producing a variant of a parent TY145 like subtilase, the variant having at least one altered property as compared to the parent TY145 like subtilase, the method comprising: a) modelling the parent TY145 subtilase on the three-dimensional structure of a TY145 subtilase to produce a three-dimensional structure of the parent TY145 subtilase; b) comparing the three-dimensional structure obtained in step a) to the three-dimensional structure of a TY145 subtilase; c) identifying on the basis of the comparison in step b) at least one structural part of the parent TY145 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 TY145 subtilase to produce a nucleic acid sequence encoding deletion or substitution of one or more amino acids at a position corresponding to said structural part, or an insertion of one or more amino acid residues in positions
• Removal of the Far site can be done by deletion(s) of or in the region K290-D300 (of SEQ ID NO:1) and subsequent insertion of one or more amino acid residues.
• a preferred variant has the whole region deleted and a subsequent insertion between I289 and Y301 of the sequence GDS or DST.
• the substitution S303Y is further added.
• Removal of the Near site can be done by deletion(s) of or in the region N212-R224 (of SEQ ID NO:1) and subsequent insertion of one or more amino acid residues.
• a preferred variant has the whole region deleted and a subsequent insertion of a proline or alanine residue between G211 and D225.
• TY145 a mesophilic-derived enzyme obtained from crystal structure
• TA41 a psychrophilic derived enzyme obtained from modelling
• Regions closest to the active site and the substrate binding site are regarded as preferred in relation to making higher activity at low temperature for TY145: 40-73, and 140-161 , preferably 65-73 and 140-150.
• the regions in TY145 should be modified to be more mobile for example by substitution with small less rigid residues, i.e. residues with smaller side chains (such as Gly, Ala, Ser, Thr or Val), into the TY145 backbone.
• 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 TY145 subtilase with CI2 bound in the active site can be found in Appendix 1. Without being limited to any theory, it is presently believed that binding between a substrate and an enzyme is supported by favor- able interactions found within a sphere 10 A from the substrate molecule, in particular within a sphere of 6 A from the substrate molecule. Examples of such favorable bonds are hydrogen bonds, strong electrostatic interaction and/or hydrophobic interactions.
• Asn-Gly sequences can be found in the following positions: B. sphaericus: 198-199, 240-241 TY145: 87-88, 109-110, 199-200 TA41 : 83-84, 198-199 TA39: 88-89, 198-199
• the present invention in this respect thus relates to modifications, such as deletions and substitutions in one or more of these positions in accordance with the principles given above.
• subtilase variant described herein may advantageously be com- bined with one or more modification(s) in any of the positions:
• 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 or eukaryotes
• fungal cells e.g. yeasts or filamentous fungi
• insect cells e.g. yeasts or filamentous fungi
• 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.
• Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.
• cellulases are the alkaline or neutral cellulases having colour care benefits.
• Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
• Other examples are cellulase variants such as 5 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.
• 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.
• AMSA wash tests were performed on variants with Segment X and Segments X and XI like described in Example 5 herein.
• the assay was conducted under the experimental conditions specified below:
• HCIC Hydrophobic Charge Induction Chromatography
• 4-MEP 4-Mercapto-Ethyl-Pyridine
• 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 incubated at pH 9.5 in Unifilter ® microplates.
• ATOM 422 C ILE A 29 28, .143 24 .586 9. .459 1, .00 14. .30 c
• ATOM 507 N ASP A 35 21, .571 9, .765 12. .490 1. .00 11, .34 N
• ATOM 604 CA HIS A 42 12 .245 17 .917 1, .419 1, .00 21, .28 C
• ATOM 1069 CA HIS A 75 12 .345 15 .653 11, ,803 1. .00 13, .53 C

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