AU2002320741B2 - Multiply-substituted protease variants - Google Patents
Multiply-substituted protease variants Download PDFInfo
- Publication number
- AU2002320741B2 AU2002320741B2 AU2002320741A AU2002320741A AU2002320741B2 AU 2002320741 B2 AU2002320741 B2 AU 2002320741B2 AU 2002320741 A AU2002320741 A AU 2002320741A AU 2002320741 A AU2002320741 A AU 2002320741A AU 2002320741 B2 AU2002320741 B2 AU 2002320741B2
- Authority
- AU
- Australia
- Prior art keywords
- protease
- positions
- amino acid
- residue
- protease variant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
- 108091005804 Peptidases Proteins 0.000 title claims description 138
- 239000004365 Protease Substances 0.000 title claims description 125
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims 15
- 108090000787 Subtilisin Proteins 0.000 claims description 75
- 238000006467 substitution reaction Methods 0.000 claims description 47
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims description 42
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 33
- 239000000203 mixture Substances 0.000 claims description 33
- 241000193422 Bacillus lentus Species 0.000 claims description 18
- 125000000539 amino acid group Chemical group 0.000 claims description 17
- 238000004140 cleaning Methods 0.000 claims description 14
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 5
- 239000004753 textile Substances 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 3
- 102200025035 rs786203989 Human genes 0.000 description 360
- 102200118280 rs33918343 Human genes 0.000 description 273
- 102000035195 Peptidases Human genes 0.000 description 115
- 235000019419 proteases Nutrition 0.000 description 87
- 239000003599 detergent Substances 0.000 description 65
- 102220245308 rs1555601006 Human genes 0.000 description 44
- 239000002243 precursor Substances 0.000 description 28
- 108090000623 proteins and genes Proteins 0.000 description 26
- 102000004190 Enzymes Human genes 0.000 description 24
- 108090000790 Enzymes Proteins 0.000 description 24
- 229940088598 enzyme Drugs 0.000 description 24
- 102220499813 Carbonic anhydrase 2_N62D_mutation Human genes 0.000 description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 18
- 229940024606 amino acid Drugs 0.000 description 17
- 238000000034 method Methods 0.000 description 17
- 108010056079 Subtilisins Proteins 0.000 description 16
- 102000005158 Subtilisins Human genes 0.000 description 16
- 150000001413 amino acids Chemical class 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 11
- 108010022999 Serine Proteases Proteins 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 102000012479 Serine Proteases Human genes 0.000 description 10
- 102220246481 rs147782267 Human genes 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 102000004157 Hydrolases Human genes 0.000 description 7
- 108090000604 Hydrolases Proteins 0.000 description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 239000000654 additive Substances 0.000 description 6
- 102220351005 c.143C>T Human genes 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 230000002797 proteolythic effect Effects 0.000 description 6
- 230000003197 catalytic effect Effects 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000003780 insertion Methods 0.000 description 5
- 230000037431 insertion Effects 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 102220146036 rs371561776 Human genes 0.000 description 5
- 102220171488 rs760746448 Human genes 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 244000063299 Bacillus subtilis Species 0.000 description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 description 4
- 108090000317 Chymotrypsin Proteins 0.000 description 4
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 4
- 229960002376 chymotrypsin Drugs 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 102220042823 rs35942089 Human genes 0.000 description 4
- 102200027868 rs62516151 Human genes 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 102220553631 CBY1-interacting BAR domain-containing protein 1_K27N_mutation Human genes 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- 102220583535 Small nuclear ribonucleoprotein F_T58S_mutation Human genes 0.000 description 3
- 102220513367 Synaptotagmin-8_H17Q_mutation Human genes 0.000 description 3
- 102220415048 c.113C>G Human genes 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 230000014616 translation Effects 0.000 description 3
- 102220495819 Actin-like protein 6A_H17L_mutation Human genes 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- -1 CA on CA Inorganic materials 0.000 description 2
- 102220499807 Carbonic anhydrase 2_N62H_mutation Human genes 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 102220493167 FGGY carbohydrate kinase domain-containing protein_N43K_mutation Human genes 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- 108010006035 Metalloproteases Proteins 0.000 description 2
- 102220484443 Myb/SANT-like DNA-binding domain-containing protein 1_L42V_mutation Human genes 0.000 description 2
- 102220569900 Phosphoenolpyruvate carboxykinase [GTP], mitochondrial_E89Q_mutation Human genes 0.000 description 2
- 101710118538 Protease Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 108700005078 Synthetic Genes Proteins 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 229940009098 aspartate Drugs 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 102220350531 c.80A>G Human genes 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 108010020132 microbial serine proteinases Proteins 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 235000019833 protease Nutrition 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 102220132395 rs1057515456 Human genes 0.000 description 2
- 102220224495 rs1060503668 Human genes 0.000 description 2
- 102220036452 rs137882485 Human genes 0.000 description 2
- 102200093472 rs1800574 Human genes 0.000 description 2
- 102220341170 rs199565868 Human genes 0.000 description 2
- 102200029981 rs28936700 Human genes 0.000 description 2
- 102200133203 rs3218619 Human genes 0.000 description 2
- 102220261561 rs374050750 Human genes 0.000 description 2
- 102220082985 rs375349172 Human genes 0.000 description 2
- 102200089607 rs398123481 Human genes 0.000 description 2
- 102200047002 rs61752065 Human genes 0.000 description 2
- 102220313493 rs746811389 Human genes 0.000 description 2
- 102220276580 rs752209909 Human genes 0.000 description 2
- 102220087975 rs777340009 Human genes 0.000 description 2
- 102200091604 rs863225072 Human genes 0.000 description 2
- 102200024254 rs864309502 Human genes 0.000 description 2
- 102220098981 rs878854155 Human genes 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 150000003355 serines Chemical class 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 150000003573 thiols Chemical class 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- INMYHLJNCIBJSD-KOKHDOHPSA-N (2s)-2-aminobutanedioic acid;(2s)-2-amino-3-hydroxypropanoic acid;(2s)-2-amino-3-(1h-imidazol-5-yl)propanoic acid Chemical compound OC[C@H](N)C(O)=O.OC(=O)[C@@H](N)CC(O)=O.OC(=O)[C@@H](N)CC1=CNC=N1 INMYHLJNCIBJSD-KOKHDOHPSA-N 0.000 description 1
- 108091005508 Acid proteases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000228257 Aspergillus sp. Species 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- 101000839068 Boana punctata Hylaseptin-P1 Proteins 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 101710140962 Capsid scaffolding protein Proteins 0.000 description 1
- 102220581852 Caspase recruitment domain-containing protein 16_S57P_mutation Human genes 0.000 description 1
- 102220582865 Cellular tumor antigen p53_S37T_mutation Human genes 0.000 description 1
- 102000005575 Cellulases Human genes 0.000 description 1
- 108010084185 Cellulases Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010091443 Exopeptidases Proteins 0.000 description 1
- 102000018389 Exopeptidases Human genes 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 108030000089 Metallocarboxypeptidases Proteins 0.000 description 1
- 102000006166 Metallocarboxypeptidases Human genes 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 241000192041 Micrococcus Species 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 102220481026 Protein FAM151A_N87D_mutation Human genes 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 102220493486 Sodium/calcium exchanger 3_N16D_mutation Human genes 0.000 description 1
- 101710135785 Subtilisin-like protease Proteins 0.000 description 1
- 101710174704 Subtilisin-like serine protease Proteins 0.000 description 1
- 102220483110 TLC domain-containing protein 4_N75D_mutation Human genes 0.000 description 1
- 101710097834 Thiol protease Proteins 0.000 description 1
- 101000621261 Xanthomonas sp. (strain T-22) Xanthomonalisin Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000004442 acylamino group Chemical group 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 229940025131 amylases Drugs 0.000 description 1
- 235000019730 animal feed additive Nutrition 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 102220363630 c.62C>T Human genes 0.000 description 1
- 238000012219 cassette mutagenesis Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000882 contact lens solution Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000002447 crystallographic data Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000002050 diffraction method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- AJIPIJNNOJSSQC-NYLIRDPKSA-N estetrol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H]([C@H](O)[C@@H]4O)O)[C@@H]4[C@@H]3CCC2=C1 AJIPIJNNOJSSQC-NYLIRDPKSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 102200093155 rs104894030 Human genes 0.000 description 1
- 102220225966 rs1064793727 Human genes 0.000 description 1
- 102200115358 rs121918322 Human genes 0.000 description 1
- 102220283756 rs1300016035 Human genes 0.000 description 1
- 102220219210 rs142454490 Human genes 0.000 description 1
- 102220215697 rs200971068 Human genes 0.000 description 1
- 102200129657 rs35278779 Human genes 0.000 description 1
- 102220005516 rs35993097 Human genes 0.000 description 1
- 102220248108 rs377129682 Human genes 0.000 description 1
- 102220015769 rs397517930 Human genes 0.000 description 1
- 102220041795 rs587780747 Human genes 0.000 description 1
- 102220075257 rs796052432 Human genes 0.000 description 1
- 102220093346 rs876661018 Human genes 0.000 description 1
- 102220103399 rs878854207 Human genes 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 108010059841 serine carboxypeptidase Proteins 0.000 description 1
- 239000002453 shampoo Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Detergent Compositions (AREA)
Description
ii .L
AUSTRALIA
Patents Act COMPLETE SPECIFICATION
(ORIGINAL)
Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Genencor International, Inc. and The Procter Gamble Company Actual Inventor(s): Volker Schellenberger, James T Kellis, Christian Paech, Joanne Nadherny, Donald P Naki, Ayrookaran J Poulose, Katherine D Collier, Robert M Caldwell, Andre Cesar Baeck Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: MULTIPLY-SUBSTITUTED PROTEASE VARIANTS Our Ref: 684711 POF Code: 2420/2420, 44135 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- WO 99/20770 PCT/US98/22572 -1A- MULTIPLY-SUBSTITUTED PROTEASE VARIANTS Related Applications The present application is a divisional application of Australian patent application no. 12762/99.
Background of the Invention Serine proteases are a subgroup of carbonyl hydrolases. They compnse a diverse class of enzymes having a wide range of specificities and biological functions. Stroud, R. Sci. Amer., 131:74-88. Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct families of enzymes: 1) the subtilisins and 2) the mammalian chymotrypsin-related and homologous bacterial serine proteases trypsin and S. gresius trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis. Kraut. J. (1977), Annu. Rev. Biochem., 46:331-358.
Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families bring together a conserved catalytic triad of amino acids consisting of serine. histidine and aspartate.
Subtilisins are serine proteases (approx. MW 27.500) which are secreted in large amounts from a wide variety of Bacillus species and other microorganisms.
The protein sequence of subtilisin has been determined from at least nine different species of Bacillus. Markland, et al. (i983), Hoppe-Seyler's Z. Physiol. Chem., 364:1537-1540. The three-dimensional crystallographic structure of subtilisins from Bacillus amyloliquefaciens, Bacillus licheniforimis and several natural variants of B.
lentus have been reported. These studies indicate that although subtilisin is genetically unrelated to the mammalian serine proteases, it has a similar active site structure. The x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, et al. (1972), Biochemistry, 11:2439-2449) or product complexes (Robertus, et al. (1976), J. Biol. Chem., 251:1097-1103) have also provided information regarding the active site and putative substrate binding cleft of subtilisin. In addition, a large number of kinetic and chemical Biol. Chem. 244:5333-5338) and extensive site-specific mutagenesis has been carried out (Wells and Estell (1988) TIBS 13:291-297) Summary of the Invention The present invention provides an isolated protease variant comprising an amino acid substitution at a residue position corresponding to position 103 and an amino acid substitution at a residue corresponding to position 245 of Bacillus amyloliquefaciens subtilisin and one or more amino acid substitutions at residue positions selected from the group consisting of residue positions corresponding to positions 1, 3, 4, 8,10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131,133, 134, 137, 140, 141,142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181,182, 183, 184, 185,188, 192, 194, 198,203, 204, 205, 206, 209, 210,211,212,213, 214,215, 216, 217,218, 222, 224, 227,228, 230,232, 236,237, 238,240,242,243, 244, 246,247,248,249, 251,252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271,272, 274 and 275 of Bacillus amyloliquefaciens subtilisin; wherein when a substitution at a position corresponding to residue position 103 is combined with a substitution at a position corresponding to residue position 76, there is also a substitution at one or more residue positions other than residue positions corresponding to positions 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265, or 274 of Bacillus amyloliquefaciens subtilisin While any combination of the above listed amino acid substitutions may be employed, the preferred protease variant enzymes useful for the present invention comprise the substitution of amino acid residues in the following combinations of positions. All of the residue positions correspond to positions of Bacillus amyloliquefaciens subtilisin: a protease variant including substitutions of the amino acid residues at position 103 and at one or more of the following positions 236 and 245; a protease variant including substitutions of the amino acid residues at positions 103 and 236 and at one or more of the following positions 1, 9, 12, 22 September 2005 -2a- 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 248, 252, 257, 260, 270 and 275; a protease variant including substitutions of the amino acid residues at positions 103 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 170, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 222, 230, 232, 248, 252, 257, 260, 261,270 and 275; or 22 September 2005 a protease variant including substitutions of the amino acid resioues-at positions 103, 236 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 243, 248, 252, 257, 260, 270 and 275.
More preferred protease variants are substitution sets selected from the group consisting of residue positions corresponding to positions in Table 1 of Bacillus amyloliquefaciens subtilisin: Even more preferred are substitution sets selected from the group consisting a residue position corresponding to positions in Table 2 of Bacillus amyloliquefaciens subtilisin.
Table 1 76 103 104 222 76 98 103 104 76 78 103 104 76 103 104 107 4 76 103 104 76 103 104 246 76 77 103 104 76 103 104 183 218 16 76 103 104 248 1 76 103 104 76 103 104 261 76 103 104 160 76 103 104 216 17 76 103 104 37 76 103 104 76 77 103 104 174 38 76 103 104 38 76 103 104 237 8 76 103 104 76 103 104 183 -4 19 76 103 104 13 76 103 104 19 76 103 104 76 103 104 184 76 103 104 252 76 103 104 259 76 103 104 251 76 86 103 104 72 76 103 104 185 76 103 104 237 274 76 103 104 160 '1 76 103 104 228 76 103 104 240 76 103 104 254 76 103 104 204 76 103 104 204 43 76 103 104 76 103 104 159 76 103 104 177 58 76 103 104 76 103 104 270
U,
00 i3 76 103 104 185 27 76 103 104 76 103 104 262 76 78 103 104 24 76 103 104 76 103 104 166 236 251 17 76 103 104 237 76 103 104 130 76 103 104 109 76 99 103 104 204 76 103 104 181 12 76 103 104 76 103 104 212 271 76 103 104 252 261 76 103 104 242 76 103 104 271 12 76 103 104 242 43 76 103 104 116 183 76 103 104 258 76 103 104 271 61 76 103 104
U'
go k4 38 76 103 104 182 263 76 103 104 182 272 76 103 104 109 246 76 87 103 104 206 249 265 76 103 104 137 238 271 103 104 228 76 103 104 182 198 21 76 103 104 182 76 103 104 119 137 76 103 104 137 248 13 76 103 104 4?06 76 103 104 206 76 103 104 212 258 58 76 103 104 271 76 103 104 206 261 4 76 103 104 206 76 77 103 104 206 76 103 104 158 76 103 104 206 4 76 103 104 159 217 251 4 76 103 104 159 217 252 '0 00 76 77 103 104 133 185 251 76 103 104 159 206 244 4 76 103 104 188 4 76 103 104 158 76 77 103 104 185 76 103 104 206 251 48 76 103 104 111 159 68 76 103 104 159 236 42 76 103 104 159 12 62 76 103 104 159 42 76 103 104 o'59 76 103 104 146 159 76 103 104 159 238 76 103 104 159 224 76 103 104 212 268 271 76 89 103 104 76 87 103 104 212 271 76 103 104 212 245 271 76 103 104 134 141 212 271 76 103 104 212 236 243 271 76 103 104 109 245
U'
-3
IJ
76 103 104 109 210 62 76 103 104 68 76 103 104 236 68 76 103 104 159 236 271 68 76 103 104 159 236 245 68 76 103 104 159 217 236 271 17 68 76 103 104 68 76 103 104 68 76 103 104 159 236 68 75 76 103 104 159 236 68 76 76 103 ,,114 121 159 236 245 12 68 76 103 104 159 236 68 76 103 104 159 209 236 253 68 76 103 104 117 159 184 236 68 76 103 104 159 236 243 68 76 103 104 159 236 245 68 76 103 104 142 159 68 76 103 104 123 159 236 249 68 76 103 104 159 236 249 76 103 104 222 245 12 76 103 104 222 249 00
U'
-4 toJ 76 103 104 173 222 76 103 104 222 263 21 76 103 104 222 237 263 76 103 104 109 222 76 103 104 109 222 271 61 76 103 104 222 76 103 104 137 222 76 103 104 109 222 248 76 103 104 222 249 68 76 103 104 159 236 245 261 68 76 103 104 141 159 236 245 255 68 76 103 104 159 236 245 247 68 76 103 104 159 174 204 236 245 68 76 103 104 159 204 236 245 68 76 103 104 133 159 218 236 245 68 76 103 104 159 232 236 245 68 76 103 104 159 194 203 236 245 12 76 103 104 222 245 76 103 104 232 245 24 68 76 103 104 159 232 236 245 68 103 104 159 232 236 245 252
I
00
LA
-J
68 76 103 104 159 213 232 236 245 260 12 76 103 104 222 244 245 12 76 103 222 210 245 12 76 103 104 130 222 245 22 68 76 103 104 68 76 103 104 184 68 103 104 159 232 236 245 248 252 68 103 104 159 232 236 245 68 103 104 140 159 232 236 245 252 43 68 103 104 159 232 236 245 252 43 68 103 104 ,159 232 236 245 43 68 103 104 159 232 236 245 252 68 87 103 104 159 232 236 245 252 275 12 76 103 104 130 222 245 248 262 12 76 103 104 130 215 222 245 12 76 103 104 130 222 227 245 262 12 76 103 104 130 222 245 261 76 103 104 130 222 245 12 76 103 104 130 218 222 245 262 269 12 57 76 103 104 130 222 245 251 12 76 103 104 130 170 185 222 243 245
I,.
'C
-4 '4 12 76 103 104 130 222 245 268 12 76 103 104 130 222 210 245 68 103 104 159 232 236 245 257 68 103 104 116 159 232 236 245 68 103 104 159 232 236 245 248 68 103 104 159 232 236 245 68 103 104 159 203 232 236 245 68 103 104 159 232 236 237 245 68 76 79 103 104 159 232 236 245 68 103 104 159 183 232 236 245 68 103 104 159 74 206 232 236 245 68 103 104 159 188 232 236 245 68 103 104 159 230 232 236 245 68 98 103 104 159 232 236 245 68 103 104 159 215 232 236 245 68 103 104 159 232 236 245 248 68 76 103 104 159 232 236 245 68 76 103 104 159 210 232 236 245 68 76 103 104 159 232 236 245 257 76 103 104 232 236 245 257 68 103 104 159 232 236 245 257 275 0
LA
-4 k'J 76 103 104 257 275 68 103 104 159 224 232 236 245 257 76 103 104 159 232 236 245 257 68 76 103 104 159 209 232 236 245 68 76 103 104 159 211 232 236 245 12 68 76 103 104 159 214 232 236 245 68 76 103 104 159 215 232 236 245 12 68 76 103 104 159 232 236 245 68 76 103 104 159 232 236 245 259 68 87 76 103 104 159 232 236 245 260 68 76 103 104 169 232 236 245 261 76 103 104 232 236 242 245 68 76 103 104 159 210 232 236 245 12 48 68 76 103 104 159 232 236 245 76 103 104 232 236 245 76 103 104 159 192 232 236 245 76 103 104 147 159 232 236 245 248 251 12 68 76 103 104 159 232 236 245 272 68 76 103 104 159 183 206 232 236 245 68 76 103 104 159 232 236 245 256 68 76 103 104 159 206 232 236 245
C,
Co
-J
27 68 76 103 104 159 232 236 245 68 76 103 104 116 159 170 185 232 236 245 61 68 103 104 159 232 236 245 248 252 43 68 103 104 159 232 236 245 248 252 68 103 104 159 212 232 236 245 248 252 68 103 104 99 159 184 232 236 245 248 252 103 104 159 232 236 245 248 252 68 103 104 159 209 232 236 245 248 252 68 103 104 109 159 232 236 245 248 252 68 103 104 159 232 236 245 248 252 68 103 104 159 209 232 236 245 248 252 68 103 104 159 232 236 245 248 252 261 68 103 104 159 185 232 236 245 248 252 68 103 104 159 210 232 236 245 248 252 68 103 104 159 185 210 232 236 245 248 252 68 103 104 159 212 232 236 245 248 252 68 103 104 159 213 232 236 245 248 252 68 103 104 213 232 236 245 248 252 68 103 104 159 215 232 236 245 248 252 68 103 104 159 216 232 236 245 248 252 68 103 104 159 232 236 245 248 252
C,
t4 4n
W
68 103 104 159 173 232 236 245 248 252 68 103 104 159 232 236 245 248 251 252 68 103 104 159 206 232 236 245 248 252 68 103 104 159 232 236 245 248 252 68 103 104 159 232 236 245 248 252 68 103 104 159 232 236 245 248 252 255 68 103 104 159 232 236 245 248 252 256 68 103 104 159 232 236 245 248 252 260 68 103 104 159 232 236 245 248 252 257 68 103 104 159 232 236 245 248 252 258 8 68 103 104 f59 232 236 245 248 252 269 68 103 104 116 159 232 236 245 248 252 260 68 103 104 159 232 236 245 248 252 261 68 103 104 159 232 236 245 248 252 261 68 76 103 104 159 232 236 245 248 252 68 103 104 232 236 245 248 252 103 104 159 232 236 245 248 252 68 103 104 159 232 236 245 248 252 18 68 103 104 159 232 236 245 248 252 68 103 104 159 232 236 245 248 252 68 76 101 103 104 159 213 218 232 236 245 260 00 b\J
-J
miJ 68 103 104 159 228 232 236 245 248 252 33 68 76 103 104 159 232 236 245 248 252 68 76 89 103 104 159 210 213 232 236 245 260 61 68 76 103 104 159 232 236 245 248 252 103 104 159 205 210 232 236 245 61 68 103 104 130 159 232 236 245 248 252 61 68 103 104 133 137 159 232 236 245 248 252 61 103 104 133 159 232 236 245 248 252 68 103 104 159 232 236 245 248 252 68 103 104 159 218 232 236 245 248 252 61 68 103 104 4*59 160 232 236 245 248 252 3 61 68 76 103 104 232 236 245 248 252 61 68 103 104 159 167 232 236 245 248 252 97 103 104 159 232 236 245 248 252 98 103 104 159 232 236 245 248 252 99 103 104 159 232 236 245 248 252 101 103 104 159 232 236 245 248 252 102 103 104 159 232 236 245 248 252 103 104 106 159 232 236 245 248 252 103 104 109 159 232 236 245 248 252 103 104 159 232 236 245 248 252 261 00, 14j 62 103 104 159 232 236 245 248 252 103 104 159 184 232 236 245 248 252 103 104 159 166 232 236 245 248 252 103 104 159 217 232 236 245 248 252 62 103 104 159 213 232 236 245 248 252 62 103 104 159 213 232 236 245 248 252 103 104 159 206 217 232 236 245 248 252 62 103 104 159 206 232 236 245 248 252 103 104 130 159 232 236 245 248 252 103 104 131 159 232 236 245 248 252 27 103 104 159 ?32 236 245 248 252 38 103 104 159 232 236 245 248 252 38 76 103 104 159 213 232 236 245 260 68 76 103 104 159 213 232 236 245 260 271 68 76 103 104 159 209 213 232 236 245 260 68 76 103 104 159 210 213 232 236 245 260 68 76 103 104 159 205 213 232 236 245 260 68 76 103 104 159 210 232 236 245 260 68 103 104 159 213 232 236 245 260 76 103 104 159 213 232 236 245 260 68 103 104 159 209 232 236 245
C',
00
U'
68 103 104 159 210 232 236 245 68 103 104 159 230 232 236 245 68 103 104 159 126 232 236 245 68 103 104 159 205 232 236 245 68 103 104 159 210 232 236 245 103 104 159 230 236 245 68 103 104 159 232 236 245 260 103 104 159 232 236 245 68 103 104 159 174 232 236 245 257 68 103 104 159 194 232 236 245 257 68 103 104 159 209 232 236 245 257 103 104 159 232 236 245 257 68 76 103 104 159 213 232 236 245 260 261 68 103 104 159 232 236 245 257 261 103 104 159 213 232 236 245 260 103 104 159 210 232 236 245 248 252 103 104 159 209 232 236 245 257 68 76 103 104 159 210 213 232 236 245 260 12 103 104 159 209 213 232 236 245 260 103 104 209 232 236 245 257 103 104 159 205 210 213 232 236 245 260
U,
a0 00 t,J 103 104 159 205 209 232 236 245 260 68 103 104 159 205 209 210 232 236 245 103 104 159 205 209 210 232 236 245 257 103 104 159 205 209 232 236 245 257 68 103 104 159 205 209 210 232 236 245 260 103 104 159 205 209 210 232 236 245 103 104 159 209 210 232 236 245 103 104 159 205 210 232 236 245 68 103 104 128 159 232 236 245 48 103 104 159 230 236 245 48 68 103 104 169 209 232 236 245 48 68 103 104 159 232 236 245 248 252 48 68 103 104 159 232 236 245 257 261 102 103 104 159 212 232 236 245 248 252 12 102 103 104 159 212 232 236 245 248 252 101 102 103 104 159 212 232 236 245 248 252 98 102 103 104 159 212 232 236 245 248 252 102 103 104 159 213 232 236 245 248 252 103 104 131 159 232 236 245 248 252 103 104 159 184 232 236 245 248 252 103 104 159 232 236 244 245 248 252 00 '1 Nl 62 103 104 159 213 232 236 245 248 252 256 12 62 103 104 159 213 232 236 245 248 252 101 103 104 159 185 232 236 245 248 252 101 103 104 159 206 232 236 245 248 252 101 103 104 159 213 232 236 245 248 252 98 102 103 104 159 232 236 245 248 252 101 102 103 104 159 232 236 245 248 252 98 102 103 104 159 212 232 236 245 248 252 98 102 103 104 159 212 232 236 248 252 62 103 104 109 159 213 232 236 245 248 252 62 103 104 159 12 213 232 236 245 248 252 62 101 103 104 159 212 213 232 236 245 248 252 103 104 159 232 245 248 252 103 104 159 230 245 62 103 104 130 159 213 232 236 245 248 252 101 103 104 130 159 232 236 245 248 252 101 103 104 128 159 232 236 245 248 252 62 101 103 104 159 213 232 236 245 248 252 62 103 104 128 159 213 232 236 245 248 252 62 103 104 128 159 213 232 236 245 248 252 101 103 104 159 232 236 245 248 252 260
C,
EZ3
CA
-J
101 103 104 131 159 232 236 245 248 252 98 101 103 104 159 232 236 245 248 252 99 101 103 104 159 232 236 245 248 252 101 103 104 159 212 232 236 245 248 252 101 103 104 159 209 232 236 245 248 252 101 103 104 159 210 232 236 245 248 252 101 103 104 159 205 232 236 245 248 252 101 103 104 159 230 236 245 101 103 104 159 194 232 236 245 248 252 76 101 103 104 159 194 232 236 245 248 252 101 103 104 159 230 232 236 245 248 252 62 103 104 159 185 206 213 232 236 245 248 252 271 %0 ,a 22 Most preferred protease variants are those shown in Table 3.
It is a further aspect to provide DNA sequences encoding such protease variants, as well as expression vectors containing such variant DNA sequences.
Still further, another aspect of the invention is to provide host cells transformed with such vectors, as well as host cells which are capable of expressing such DNA to produce protease variants either intracellularly or extracellularly.
There is further provided a cleaning composition comprising a protease variant of the present invention.
Additionally, there is provided an animal feed comprising a protease variant of the present invention.
Also provided is a composition for the treatment of a textile comprising a protease variant of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1 A-C depict the DNA and amino acid sequence for Bacillus amyloliquefaciens subtilisin and a partial restriction map of this gene.
Fig. 2 depicts the conserved amino acid residues among subtilisins from Bacillus amyloliquefaciens (BPN)' and Bacillus lentus (wild-type).
Figs. 3A and 3B depict the amino acid sequence of four subtilisins. The top line represents the amino acid sequence of subtilisin from Bacillus amyloliquefaciens subtilisin (also sometimes referred to as subtilisin BPN'). The second line depicts the amino acid sequence of subtilisin from Bacillus subtilis. The third line depicts the amino acid sequence of subtilisin from B. licheniformis. The fourth line depicts the amino acid sequence of subtilisin from Bacillus lentus (also referred to as subtilisin 309 in PCT WO89/06276). The symbol denotes the absence of specific amino acid residues as compared to subtilisin
BPN'.
Detailed Description of the Invention Proteases are carbonyl hydrolases which generally act to cleave peptide bonds of proteins or peptides. As used herein, "protease" means a naturally-occurring protease or a recombinant protease. Naturally-occurring proteases include a-aminoacylpeptide hydrolase, peptidylamino acid hydrolase, acylamino hydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiol proteinase, carboxylproteinase and metalloproteinase.
Serine, metallo, thiol and acid proteases are included, as well as endo and exo-proteases.
WO 99/20770 PCT/US98/22572 23 The present invention includes protease enzymes which are non-naturally occurring carbonyl hydrolase variants (protease variants) having a different proteolytic activity, stability, substrate specificity, pH profile and/or performance characteristic as compared to the precursor carbonyl hydrolase from which the amino acid sequence of the variant is derived. Specifically, such protease variants have an amino acid sequence not found in nature, which is derived by substitution of a plurality of amino acid residues of a precursor protease with different amino acids. The precursor protease may be a naturally-occurring protease or a recombinant protease.
The protease variants useful herein encompass the substitution of any of the nineteen naturally occurring L-amino acids at the designated amino acid residue positions.
Such substitutions can be made in any precursor subtilisin (procaryotic, eucaryotic, mammalian, etc.). Throughout this application reference is made to various amino acids by way of common one and three-letter codes. Such codes are identified in Dale, M.W.
(1989), Molecular Genetics of Bacteria, John Wiley Sons, Ltd., Appendix B.
The protease variants useful herein are preferably derived from a Bacillus subtilisin.
More preferably, the protease variants are derived from Bacillus lentus subtilisin and/or subtilisin 309.
Subtilisins are bacterial or fungal proteases which generally act to cleave peptide bonds of proteins or peptides. As used herein, "subtilisin" means a naturally-occurring subtilisin or a recombinant subtilisin. A series of naturally-occurring subtilisins is known to be produced and often secreted by various microbial species. Amino acid sequences of the members of this series are not entirely homologous. However, the subtilisins in this series exhibit the same or similar type of proteolytic activity. This class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases. The subtilisins and chymotrypsin related serine proteases both have a catalic triad comprising aspartate, histidine and serine. In the subtilisin related proteases the relative order of these amino acids, reading from the amino to carboxy terminus, is aspartate-histidine-serine. In the chymotrypsin related proteases, the relative order, however, is histidine-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases.
Examples include but are not limited to the subtilisins identified in Fig. 3 herein. Generally and for purposes of the present invention, numbering of the amino acids in proteases corresponds to the numbers assigned to the mature Bacillus amyloliquefaciens subtilisin sequence presented in Fig. 1.
WO 99/20770 PCT/US98/22572 24 "Recombinant subtilisin" or "recombinant protease" refer to a subtilisin or protease in which the DNA sequence encoding the subtilisin or protease is modified to produce a variant (or mutant) DNA sequence which encodes the substitution, deletion or insertion of one or more amino acids in the naturally-occurring amino acid sequence. Suitable methods to produce such modification, and which may be combined with those.disclosed herein, include those disclosed in US Patent RE 34,606, US Patent 5,204,015 and US Patent 5,185,258, U.S. Patent 5,700,676, U.S. Patent 5,801,038, and U.S. Patent 5,763,257.
"Non-human subtilisins" and the DNA encoding them may be obtained from many procaryotic and eucaryotic organisms. Suitable examples of procaryotic organisms include gram negative organisms such as E. coli or Pseudomonas and gram positive bacteria such as Micrococcus or Bacillus. Examples of eucaryotic organisms from which subtilisin and their genes may be obtained include yeast such as Saccharomyces cerevisiae, fungi such as Aspergillus sp.
A "protease variant" has an amino acid sequence which is derived from the amino acid sequence of a "precursor protease". The precursor proteases include naturallyoccurring proteases and recombinant proteases. The amino acid sequence of the protease variant is "derived" from the precursor protease amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is of the "precursor DNA sequence" which encodes the amino acid sequence of the precursor protease rather than manipulation of the precursor protease enzyme perse. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein, as well as methods known to those skilled in the art (see, for example, EP 0 328299, W089/06279 and the US patents and applications already referenced herein).
Specific substitutions corresponding o position 103 in combination with one or more of the following substitutions corresponding to positions 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61,62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens subtilisin are identified herein.
WO 99/20770 PCTIUS98/22572 25 Preferred variants are those having combinations of substitutions at residue positions corresponding to positions of Bacillus amyloliquefaciens subtilisin in Table 1.
More preferred variants are those having combinations of substitutions at residue positions corresponding to positions of Bacillus amyloliquefaciens subtilisin in Table 3.
Further preferred variants are those having combinations of substitutions at residue positions corresponding to positions of Bacillus amyloliquefaciens subtilisin in Table 2.
Table 2 76 103 104 222 245 76 103 104 222 249 68 103 104 159 232 236 245 252 68 76 103 104 159 213 232 236 245 260 22 68 76 103 104 68 103 104 159 232 236 245 248 252 68 103 104 159 232 236 245 68 103 104 140 159 232 236 245 252 43 68 103 104 159w 232 236 245 252 43 68 103 104 159 232 236 245 12 76 103 104 130 222 245 261 76 103 104 130 222 245 68 103 104 159 232 236 245 257 68 76 103 104 159 210 232 236 245 68 103 104 159 224 232 236 245 257 76 103 104 159 232 236 245 257 68 76 103 104 159 211 232 236 245 12 68 76 103 104 159 214 232 236 245 68 76 103 104 159 215 232 236 245 00 -a 12 68 76 103 104 159 232 236 245 68 76 103 104 159 232 236 245 259- 68 76 87 103 104 159 232 236 245 260 68 76 103 104 159 232 236 245 261 12 48 68 76 103 104 159 232 236 245 76 103 104 159 192 232 236 245 76 103 104 147 159 232 236 245 248 251 12 68 76 103 104 159 232 236 245 272 68 76 103 104 159 183 206 232 236 245 68 76 103 104 150V 232 236 245 256 68 76 103 104 159 206 232 236 245 27 68 76 103 104 159 232 236 245 68 103 104 159 212 232 236 245 248 252 103 104 159 232 236 245 248 252 68 103 104 159 209 232 236 245 248 252 68 103 104 109 159 232 236 245 248 252 68 103 104 159 232 236 245 248 252 68 103 104 159 209 232 236 245 248 252 68 103 104 159 210 232 236 245 248 252 68 103 104 159 212 232 236 245 248 252
C,)
cc
U'
4 68 103 104 159 213 232 236 245 248 252 68 103 104 213 232 236 245 248 252 68 103 104 159 215 232 236 245 248 252 68 103 104 159 216 232 236 245 248 252 68 103 104 159 232 236 245 248 252 68 103 104 159 232 236 245 248 252 255 68 103 104 159 232 236 245 248 252 256 68 103 104 159 232 236 245 248 252 260 68 103 104 159 228 232 236 245 248 252 68 76 89 103 104,, 159 210 213 232 236 245 260 68 103 104 159 218 232 236 245 248 252
C,
0 -a WO 99/20770 PCT/US98/22572 29 These amino acid position numbers refer to those assigned to the mature Bacillus amyloliquefaciens subtilisin sequence presented in Fig. 1. The invention, however, is not limited to the mutation of this particular subtilisin but extends to precursor proteases containing amino acid residues at positions which are "equivalent" to the particular identified residues in Bacillus amyloliquefaciens subtilisin. In a preferred embodiment of the present invention, the precursor protease is Bacillus lentus subtilisin and the substitutions are made at the equivalent amino acid residue positions in B. lentus corresponding to those listed above.
A residue (amino acid) position of a precursor protease is equivalent to a residue of Bacillus amyloliquefaciens subtilisin if it is either homologous corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in Bacillus amyloliquefaciens subtilisin having the same or similar functional capacity to combine, react, or interact chemically).
In order to establish homology to primary structure, the amino acid sequence of a precursor protease is directly compared to the Bacillus amyloliquefaciens subtilisin primary sequence and particularly to a set of residues known to be invariant in subtilisins for which sequence is known. For example, Fig. 2 herein shows the conserved residues as between B. amyloliquefaciens subtilisin and B. lentus subtilisin. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of Bacillus amyloliquefaciens subtilisin are defined. Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than or as little as 50% of conserved residues is also adequate to define equivalent residues. Conservation of the catalytic triad, Asp32/His64/Ser221 should be maintained.
Siezen et al. (1991) Protein Enq. 4(7):719-737 shows the alignment of a large number of serine proteases. Siezen et al. refer to the grouping as subtilases or subtilisin-like serine proteases.
For example, in Fig. 3, the amino acid sequence of subtilisin from Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis (carlsbergensis) and Bacillus lentus are aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences shows that there are a number of conserved residues contained in each sequence. These conserved residues (as between BPN' and B. lentus) are identified in Fig. 2.
WO 99/20770 PCT/US98/22572 30 These conserved residues, thus, may be used to define the corresponding equivalent amino acid residues of Bacillus amyloliquefaciens subtilisin in other subtilisins such as subtilisin from Bacillus lentus (PCT Publication No. W089/06279 published July 13, 1989), the preferred protease precursor enzyme herein, or the subtilisin referred to as PB92 (EP 0 328 299), which is highly homologous to the preferred Bacillus lentus subtilisin. The amino acid sequences of certain of these subtilisins are aligned in Figs.
3A and 3B with the sequence of Bacillus amyloliquefaciens subtilisin to produce the maximum homology of conserved residues. As can be seen, there are a number of deletions in the sequence of Bacillus lentus as compared to Bacillus amyloliquefaciens subtilisin. Thus, for example, the equivalent amino acid for Va1165 in Bacillus amyloliquefaciens subtilisin in the other subtilisins is isoleucine for B. lentus and B.
licheniformis.
"Equivalent residues" may also be defined by determining homology at the level of tertiary structure for a precursor protease whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the precursor protease and Bacillus amyloliquefaciens subtilisin (N on N, CA on CA, C on C and O on 0) are within 0.13nm and preferably 0.1nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the protease in question to the Bacillus amyloliquefaciens subtilisin. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.
SI, FZho(h)Ji-Fc(h)\ R factor I, I Fo(h)l Equivalent residues which are functionally analogous to a specific residue of Bacillus amyloliquefaciens subtilisin are defined as those amino acids of the precursor protease which may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the Bacillus amyloliquefaciens subtilisin. Further, they are those residues of the precursor protease (for which a tertiary structure has been obtained by xray crystallography) which occupy an analogous position to the extent that, although the WO 99/0770 PCT/US98/22572 31 main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13nm of the corresponding side chain atoms of Bacillus amyloliquefaciens subtilisin. The coordinates of the three dimensional structure of Bacillus amyloliquefaciens subtilisin are set forth in EPO Publication No. 0 251 446 (equivalent to US Patent 5,182,204, the disclosure of which is incorporated herein by reference) and can be used as outlined above to determine equivalent residues on the level of tertiary structure.
Some of the residues identified for substitution are conserved residues whereas others are not. In the case of residues which are not conserved, the substitution of one or more amino acids is limited to substitutions which produce a variant which has an amino acid sequence that does not correspond to one found in nature. In the case of conserved residues, such substitutions should not result in a naturally-occurring sequence. The protease variants of the present invention include the mature forms of protease variants, as well as the pro- and prepro-forms of such protease variants. The prepro-forms are the preferred construction since this facilitates the expression, secretion and maturation of the protease variants.
"Prosequence" refers to a sequence of amino acids bound to the N-terminal portion of the mature form of a protease which when removed results in the appearance of the "mature" form of the protease. Many proteolytic enzymes are found in nature as translational proenzyme products and, in the absence of post-translational processing, are expressed in this fashion. A preferred prosequence for producing protease variants.
is the putative prosequence of Bacillus amyloliquefaciens subtilisin, although other protease prosequences may be used.
A "signal sequence" or "presequence" refers to any sequence of amino acids bound to the N-terminal portion of a'protease or to the N-terminal portion of a proprotease which may participate in the secretion of the mature or pro forms of the protease. This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the protease gene which participate in the effectuation of the secretion of protease under native conditions.
The present invention utilizes such sequences to effect the secretion of the protease variants as defined herein. One possible signal sequence comprises the first seven amino acid residues of the signal sequence from Bacillus subtilis subtilisin fused to the remainder of the signal sequence of the subtilisin from Bacillus lentus (ATCC 21536).
WO 99/20770 PCT/US98/22572 32 A "prepro" form of a protease variant consists of the mature form of the protease having a prosequence operably linked to the amino terminus of the protease and a "pre" or "signal" sequence operably linked to the amino terminus of the prosequence.
"Expression vector" refers to a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, "plasmid" and "vector" are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.
The "host cells" used in the present invention generally are procaryotic or eucaryotic hosts which preferably have been manipulated by the methods disclosed in US Patent RE 34,606 to render them incapable of secreting enzymatically active endoprotease. A preferred host cell for expressing protease is the Bacillus strain BG2036 which is deficient in enzymatically active neutral protease and alkaline protease (subtilisin). The construction of strain BG2036 is described in detail in US Patent 5,264,366. Other host cells for expressing protease include Bacillus subtilis 1168 (also described in US Patent RE 34,606 and US Patent 5,264,366, the disclosure of which are incorporated herein by reference), as well as any suitable Bacillus strain such as B.
licheniformis, B. lentus, etc.
Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of either replicating vectors encoding the protease variants or expressing the desired protease variant. In the case of vectors which encode the pre- or prepro-form of the protease variant, such variants, when expressed, are typically secreted from the host cell into the host cell medium.
"Operably linked, when describing the relationship between two DNA regions, simply means that they are functionally related to each other. For example, a presequence is operably linked to a peptide if it functions as a signal sequence, WO 99/20770 PCT/US98/22572 33 participating in the secretion of the mature form of the protein most probably involving cleavage of the signal sequence. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
The genes encoding the naturally-occurring precursor protease may be obtained in accord with the general methods known to those skilled in the art. The methods generally comprise synthesizing labeled probes having putative sequences encoding regions of the protease of interest, preparing genomic libraries from organisms expressing the protease, and screening the libraries for the gene of interest by hybridization to the probes. Positively hybridizing clones are then mapped and sequenced.
The cloned protease is then used to transform a host cell in order to express the protease. The protease gene is then ligated into a high copy number plasmid. This plasmid replicates in hosts in the sense that it contains the well-known elements necessary for plasmid replication: a promoter operably linked to the gene in question (which may be supplied as the gene's own homologous promoter if it is recognized, i.e., transcribed, by the host), a transcription termination and polyadenylation region (necessary for stability of the mRNA transcribed by the host from the protease gene in certain eucaryotic host cells) which is exogenous or is supplied by the endogenous terminator region of the protease gene and, desirably, a selection gene such as an antibiotic resistance gene that enables continuous cultural maintenance of plasmidinfected host cells by growth in antibiotic-containing media. High copy number plasmids also contain an origin of replication for the host, thereby enabling large numbers of plasmids to be generated in the cytoplasm without chromosomal limitations. However, it is within the scope herein to integrate multiple copies of the protease gene into host genome. This is facilitated by procaryoticend eucaryotic organisms which are particularly susceptible to homologous recombination.
The gene can be a natural B. lentus gene. Alternatively, a synthetic gene encoding a naturally-occurring or mutant precursor protease may be produced. In such an approach, the DNA and/or amino acid sequence of the precursor protease is determined. Multiple, overlapping synthetic single-stranded DNA fragments are thereafter synthesized, which upon hybridization and ligation produce a synthetic DNA encoding the precursor protease. An example of synthetic gene construction is set forth in Example 3 of US Patent 5,204,015, the disclosure of which is incorporated herein by reference.
WO 99/20770 PCT/US98/22572 34 Once the naturally-occurring or synthetic precursor protease gene has been cloned, a number of modifications are undertaken to enhance the use of the gene beyond synthesis of the naturally-occurring precursor protease. Such modifications include the production of recombinant proteases as disclosed in US Patent RE 34,606 and EPO Publication No. 0 251 446 and the production of protease variants described herein.
The following cassette mutagenesis method may be used to facilitate the construction of the protease variants of the present invention, although other methods may be used. First, the naturally-occurring gene encoding the protease is obtained and sequenced in whole or in part. Then the sequence is scanned for a point at which it is desired to make a mutation (deletion, insertion or substitution) of one or more amino acids in the encoded enzyme. The sequences flanking this point are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide pool which when expressed will encode various mutants. Such restriction sites are preferably unique sites within the protease gene so as to facilitate the replacement of the gene segment. However, any convenient restriction site which is not overly redundant in the protease gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction. Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by M13 primer extension in accord with generally known methods. The task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the ger'etic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site is available, the above method need be used only in connection with the flanking region which does not contain a site.
Once the naturally-occurring DNA or synthetic DNA is cloned, the restriction sites flanking the positions to be mutated are digested with the cognate restriction enzymes and a plurality of end termini-complementary oligonucleotide cassettes are ligated into the gene. The mutagenesis is simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites.
WO 99/20770 PCT/US98/22572 35 As used herein, proteolytic activity is defined as the rate of hydrolysis of peptide bonds per milligram of active enzyme. Many well known procedures exist for measuring proteolytic activity M. Kalisz, "Microbial Proteinases," Advances in Biochemical Engineering/Biotechnology, A. Fiechter ed., 1988). In addition to or as an alternative to modified proteolytic activity, the variant enzymes of the present invention may have other modified properties such as kt, km/Km ratio and/or modified substrate specificity and/or modified pH activity profile. These enzymes can be tailored for the particular substrate which is anticipated to be present, for example, in the preparation of peptides or for hydrolytic processes such as laundry uses.
In one aspect of the invention, the objective is to secure a variant protease having altered, preferably improved wash performance as compared to a precursor protease in at least one detergent formulation and or under at least one set of wash conditions.
There is a variety of wash conditions including varying detergent formulations, wash water volume, wash water temperature and length of wash time that a protease variant might be exposed to. For example, detergent formulations used in different areas have different concentrations of their relevant components present in the wash water.
For example, a European detergent typically has about 4500-5000 ppm of detergent components in the wash water while a Japanese detergent typically has approximately 667 ppm of detergent components in the wash water. In North America, particularly the United States, a detergent typically has about 975 ppm of detergent components present in the wash water.
A low detergent concentration system includes detergents where less than about 800 ppm of detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration system as they have approximately 667 ppm of detergent components present in the wash water.
A medium detergent concentration includes detergents where between about 800 ppm and about 2000ppm of detergent components are present in the wash water. North American detergents are generally considered to be medium detergent concentration systems as they have approximately 975 ppm of detergent components present in the wash water. Brazil typically has approximately 1500 ppm of detergent components present in the wash water.
A high detergent concentration system includes detergents where greater than about 2000 ppm of detergent components are present in the wash water. European detergents are generally considered to be high detergent concentration systems as they have approximately 4500-5000 ppm of detergent components in the wash water.
WO 99/2070 PCT/US98/22572 36 Latin American detergents are generally high suds phosphate builder detergents and the range of detergents used in Latin America can fall in both the medium and high detergent concentrations as they range from 1500 ppm to 6000 ppm of detergent components in the wash water. As mentioned above, Brazil typically has approximately 1500 ppm of detergent components present in the wash water. However, other high suds phosphate builder detergent geographies, not limited to other Latin American countries, may have high detergent concentration systems up to about 6000 ppm of detergent components present in the wash water.
In light of the foregoing, it is evident that concentrations of detergent compositions in typical wash solutions throughout the world varies from less than about 800 ppm of detergent composition ("low detergent concentration geographies"), for example about 667 ppm in Japan, to between about 800 ppm to about 2000 ppm ("medium detergent concentration geographies"), for example about 975 ppm in U.S. and about 1500 ppm in Brazil, to greater than about 2000 ppm ("high detergent concentration geographies"), for example about 4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high suds phosphate builder geographies.
The concentrations of the typical wash solutions are determined empirically. For example, in the a typical washing machine holds a volume of about 64.4 L of wash solution. Accordingly, in order to obtain a concentration of about 975 ppm of detergent within the wash solution about 62.79 g of detergent composition must be added to the 64.4 L of wash solution. This amount is the typical amount measured into the wash water by the consumer using the measuring cup provided with the detergent.
As a further example, different geographies use different wash temperatures.
The temperature of the wash water in Japan is typically less than that used in Europe.
Accordingly one aspect of the present invention includes a protease variant that shows improved wash performance in at Ieast one set of wash conditions.
In another aspect of the invention, it has been determined that substitutions at a position corresponding to 103 in combination with one or more substitutions selected from the group consisting of positions corresponding 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181,182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, WO 99/20770 PCT/US98/22572 37 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens subtilisin are important in improving the wash performance of the enzyme.
These substitutions are preferably made in Bacillus lentus (recombinant or nativetype) subtilisin, although the substitutions may be made in any Bacillus protease.
Based on the screening results obtained with the variant proteases, the noted mutations in Bacillus amyloliquefaciens subtilisin are important to the proteolytic activity, performance and/or stability of these enzymes and the cleaning or wash performance of such variant enzymes.
Many of the protease variants of the invention are useful in formulating various detergent compositions or personal care formulations such as shampoos or lotions. A number of known compounds are suitable surfactants useful in compositions comprising the protease mutants of the invention. These include nonionic, anionic, cationic, or zwitterionic detergents, as disclosed in US 4,404,128 to Barry J. Anderson and US 4,261,868 to Jiri Flora, et al. A suitable detergent formulation is that described in Example 7 of US Patent 5,204,015 (previously incorporated by reference). The art is familiar with the different formulations which can be used as cleaning compositions. In addition to typical cleaning compositions, it is readily understood that the protease variants of the present invention may be used for any purpose that native or wild-type proteases are used. Thus, these variants can be used, for example, in bar or liquid soap applications, dishcare formulations, contact lens cleaning solutions or products, peptide hydrolysis, waste treatment, textile applications, as fusion-cleavage enzymes in protein production, etc. The variants of the present invention may comprise enhanced performance in a detergent composition (as compared to the precursor). As used herein, enhanced performance in a detergent is defined as increasing cleaning of certain enzyme sensitive stains such as grass or blood, as determined by usual evaluation after a standard wash cycle.
Proteases of the invention can be formulated into known powdered and liquid detergents having pH between 6.5 and 12.0 at levels of about 0.01 to about (preferably 0.1% to by weight. These detergent cleaning compositions can also include other enzymes such as known proteases, amylases, cellulases, lipases or endoglycosidases, as well as builders and stabilizers.
The addition of proteases of the invention to conventional cleaning compositions does not create any special use limitation. In other words, any temperature and pH suitable for the detergent is also suitable for the present compositions as long as the pH 38 is within the above range, and the temperature is below the described protease's denaturing temperature. In addition, proteases of the invention can.be used in a cleaning composition without detergents, again either alone or in combination with builders and stabilizers.
The present invention also relates to cleaning compositions containing the protease variants of the invention. The cleaning compositions may additionally contain additives which are commonly used in cleaning compositions. These can be selected from, but not limited to, bleaches, surfactants, builders, enzymes and bleach catalysts. It would be readily apparent to one of ordinary skill in the art what additives are suitable for inclusion into the compositions. The list provided herein is by no means exhaustive and should be only taken as examples of suitable additives. It will also be readily apparent to one of ordinary skill in the art to only use those additives which are compatible with the enzymes and other components in the composition, for example, surfactant When present, the amount of additive present in the cleaning composition is from about 0.01% to about 99.9%, preferably about 1% to about 95%, more preferably about 1% to about The variant proteases of the present invention can be included in animal feed such as part of animal feed additives as described in, for example, US 5,612,055;
US
5,314,692; and US 5,147,642.
One aspect of the invention is a composition for the treatment of a textile that includes variant proteases of the present invention. The composition can be used to treat for example silk or wool as described in publications such as RD 216,034;
EP
134,267; US 4,533,359; and EP 344,259.
The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.
All publications and patents refere'iced herein are hereby incorporated by reference in their entirety.
The above discussion of background art is included to explain the context of the invention. It is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in Australia at the priority date of any of the claims of this specification.
Throughout the description and claims of this specification the word "comprise" and variations of that word such as "comprises" and "comprising" are not intended to exclude other additives, components, integers or steps.
Example 1 A large number of protease variants were produced and purified using methods well known in the art. All mutations were made in Bacillus lentus GG36 subtilisin. The variants are shown in Table 3.
Table 3 N76D S103A V1041 M222S N76D A98E S103A V1041 N76D S7BT S103A V1041 N76D S103A V1041 11 07V V4E N76D S103A V1041 N76D S103A V1041 1246V N76D N77D S103AI V1041 N76D S103A V1041 N183D N2181 A16T N76D S103A V1041 N248D AME N76D S103A V1041 N76D S103A V1041 N261D N76D S103A V1041 S160T N76D S103A V1041 S216C H17Q N76D S103A V1041 S37T N76D S103A V1041 N76D N77D IS103A V1041 A174 V T3SN76D S103A V1041
U,
00
N
U,
-4
N
T38S N76D S103A V1041 K237Q I8V N76D S103A V1041 N76D S103A V1041 N183D R19L N76D S103A V1041 A13V N76D S103A V1041 R19C N76D S103A V1041 N76D S103A V1041 N184D N76D S103A V1041 N252D N76D S103A V1041 S259C N76D S103A V1041 K251T N76D P86S S103AI V1041 172V N76D S103A V1041 N185D N76D S103A V1041 K237E T274A N76D S103A V1041 S160L N76D S103A V1041 A228V N76D S103A V1041 S240T N76D S103A V1041 A254T N76D S103A 11 04N N204T N76D S103A V1041 N204DI
U,
'0 00
U,
.4 N43S N76D S103AI V1041 N76D S103A V11041 IG159D N76D S103A I'V1041 V177A T58S N76D S103AI V1041 N76D S103A V110411A270V N76D S103A V11041IN185D K27N N76D S103A V11041 N76D S103A V1041 L262M N76D S78P 013A Vt041 S24P N76D S103A V1041 N76D S103A V1041 S166G Q236R K251R H17L N76D S103AI V1041 K237E N76D S103A V11041 S130L N76D S103A V11041 Q109R N76D S99R S103A V11041 N204T N76D S103A V1041 D181N Q12R N76D S103A V11041 N76D S103A V11041 S212P E271V IN76D IS103AI '11041 IN252KI N261YI
C,,
'0 1 N76D S103A V1041 IS242T N76D S103A V1041JE271Q Q12R N76D S103AI V1041 S242T N43S N76D S103AI V1041 N116K N1831 N76D S103A V1041 G258R N76D 8103A V1041 E271 G G61 R N76D S103A V1041 T38S N76D S103A V1041 Q182R Y263H N76D S103A V1041 Ql182R A272S N76D S103A V1041 Q109R 1246V N76D S87G S.1 03A V1041 Q206R H249Q S265G N76D S103A V1041 Q137R N238Y E271V S103A V1041 A228T N76D S103A V1041 Q182R 1198V L21 M N76D S103A V1041 Q182R N76D S103A V1041 Ml1191 0137R N76D S103A V1041 Q137R N248S A13T N76D S103A V1041 Q206R N76D S103A V1041 Q206R 0 '0
GO
U'
-i N76D S103A V1041 S212P G258R T58S N76D S103A V1041 E271G N76D S103A V1041 Q206E N261 D V4E N76D S103A V1041 Q206E N76D N77D S103A V1041 Q206E N76D S103A V1041 A158E N76D S103A V1041 Q206E V4E N76D S103A V1041 G159D L217E K251Q V4E N76D S103A A!041 G159D L21.7E N252D N76D N77D S103A V1041 A133T N185D K251T N76D S103A V1041 G159D Q206E V244A V4E N76D S103A V1041 S188E V4E N76D S103A V1041 A158E N76D N77D S103A V1041 N 185D N76D S103A V1041 Q206E K251T A48T N76D S103A V1041 L111M G159D V68A N76D S103AI V1041 G159D Q236H L42V N76D S103A V1041 G159D Q12H N62H N76D S103A V1041 G159D ~0 t 1 L421 N76D S103A V1041 G159D N76D S103A V1041 G146S G159D N76D S103A V1041 G159D N238S N76D S103A V1041 G159D T224A N76D S103A V1041 S212P V268F E271.V N76D E89A S103A V1041 N76D S87R S103A V1041 S212P E271V N76D S103A V1041 S212P Q245L E271V N76D S103A V1041 T1l4S S141N S212P E271V N76D S103A V1041 S212P Q236L N243S E271V N76D S103A V1041 Q109R Q245R N76D S103A V1041 Q109R P210L N 62S N76D S103A V1041 V68A N76D S103A V1041 Q236H V/68A N76D S103AI V1041 G159D IQ236H E271V V68A N76D S103A V1041 G159D Q236H Q245R V68A N76D S103A V1041 G159D L2171 Q236H E271V H17Q V68A N76D S103A V1041 V68A N76D S103A V1041 00 k4 ul V68A N76D S103AI V1041 G159D Q236R V68A L75R N76D S103A V1041 G159D Q236H V68A N76D N76D S103A A114V V1211 G159D Q236H Q245R Q12R V68A N76D S103A V1041 G159D Q236H V68A N76D S103A V1041 G159D Y209S Q236H T253K V68A N76D S103A V1041 N117K G159D N184S Q236H V68A N76D S103A V1041 G159D Q236H N2431 V68A N76D S103A V1041 G159D Q236H 0245L V68A N76D S103A vib41 A142V G159D V68A N76D S103A V1041 N123S G159D Q236H H249Y V68A N76D S103A V1041 G159D Q236H H249Q N76D S103A V1041 M222S Q245R N76D S103A V1041 Q12R M222S H249R N76D S103A V1041 N173R M222S N76D S103A V1041 M222S Y263F L21M N76D S103A V1041 M222S K237R Y263F N76D S103A V1041 Q109R M222S N76D S103A V1041 Q109R M222S E271D G61 R N76D S103A V1041 M222S k-n N76D S103A V1041 Q137R M222S N76D S103A V1041 Q109R M222S N248S N76D S103A V1041 M222S H249R V68A N76D S103A V1041 G159D Q236H Q245R N261D V68A N76D S103A V1041 S141N G159D Q236H Q245R T255S V68A N76D S103A V1041 G159D Q236H Q245R R247H V68A N76D S103A V1041 G159D A174V N204D Q236H 0245R V68A N76D S103AI V1041 G159D N204D Q236H Q245R V68A N76D S103AI Vi041 A133V G1519D N218D Q236H Q245R V68A N76D S103A V1041 G159D A232V Q236H Q245R V68A N76D S103A V1041 G159D A1941 V203A Q236H Q245R Q12R N76D S103A V1041 M222S Q245R N76D S103A V1041 A232V Q245R S24T V68A N76D S103A V1041 G159D A232V Q236H Q245R V68A S103A V1041 G159D A232V Q236H Q245R N252K V68A N76D S103A V1041 G159D T213R A232V Q236H Q245R T260A Q12R N76D S103A 1104T M222S V2441 0245R Q12R N76D S103A M222S P210T Q245R Q512-R N76 S13A 11 04T IS1 3OT -M222S Q245R 00 h~J -a N76D S103A V1041 Q137R M222S N76D S103A V1041 Q109R M222S N248S N76D S103A V1041 M222S H249R V68A N76D S103A V1041 G159D Q236H Q245R N261D V68A N76D S103A V1041 S141N G159D Q236H Q245R T255S V68A N76D S103A V1041 G159D Q236H Q245R R247H V68A N76D S103A V1041 G159D A174V N204D Q236HIQ245R V68A N76D S103A V1041 G159D N204D Q236H Q245R V68A N76D S103A V1041 A133V G159D N218D Q236H Q245R V68A N76D S103A V1041 G159D A232V IQ236H Q245R V68A N76D S103A V1041 G159D A1941 V203A Q236H Q245R Q12R N76D S103A V1041 M222S Q245R N76D S103A V1041 A232V Q245R S24T V68A N76D S103A V1041 G159D A232V Q236H Q245R V68A S103A V1041 G159D A232V Q236H Q245R N252K V68A N76D S103A V1041 G159D T213R A232V Q236H Q245R T260A Q12R N76D S103A 11 04T M222S V2441 Q245R Q12R N76D S103A M222S P210T Q245R Q12R N76D S103A 1104T S130T M222S Q245R 00J T22K V68A N76D IS103A V1041 V68A N76D S103A N184D VJ68A S103A V1041 G159D A232V Q236H Q245R N248D N252K V68A I3 14 19 22 26 25 EV68A IS103A V1041N1 G159D A232V Q236H Q245R N2K N4V68A S13A V104110 G159D A232V Q236H Q245R N252K 68KV8 13 V01G5DA32 26 N43S V68A S103A V1041 G159D A232V Q236H Q245R N252K N43K V8A S103A V1041 G159D A232V Q236H Q245RN2KR75 N43D V68A S103A V1041 G15D M2322S Q236H N248S L262M V68A N87D S103A V1041 G159D A232V Q236H 0245RN2KR75 Q12R N76D S103A 1104T S130T M222S Q245R N248S L262S Q12R N76D S103A 1104T S130T A215V M222S 0245R Q1RN6c13 14 1O 22 25 21 Q1N76D S103A 1104T S130T M222S 27 Q245RL2S Qd1 2R N76D S103A 1104T S130T A215T M222S Q245RL2SN69 Q12R S7 N76D S13A 1104 S13 T M222S Q245R Q12R N76D S103A 1104T S130T R170S N185D M222S N23 245R Q12R IN76D IS103A 1104T S130T M222S Q245R V268A
U,
00
U,
-J
t~J Q12R N76D S103A 1104T S130T M222S P210S Q245R V68A S103A V1041 G159D A232V Q236H Q245R L257V V68A S103A V1041 N116D G159D A232V Q236H Q245R V68A S103A V1041 G159D A232V Q236H Q245R N248D V68A S103A V1041 G159D A232V Q236H Q245R V68A S103A V1041 G159D V203E A232V Q236H Q245R V68A S103A V1041 G159D A232V Q236H K237E Q245R V68A N76D 179N S103A V1041 G159D A232V Q236H Q245R V68A S103A V1041 G9DN183D A232V Q236H Q245R V68A S103A V1041 G159D A174V Q206L A232V Q236H Q245R V68A S103A V1041 G159D S188C A232V Q236H Q245R V68A S103A V1041 G159D A230T A232V Q236H Q245R V68A A98T S103A V1041 G159D A232V Q236H Q245R V68A S103A V1041 G159D A215T A232V Q236H Q245R V68A S103A V1041 G159D A232V Q236H Q245R N248S V68A N76D S103A V1041 G159D A232V Q236H Q245R V68A N76D S103A V1041 G159D P21OR A232V Q236H Q245R V68A N76D S103A V1041 G159D A232V Q236H Q245R L257V N76D S103A IV1041 IA232V IQ236H IQ245RL27
C,)
'C
00 V68A S103A V1041 G159D A232V Q236H Q245R L257V R275H N76D S103A V1041 L257V R275H V68A S103A V1041 G159D T224A A232V Q236H Q245R L257V N76D S103A V1041 G159D A232V Q236H Q245R L257V V68A N76D S103A V1041 G159D Y209W A232V Q236H Q245R V68A N76D S103A V1041 G159D G211R A232V Q236H Q245R V68A N76D S103A V1041 G159D G211V A232V Q236H Q245R Q12R V68A N76D S103A V1041 G159D Y214L A232V Q236H Q245R V68A N76D S103A V ?0'41 G159D A215R A232V Q236H 0245R Q12R V68A N76D S103A V1041 G159D A232V Q236H Q245R V68A N76D S103A V1041 G159D A232V Q236H Q245R S259G V68A S87R N76D S103A V1041 G159D A232V Q236H Q245R T260V V68A N76D S103A V1041 G159D A232V Q236H Q245R N261G V68A N76D S103A V1041 G159D A232V 0236H Q245R N261W N76D S103A V1041 A232V Q236H S242P Q245R V68A N76D S103A V1041 G159D P210L A232V Q236H Q245R Q12R A48V V68A N76D S103A V1041 G159D A232V Q236H Q245R N7DS103A V1041 A232V Q236H Q245R N7DS103A V104 1 G159DI Y192F IA232V IQ236H IQ245RI tA N76D S103A V1041 V1471 G159D A232V Q236H Q245R N248S K251 R Q12R V68A N76D S103A V1041 G159D A232V Q236H Q245R A272S V68A N76D S103A V1041 G159D N183K Q206L A232V Q236H Q245R V68A N76D S103A V1041 G1 59D A232V Q236H Q245R S256R V68A N76D S103A V1041 G159D Q206R A232V Q236H Q245R K27R V68A N76D S103A V1041 G159D A232V Q236H 0245R V68A N76D S103A V1041 N1 16T G159D R170S N185S A232V Q236H Q245R G61 E V/68A S103A V1041 G159D A232V Q236H 0245R N248D N252K N43D V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D S212P A232V Q236H Q245R N248D N252K V68A S103A V1041 S99N G159D N184D A232V Q236H Q245R N248D N252K S103A V1041 G159D A232V Q 36H Q245R N248D N252K V68A S103A V1041 G159D Y209W A232V Q236H Q245R N248D N252K V68A S103A V1041 Q109R G159D A232V Q236H 0245R N248D N252K V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D Y209F A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K N2610D V68A S103A V1041 G159D N185D A232V Q236H Q245R N248D N252K V68A IS103A V/1041 IG159D P21OR A232V IQ236H Q245R IN248D N252K cn '0 00 m~J
U'
-J
V68A S103A V1041 G159D P210T A232VIQ236H Q245R N248D N252K V68A S103A V1041 G159D P21 OS A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D N185D P21 OL A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D P21 OL A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D S212A A232V Q236H 0245R N248D N252K V68A S103A V1041 G159D S212G A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D S212E A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D T213E A232V Q236H Q245R N248D N252K V68A S103A V1041 T21 3S A232V Q236H Q245R N248D N252K V68A A103V V1041 G159D T213E A232V 0236H Q245R N248D N252K V68A S103A V1041 G159D T213R A232V Q236H Q245R N248D N252K V68A S103A V1041 IG159D T213G A232V Q236H Q245RIN248D N252K V68A S103A V1041 G159D A215V A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D A215R A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D S216T A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D S216V A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D S216C A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K V6A )13A IV1041 IG159D IN173D A232V Q236H IQ245R N248D N252KI 0
C,,
'0 00 t.J
U'
-J
V68A S103A V1041 G159D A232V Q236H Q245R N248D K251V N252K V68A S103A V1 041 G159D Q206R A232V 0236H 0245R N248D N252K V68A S103A V1041 G159D A232V Q236H Q245R N248D N252F V68A S103A V1041 G159D A232V Q236H Q245R N248D N252L V68A S103A V1041 G159D A232V Q236H Q245R N248D N252F V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K T255V V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K S256N V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K S256E V68A S103A V1041 Git9D A232V Q236H Q245R N248D N252K S256R V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K T260R V68A S103A V1041 G159D A232V IQ236H Q245R N248D IN252K L257R V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K G258D 18V V68A S103A V1041 G159D A232V Q236H 0245R N248D N252K N269D V68A S103A V1041 N116S G159D A232V Q236H Q245R N248D N252K T260E V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K N261R V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K N261D V68A N76D S103A V1041 G159D A232V Q236H Q245R N248D N252K V68A S103A V1041 A232V Q236H Q245R N248D N252K S103A V1041 G159D A232S Q236H Q245R N248D N252K '0 00
-J
t.J V68A S103A V1041 G159D A232V Q236R Q245R N248D N252K N18S V68A S103A V1041 G159D A232V 0236H Q245R N248D N252K V68A S103A V1041 G159D A232V Q236H Q245V N248D N252K V68A N76D S101T S103A V1041 G159D T213R N218S A232V Q236H Q245R T260A V68A S103A V1041 G159D A228V A232V Q236H Q245R N248D N252K T33S V68A N76D S103A V1041 G159D A232V Q236H Q245R N248D N252K V68A N76D E89D S103A V1041 G159D P210L T213R A232V Q236H Q245R T260A G61 E V68A N76D S103A V1041 G159D A232V Q236H Q245R N248D N252K S103A V1041 G159D V2651 P2 101 A232V Q236H Q245R G61 E V68A S103A V1041 S130A G159D A232V Q236H Q245R N248D N252K G61 E V68A S103A V1041 A133S Q137R G159D A232V Q236H Q245R N248D N252K G61 E S103A V1041 A133V G159D A232V Q236H Q245R N248D N252K V68A S103A V1041 G159D A232V Q236H Q245R N248G N252K V68A S103A V1041 G159D N218S A232V Q236H Q245R N248D N252K G61 E V68A S103A V1041 G159D S160V A232V Q236H Q245R N248D N252K S3L G61E V68A N76D S103A V1041 A232V 0236H Q245R N248D N252K G61E V68A S103A V1041 G159D S167F A232V Q236H Q245R N248D N252K G97E S103A V1041 G159D A232V Q236H Q245R N248D N252K A98D S103A V1041 G159D A232V Q236H Q245R N248D N252K '0 -4 t'J S99E S103A V1041 G159D A232V Q236H Q245R N248D N252K Sl0lE S103A V1041 G159D A232V Q236H Q245R N248D N252K S101G S103A V1041 G159D A232V Q236H Q245R N248D N252K G102A S103A V1 041 G159D A232V Q236H Q245R N248D N252K S103A V1041 S106E G159D A232V Q236H Q245R N248D N252K S103A V1041 Q109E G 159D A232V Q236H Q245R N248D N252K S103A V1041 G159D A232V Q236H Q245R N248D N252K N261 R S103A V1 041 Q109R G159D A232V Q236H Q245R N248D N252K N62D S103A V1041 G gDA232V Q236H Q245R N248D N252K S103A V1 041 G159D N184D A232V Q236H 0245R N248D N252K S103A Vl 041 G159D S166D A232V Q236H Q245R N248D N252K S103A V1 041 G159D L217E A232V Q236H Q245R N248D N252K N62D S103A V1041 G159D T213R A232V Q236H Q245R N248D N252K N62D S103A V1041 G159D T213R A232V Q236H Q245R N248D N252K S103A V1 041 G159D Q206R L217E A232V Q236H Q245R N248D N252K N62D S103A V1041 G159D Q206R A232V Q236H Q245R N248D N252K S103A V1041 S13OG G159D A232V Q236H Q245R N248D N252K S103A V1041 P131V G159D A232V Q236H Q245R N248D N252K K27N S103A V1041 G159D A232V Q236H Q245R N248D N252K Cl) 00
U'
'-4 T38G S103A V1041 G159D A232V Q236H Q245R N248D N252K T38A N76D S103A V1041 G159D T213R A232V Q236H Q245R T260A V68A N76D S103A V1041 G159D T213R A232V Q236H 0245R T260A E271G V68A N76D S103A V1041 G159D Y209W T213R A232V Q236H Q245R T260A V68A N76D S103A V1041 G159D P2101 T213R A232V Q236H Q245R T260A V68A N76D S103A V1041 G159D V2051 T213R A232V Q236H Q245R T260A V68A N76D S103A V1041 G159D P2 101 A232V Q236H Q245R T260A V68A S103A V1041 G159D T213R A232V Q236H Q245R T260A N76D S103A V1041 G t§5- T213R A232V Q236H Q245R T260A V68A S103A V1041 G159D Y209W A232V Q236H Q245R V68A S103A V1041 G159D P2101 A232V Q236H Q245R V68A S103A V1041 G159D A230V A232V Q236H Q245R V68A S103A V1041 G159D L126F A232V Q236H 0245R V68A S103A V1041 G159D V2051 A232V Q236H Q245R V68A S103A V1041 G159D P210L A232V Q236H 0245R S103A V1041 G159D A230V Q236H Q245R V68A S103A V1041 G159D A232V Q236H Q245R T260A S103A V1041 G159DIA232V Q236H Q245R V68A S103A 11041G159D A174V A232V Q236H Q245R L257V ov 0', 00 t4J V68A S103A V1041 G159D A194S A232V Q236H Q245R L257V V68A S103A V1041 G159D Y209W A232V Q236H Q245R L257V S103A V1041 G159D A232V 0236H Q245R L257V V68A N76D S103A V1041 G159D T213R A232V Q236H Q245R T260A N261WI V68A S103A V1041 G159D A232V Q236H 0245R L257V N261W S103A V1041 G159D T213R A232V Q236H Q245R T260A S103A V1041 G159D P2101 A232V Q236H Q245R N248D N252K S103A V1041 G159D Y209W A232V Q236H Q245R L257V V68A N76D S103A V11041 G159D P210L T213R A232V Q236H Q245R T260A Q12R S103A V1041 G159D Y209W T213R A232V Q236H 0245R T260A S103A V1041 Y209W A232V Q236H Q245R L257VIII S103A V1041 G159D V2051 P21 01 T213R A232V Q236H 0245R T260A S103A V1041 G159D V2051 Y209W A232V Q236H Q245R T260A V68A S103A V1041 G159D V2051 Y209W P21 01 A232V Q236H Q245R S103A V1041 G159D V2051 Y209W P2101 A232V Q236H Q245R L257V S103A V1041 G159D V2051 Y209W A232V Q236H Q245R L257V V68A S103A V1041 G159D V2051 Y209W P2101 A232V Q236H Q245R T260A S103A V1041 G159D V2051 Y209W P2101 A232V Q236H Q245R S103A V1041 G159D Y209W P2101 A232V Q236H Q245R '0 t4I -4 S103A V1041 G159DI V2051 P2101 A232V Q236H Q245R V68A S103A V1041 S128L G159D A232V Q236H Q245R A48V S103A V1041 G159D A230V Q236H Q245R A48V V68A S103A V1041 G159D Y209W A232V Q236H IQ245R A48V V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K A48V V68A S103A V1041 G159D A232V Q236H Q245R L257V N261W G102A S103A V1041 G159D S212G A232V Q236H Q245R N248D N252K Q12R G102A S103A [V1041 G159D S212G A232V 0236H Q245R N248D N252K S101G G102A S103A V 04 1 G159D S212G A232V Q236H Q245R N248D N252K A98L G102A S103A V1041 G159D S212G A232V Q236H Q245R N248D N252K G102A S103A V1041 G159D T213R A232V Q236H Q245R N248D N252K S103A V1041 P131V G159D A232V Q236H Q245R N248D N252K S103A V1041 G159D N184S A232V Q236H Q245R N248D N252K S103A V1041 G159D N184G A232V Q236H Q245R N248D N252K S103A V1041 G159D A232V Q236H V244T Q245R N248D N252K S103A V1041 G159D A232V Q236H V244A Q245R N248D N252K N62D S103A V1041 G159D T213R A232V Q236H Q245R N248D N252k S256R Q12R N62D S103A V1041 G159D T213R A232V Q236H Q245R N248D N252K S101G S103A V1041 G159D N185D IA232V Q236H Q245R IN248D N252KI 00 S101G S103A V1041 G159D Q206E A232V Q236H Q245R N248D N252K S101G S103A V1041 G159D T213Q A232V Q236H Q245R N248D N252K A98L G102A S103A V1041 G159D A232V Q236H Q245R N248D N252K S101G G102A S103A V1041 G159D A232V Q236H Q245R N248D N252K A98L G102A S103A V1041 G159D S212G A232V Q236H Q245R N248D N252K A98L G102A S103A V1041 G159D S212G A232V Q236H N248D N252K N62D S103A V1041 Q 109R G159D T213R A232V Q236H Q245R N248D N252K N62D S103A V1041 G159D S212G T213R A232V Q236H Q245R N248D N252K N62D S101G S103A Vlb4I G159D S212G T213R A232V Q236H Q245R N248D N252K S103A V1041 G159D A232V Q245R N248D N252K S103A V1041 G159D A230V Q245R N62D S103A V1041 S13OG G159D T213R A232V Q236H Q245R N248D N252K S101G S103A V1041' S13OG G159D A232V Q236H Q245R N248D N252K S101G S103A V1041 S128G G159D A232V Q236H Q245R N248D N252K S101G S103A V1041 S128L G159D A232V Q236H Q245R N248D N252K N62D S101G S103A V1041 G159D T213R A232V Q236H Q245R N248D N252K N62D S103A V1041 S128G G159D T213R A232V Q236H Q245R N248D N252K N62D S103A V1041 S128L G159D T213R A232V Q236H Q245R N248D N252K S101G S103A V1041 G159D A232V IQ236H Q245R N248D IN252K T260A '0 S101G S103A V1041 P131V G159D A232V Q236H Q245R N248D N252K A98V S101G S103A V1041 G159D A232V Q236H Q245R N248D N252K S99G S101G S103A V1041 G159D A232V Q236H Q245R N248D N252K S101G S103A V1041 G159D S212G A232V Q236H Q245R N248D N252K S101G S103A V1041 G159D Y209W A232V Q236H Q245R N248D N252K S101G S103A V1041 G159D P2101 A232V Q236H Q245R N248D N252K S101G S103A V1041 G159D V2051 A232V 0236H Q245R N248D N252K S101G S103A V1041 G159D A230V Q236H Q245R S101G S10O3A V1041 G16 D A194P A232V Q236H Q245R N248D N252K N76D S101G S103A V1041 G159D A194P A232V Q236H Q245R N248D N252K S101G S103A V1041 G159D A230V A232V Q236H Q245R N248D N252K N620 S103A V1041 G159D N185D Q206E T213R A232V Q236H Q245R N248D N252K E271Q ~0 t4 WO 99/20770 PCT/US98/22572 Example 2 A large number of the protease variants produced in Example 1 were tested for performance in two types of detergent and wash conditions using a microswatch assay described in "An improved method of assaying for a preferred enzyme and/or preferred detergent composition", U.S. Serial No. 60/068,796.
Table 4 lists the variant proteases assayed and the results of testing in two different detergents. For column A, the detergent was 0.67 g/l filtered Ariel Ultra (Procter Gamble, Cincinnati, OH, USA), in a solution containing 3 grains per gallon mixed Ca2+/Mg 2 hardness, and 0.3 ppm enzyme was used in each well at 20"C. For column B, the detergent was 3.38 g/l filtered Ariel Futur (Procter Gamble, Cincinnati, OH, USA), in a solution containing 15 grains per gallon mixed Ca2+/Mg 2 hardness, and 0.3 ppm enzyme was used in each well at Table 4 N76D S103A V10411 1 S103A V1041 A228T 0.56 1.11 V6 I8A S103A V1041 G159D A232V Q236H Q245R N252KI 1.41 1.85 V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K 2.77 1.201 V68A S103A V1041 G159D A232V Q236H Q245R 2.26 1.671 V68A S103A V1041 N140D G159D A232V Q236H Q245R N252K 2.96 1.42 N43S V68A S103A IV1041 G159D A232V Q236H Q245R IN252K 1.91 1.80 N43K V68A S103A V104,1 G159D A232V Q236H Q245R 2.05 1.78 N43D V68A S103A V1 041 G159D A232V Q236H Q245R N252K 2.00 1.341 V68A S103A V1041 G159D A232V Q236H Q245R L1257V 2.3-8 1.67 V68A S103A V1041 G159D A232V Q236H Q245R N248D 2.83 0.53 V68A S103A V1041 G159D A232V Q236H K237E Q245R 2.87 0.20 V68A S103A V1041 G159D A232V Q236H Q245R N252S 12.56 1.411 V68A S103A V1041 G159D A232V Q236H Q245R L257V R275H 3.97 0.471 V68A S103A V1041 G159D T224A A232V Q236H Q245R L257V 3.35 1.28 G61 E V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K 3.77 0.09 N43D V68A S103A V1041 G159D A232V Q236H Q245R N248 252K 3.50 0.47 V68A S103A V1041 G159D S212P A232V Q236H -Q245R N248D N252K 2.81 1.46 00
U'
.4 N76D A98E S103A V1041 1.56 0.28 W4E N76D S103A V1041 1.22 0.33 N76D N77D S103A V1041 1.13 0.36 A16T N76D S1O3A. V1041 N248D 1.22 0.431 AlE N76D S103A V1041 1.12 0.32 N76D S103A V1041 N261 D 1.54 0.33 N76D S103A V1041 S216C 1.04 0.13 N76D N77D S103A V1041 Al174V 1.09 0.351 T38S N76D S103A V1041 K237Q 1.11 0.551 N76D S103A V1041 N1830 1.50 0.25 R19L N76D S103A V1041 1.11 0.48 R19C N76D S103A V1041 1.05 0.19 N76D S103A V1041 N184D 1.32 0.29 N76D S103A V1041 N252D 1.19 0.53 N76D S103A V1041 S259C 0.92 0.12 N76D S103A V1041 K251 T 1.31 0.431 N76D P86S S103A V1041 1.00 0.981 172V N76D S103A V1041 N185D 1.70 0.37 N76D S103A V1041 K237E T274A 1.12 0.16 N76D IS103A V1041 IA228V 1. 131 0.99, 00 N76D S103A V1041 G159D 1.88 0.23 H17L N76D S103A V1041 K237E 1.29 0.28 N76D S103A V1041 S130L 0.52 0.71 N76D S103A V1041 Q109R 0.23 1.261 N76D S99R S103A V1041 N204T 0.21 0.871 N76D S103A V1041 D181N 0.24 1.071 Q12R N76D S103A V1041 0.61 1.311 N76D S103A V1041 S212P E271V 0.69 1.35; N76D S103A V1041 N252K N261Y 0.37 1.02 N76D S103A V1041 S2421 0.98 0.92 N76D S103A V1041 E271Q 0.63 1.25 Q12R N76D S103A V1041 S242T 0.49 1.32 N43S N76D S103A V1041 N116K N1831 0.39 1.101 N76D S103A V1041 G258R 0.34 1.17 N76D S103A V1041 E271G 0.57 1.25 N76D S103A V1041 Q182R 1198V 0.22 0.95 L21 M N76D S103A V1041 0182R 0.24 0.98 N76D S103A V1041 Ml1191 Q137R 0.13 0.91 N76D S103A V1041 0137R N248S 0.16 1.02 A13T N76D S103A V1041 Q206R 0.31 1.01 '.0
-J
N76D S103A V1041 Q206R 0.33 1.02 N76D S103A V1041 S212P G258R 0.38 1.061 T58S N76D S103A V1041 E271 G 0.84 1.26 N76D S103Ai V1041 Q206E N261 D 1.97 0.04 V4E N76D S103A V1041 Q206E 1.51 0.05 N76D N77D S103A V1041 Q206E 1.40 0.04j N76D S103A V1041 A1SE 1.95 0.16 N76D S103A V1041 Q206E 2.41 0.88 N76D N77D S103A V1041 A133T N185D K251T 1.34 0.03 N76D S103A V1 041 Q205F N261 D 1.78 0.041 N76D S 103A V1041 01590 Q206E V244A 2.16 0.041 V4E N76D S103A V1041 S188E 1.91 0.041 V4E N76D S103A V1041 A158E 2.06 0.041 N76D S103A V1041 Q206E K251T 1.73 0.06 A48T N76D S103A V1041 LliM 01590 2.04 0.16 V68A N76D IS103A V1041 0 1590 Q236H 3.20 0.091 L42V N76D S103A V1041 01590 1.83 0.17 Q12H N62H N76D S103A V1041 01590 1.42 0.14 L41 N76D S103A V1041 G159D 1.86 0.18 N70S103A V1041 G146S 01590 1.87 0.191 V4 aJ N76D S103A V1041 G159D N238S 1-T 90 0.15 N76D S103A V1041 G159D T224A 1.61 0.07 N76D S103A V1041 S212P V268F E271V 0.44 1.42 N76D S87R S103A V1041 S212P E271V 0.39 2.03 N76D S103A V1041 S212P Q245L E271V 0.62 1.79 N76D S1O03A V1041 Q109R Q245R 0.11 1.78 N76D S103A V1 041 Q109R P21 OL 0.12 1.21 N62S N76D S103A V1041 1.63 0.78 V68A N76D S103A V1041 Q236H 2.37 0.44 V68A N76D S103A V104J. G159D Q236H E271V 2.97 0.45 V68A N76D S103A V1041 G159D Q236H Q245R 3.00 0.61 V68A N76D S103A V1041 G159D L2171 Q236H E271V 2.71 0.12 H17Q V68A N76D S103A V1041 2.461 0.38 V68A N760 S103A V1041 2.46 0.61 V/68A N76D S103A V1041 G159D Q236R 3.31 0.11 V68A L75R N76D S103A V1041 G159D Q236H 3.06 0.14 V68A N76D S103A V1041 Al114V IV1211 G159DIQ236H Q245R -3.11 0.40 Q12R V68A N76D S103A V1041 G159D Q236H 3.12 0.34 V68A N76D IS103A IV1041 G159D Y209S Q236H T253K 3.18 0.03
(A
-4 V68A N16D I 1U3A I V1041 INl17KI(3159D1 N184S Q236H 2.781 0.06 L I L 1 J V68A N76D S103A V1041 Q236H 2.49 0.57 V68A N76D S103A V1041 G159D Q236H Q245L 3.37 0.03 V68A N76D S103A V1041 N123S G159D Q236H H249Y 3.11 0.03 V68A N76D S103A V1041 G159D Q236H H249Q 3.15 0.041 V68A N76D S103A V1041 G159D Q236H Q245R N261D 3.31 0.03 V68A N75D S103A V1041 S141N G159D Q236H Q245R T255S 3.26 0.62 V68A N76D S103A V1041 G159D Q236HIQ245R R247H 2.78 0.03 V68A N76D S103A V1041 G159D A174V N204D Q236H Q245R 3.28 0.02 V68A N76D S103A V1041 G159D N204D Q236H Q245R 3.34 0.02 V68A N76D S103A V1044 A133V G159D N218D Q236H Q245R 3.281 0.03 V68A N76D S103A V1041 G159D A232V Q236H Q245R 2.91 0.58 V68A N76D S103A V1041 G159D A1941 V/203A Q236H Q245R 2.86 0.13 V68A N76D S103A V1041 G159D T213R IA232V Q236H Q245RIT260A 1.30 1.73 T22K V68A N76D S103A V1041 1 1.131 V68A N76D S103A V1041 G159D P21OR A232V Q236H Q245R 1.28 1.54 V68A N76D S103A V/1041 G159D A232V Q236H Q245R L257V 3.72 0.81 N76D S103A V1041 A232V Q236H Q245R L257V 0.6 N76D S103A V/1041 L257V R275H 1.91 0.15 N76D S103A V1041 G159D A232V Q236H Q245R L257V 1.92 11.09 V68A N76D S103A V/1041 G159D Y209WI A232V Q236H Q245R 3.57 0.99 00 t.J -3 V68A N76D S103A V1041 G159D G21IR A232V Q236H Q245R 1.74 1.76 V68A N76D S103A V1 041 G159D G211V A232V Q236H Q245R 3.15 1.06 Q12R V68A N76D S103A V1041 G159D Y214L A232V 0236H Q245R 2.33 1.92 V68A N76D S103A V1041 G159D A215R A232V Q236H Q245R 1.671 1.45 Q12R V68A N76D S103A V1041 G159D A232V Q236H Q245R 2.161 1.72 .G2OR V68A N76D S103A V1041 G159D A232V Q236H Q245Rx S259G 2.77 1.59 V68A N76D S87R S103A V1041 G159D A232V Q236H Q245R T260V 2.62 1.491 V68A N76D S103A V1041 G159D A232V Q236H Q245R N261G 2.92 0.68 V68A N76D S103A V1041 G159D A232V Q236H Q245R N261W 2.17 1.37 N76D S103A V1041 A23 1 V Q236H S242P Q245R 0.48 1.2 V68A N76D S103A V1041 G159D P210L A232V Q236H Q245R 12.92 0.761 Q12R A48V V68A N76D S103A V1041 G159D A232V Q236H Q245R 2.09 1.86 N76D S103A V1041 A232V Q236H Q245R 0.51 1.44 N76D S103A V1041 G159D Y192F A232V Q236H Q245R 1.60 1.14 N76D S103A V1041 V1471 G159D A232V Q236H Q245R N248S K251R 1.35 1.29 Q12R V68A N76D S103A V1041 G159D A232V Q236H Q245R A272S 1.92 1.81 V68A N76D S103AI V1041 G159D IN183K Q206L A232V Q236H Q245R 1.17 1.53 V68A N76D S103A V1041 G159D A232V Q236H Q245R S256R 2.01 1.721 V68A N76D S103A V1041 G159D Q206R A232V Q236H Q245R 2.09 1.62 K27R V68A N76D S103A V1041 G159D A232V Q236H Q245R 3.00 1.08 00 -4 t.J V68A N76D S103A V1041 N116T G159D R170S N185S A232V Q236H Q245R ND ND N76D S103A V1041 M222S Q245R 1.01 1.23 Q12R N76D S103A V1041 M222S H249R 0.57 1.65 N76D S103A V1041 N173R M222S 0.86 0.46 N76D S103A V1041 M222S Y263F 1.24 0.77 L21 M N76D S103A V1041 M222S K237R Y263F 1.18 0.76 N76D S103A V1041 Q109R M222S 0.52 1.16 N76D S1O3AI V1041 Q109R M222S E271D 0.56 1.12 G61IR N76D S103A V1041 M222S 0.43 0.96 N76D S103A V1041 Q 1 3-R M222S 0.42 1.25 N76D S103A V1041 M222S H249R 1.15 1.01 Q12R N76D S103A V1041 M222S Q245R 0.53 1.46 N76D S103A V1041 A232V Q245R 0.69 1.56 Q12R N76D S103A 1104T M222S V2441 Q245R 0.66 1.74 Q12R N76D S103A V1041 M222S P21 OT Q245R 0.52 1.56 Q12R N76D S103A 11 04T S130T M222S Q245R 0.70 1.61 Q12R N76D S103A 1104T S130T A215V M222S Q245R 0.79 1.85 Q12R N76D S103A 11 04T S130T M222S V227A Q245R L262S 0.78 1.56 Q12R N76D S103A 1104T S130T M222S Q245R N261 D 1.25 1.30 N760D S103A I1104T S130T M222S Q245R 1.29 1.30 a0 00 tA Q12R S57P N76D S103A 11 04T S130T M222S Q245R K251Q 1.44 0.16 Q12R N76D S103A 1104T S130T R170S N185D M222S N243D Q245R 2.01 0.04 Q12R N76D S103A 11 04T S130T M222S Q24SR V268A 0,77 1.60 Q12R N76D S103A 11 04T S130T M222S P210S Q245R 0.731 1.66 V68A N76D S103A V1041 G159D A232V Q236H 0245R 12.09 0.86 '00
LA
WO 99/20770 PCT/US98/22572 Example 3 Table 5 lists the variant proteases assayed from Example 1 and the results of testing in four different detergents. The same performance tests as in Example 2 were done on the noted variant proteases with the following detergents. For column A, the detergent was 0.67 g/l filtered Ariel Ultra (Procter Gamble, Cincinnati, OH, USA), in a solution containing 3 grains per gallon mixed Ca2+/Mg 2 hardness, and 0.3 ppm enzyme was used in each well at 20 0 C. For column B, the detergent was 3.38 g/l filtered Ariel Futur (Procter Gamble, Cincinnati, OH, USA), in a solution containing 15 grains per gallon mixed Ca 2 +/Mg 2 hardness, and 0.3 ppm enzyme was used in each well at For column C, 3.5g/l HSP1 detergent (Procter Gamble, Cincinnati, OH, USA), in a solution containing 8 grains per gallon mixed Ca2+/Mg 2 hardness, and 0.3 ppm enzyme was used in each well at 20°C. For column D, 1.5 mi/I Tide KT detergent (Procter Gamble, Cincinnati, OH, USA), in a solution containing 3 grains per gallon mixed Ca 2 +/Mg 2 hardness, and 0.3 ppm enzyme was used in each well at Table A BC D N76D S103A V1 041 1 1 1 S1 03A V1041 G159D A232V 0236H Q245R N248D N252K 1.44 1.41 1.39 1.26 V68A S103A V1 041 G159D Y209W A232V 0236H Q245R N248D N252K 23 .916123 V68A S103A_ 2101Q0R G5D A3V Q26 25 28 22.34 1.49 1.65 2.35 G2RV68A S103A V104 41 G159D A232V Q236H Q245R N248D N252K 1081 1.41 1.20 1.1 G2RV68A S13A V1 041 G159D29 A232V Q236H 0245R N248D N252K 2181 1.72 1.66 1.31 V68A S103A V1 041 G159D N185D A232V Q236H 0245R N248D N252K 21 01 1.60 V68A S103A V1041 G159D P21Oil A232V 0236H Q245R N248D N252K 0.9 1 1.48 123 V68A S103A VI1041 G159D 5D P210 A232V 236H Q245R N248D N252K 26 0.8 1.4 123T08 V68A S103A V1 041 G159D 8D P210 L A232V Q236H Q245R N248 N252K 0.2 .436 145512.8 V68A S103A V1041 G159D S212C A232V Q236H Q245R N248D N252K 2311.431.55 1.7 V68A S103A V1 041 G159D S212G A232V 0236H Q245R N248D N252K 230 1.43 1.63 207 V68A S103A V1 041 G159D S212E A232V Q236H Q245R N248D N252K 23 1.47 1.6 2016 V68A S103A V1 041 G159D T213E A232V Q236H Q245R IN248D N252K 2.63 0.56 1.36 2.66 V68A S103A Vi 041 T213S A232V Q236H Q245R N248D N252K 1.111 1.38 1.311 0.75 V68A A103V V1 041 G159D T213E A232V Q236H 0245R N248D N252K 2.27 0.15 1.12 2.01 V68A S103A V1041 G159D T213R A232V Q236H Q245R N248DEN252K 1.7142 1.37 1.06 V68A S103A V1041 G159D A215V A232V 0236H IQ245R N248D N252K 2.14 1.40 1.53 1.54 V68A S103A V1 041 G159D A215R A232V Q236H Q245R N248D N252K 1.22 1.58 1.47 1.20 V68A S103A V1 041 G159D S216T A232V Q236H 0245R N248D N252K 2.12 1.36 1.56 1.56 V68A S1O03A V1 041 G159D S216V A232V Q236H Q245R N248D N252K 1.88 1.36 1.47 1.87 V68A S103A VI1041 G159D S216C A232V 0236H Q245R N248D N252K 12.24 0.33 11.07 2.891 V68A S103A VI1041 G159D N173D A232V 0236H 0245R N248D N252K 2.43 0.461 1.29 2.421 V68A S103A V1 041 G159D 0206R A232V Q236H 0245R N248D N252K 0.98 1.46 1.24 0.95 V68A S103A VI 041 G159D A232V Q236H 0245R N248D N252F 2.52 1.00 1.42 2.42 V68A S103A V1 041 G159D A232V Q236H Q245R N248D N252L 2.05 1.13 1.30 1.85 V68A S103A V1041 G159D A232V Q236H Q245R N248D N252F 2.61 0.91 1.43 3.221 V68A S103A V1 041 G159D A232V Q236H 0245R N248D N252K T255V 2.18 1.36 1.58 1.72 V68A S103A VI 041 G159D A232V Q236H Q245R N248D N252K S256N 2.14 1.46 1.59 1.65 V68A S103A V1 041 G159D A2321V Q236H Q245R N248D N252K S256E 2.46 0.77 1.33 258 V68A S103A V1 041 G159D _A232V Q236H Q245R N248D 1N252K S256R 1.311 1.52 1.46 0.94 V68A S103A V1 041 G159D A232V Q236H Q245R N248D N252K T260R 1.211 1.41 1.31 1.05 V68A S103A V1 041 G159D A232V Q236H Q245R N248D N252K L257R 1.51 1.41 0.85 1.18 V68A S103A V1 041 G159D A232V Q236H Q245R N248D N252K G258D 2.56 0.59 1.30 2.64 V68A S103A V1 041 G159D A232V Q236H Q245R N248D N252K N261 R 1.02 1.47 1.37 0.84 V68A S103A V1 041 A232V Q236H Q245R N248D N252K 1.04 1.50 1.32 0.73 V68A S103A V1041 G159D A232V Q236H Q245V N248D N252K 2.60 0.93 1.41 12.67 V68A S103A V1 041 G159D A228V A232V Q236H Q245R N248D N252K 2.31 1.38 1.53 1.57 G61 E V68A S103A V1041 SI 30A G159D A232V Q236H Q245R N248D_ N252K 2.83 0.25 1.33[ 2 .44 0 '0 '.0 0
-J
1 0 '00 Ln kJ G61 E S103A V1 041 Al133V G159D A232V Q236H Q245R N248D N252K 2.10 0.971 1.36 2.29 V68A S103A V1041 G159D A232V Q236H Q245R N248G N252K 1.37 1.54 0.89 1.27 V68A S103A V1041 G159D N218S A232V Q236H Q245R N248D N252K 2.30 -1.50 1.62 1.56 V68A S103A V1 041 G159D A232V Q236H Q245R N248D N252K 1.72 1.72 1.671 1.15 V68A N76D E89D S103A VI 041 G159D P21L T213R A232V 0236H Q245R T260A 1.32 1.30 1.111 1.28 V68A N76D S103A V1 041 G159D A232V Q236H Q245R N248D N252K 2.50 0.83 1.431 2.25 G61lE V68A S103A V1041 G159D S160V A232V Q236H Q245R N248D N252K 4.20 0.07 ND 1.28 S3L G61 E V68A N76D S103A V1 041 A232V Q236H Q245R N248D N252K 3.47 0.60 ND 1.45 G61 E V68A S103A V1041 G159D Y167F A232V Q236H Q245R N248D N252K 4.32 0.79 ND 1.55 G97E S103A V1041 G159D A232V Q236H Q245R N248D N252K 3.14 0.41 ND 1.40 A98D S103A V1 041 G159D A232\6- ,Q236H Q245R N248D N252K 2.71 0.68 ND 1.72 S99E S103A V1 041 G159D A232V Q236H Q245R N248D N252K 2.97 0.68 ND 1.71- SlOIE S103A V1 041 G159D A232V Q236H Q245R N248D N252K 3.50 0.27 ND 1.90 S101G S103A V1 041 G159D A232V Q236H 0245R N248D N252K 2.24 1.80 ND 1.33 G102A S103A VI1041 G159D A232V Q236H 0245R N248D N252K 3.35 1.33 ND 1.69 S1O03A V1041 S106E G159D A232V Q236H 0245R N248D N252K 4.88 0.55 ND 2.71 S1O03A V1 041 Q109E G159D A232V Q236H 0245R N248D N252K 4.22 1.05 ND 2.40 S103A VI1041 G159D A232V Q236H Q245R N248D N252K N261 R 5.45 2.19 ND 2.58 S1O03A V1041 Q109R G159D A232V Q236H Q245R N248D N252K 3.76 2.16 ND 1.82 N62D S103A V1 041 G159D A232V Q236H 0245R N248D N252K 7.42 0.13 ND 2.46a S13A V141 G19D N184D A232V 0236H Q245R N248D N252K 5416 N 2.84 S103 V10 1 G1 9D _L 5.3 1.6 1 D 4: ul S103A VI1041 G159D S166D A232V Q236H Q245R N248D N252K 5.12 1.21 ND 3.97 S103A V1 041 G159D L217E A232V Q236H Q245R N248D N252K 6.38 0.95 ND 3.09 N62 S13 V14 G19 23 22 26 2S 28 22 .728 D 26 G2RN62D S O3A VI1041 G159D T213R A232V Q236H Q245R N248D N252K 438 2 ND 2.60 N6210 13A V 1 041 G159 T26 217E A232V 0236H Q245R N248D N252K 4.38 1.92 ND- 2.54 N621 13A V1 041 G159D Q206R27 A232V Q236H Q245R N248D N252K 3.05 2.6 ND 1.10 13A V 1 041 G159D 84G A232V 236H Q245R N248D N252K 4.09 2.46 ND 2.55 S103A V1 041 G159D A232V 0236HVQ236H 0245R N248D N252K 2.32 2.08 ND 2.40 S103A V1 041 G159D A232V Q236H V244A Q245R N248D N252K 2.34 2.04 N D 1.86 K271 13A V I 041 G159D A232V Q236H24 Q245R N248D N252K 2.24 2.11 ND 1.957 K27N S103A VI1041 G159D A232V 0236H Q245R N248D N252K 2810 1.56 ND 2472 T38G S103A V1041 G159D T23 A23 2V 0236H Q245R N248D N252K S262.3 .6ND 14 01RN62D S103A V1041 G159D T213R A232V 236H Q245R N248D N252K26 2.63 2.66 ND 1.44 N62D S103A V1041 G159D N185D Q206E T213R A232V 0236H Q245R N248D N252K E271Q 7.74 0.94 ND 5.39 S101G S103A V1041 G159D N185D A232V Q236H Q245R N248D N252K 5.14 1.41 ND 1.92 S101G SI03A V1041 G159D Q206E A232V Q236H Q245R N248D N252K 4.97 0.57 ND 1.36 S103A V1041 G159D T213Q A232V Q236H Q245R N248D N252K 2.41 1.86 ND 1.01 A98L G102A S103A V1041 G159D A232V Q236H Q245R N248D N252K 4.42 0.50 ND 2.88 .iG G102A S103A V1041 G159D A232V 0236H 0245R N248D N252K 5.86 1.20 ND% 3.84 G1o2A S103A V1041 G159D S212G A232V Q236H Q245R N248D N252K 5.87 2.10 ND 3.19 Q12R G102A 013A V1041 G1 59D S212G A232V Q236H Q245R N248 N252K ND 2.1 7 tJI
I
A98L G102A S103A V1041 G159D S212G A232V Q236H Q245R N248D N252K 4.02 0.41 ND 2.25 SlOIG G102A S103A V1041 G159D S212G A232V Q236H Q245R N248D N252K 6.63 2.07 ND 2.08 G1O2A S1O3A V1041 G159D T213R A232V 1236H Q245R N248D N252K 2.03 248 ND 2.25 N62D S103A V1041 Q109R G159D T213R A232V Q236H Q245R N248D N252K 2.96 2.76 ND 2.34 S103A V1041 G159D A232V Q245R N248D N252K 2 20 1 2.74 2.10 ND 1.86 S103A V1041 G159D A230V Q245R 2.11 2.35 ND 1.49 N62D S103A V1041 S13OG G159D T213R A232V Q236H Q245R N248D N252K 3.42 0-71 N-Rr D 2.58 SIOIG S103A V1041 S13OG G159D A232V Q236H Q245R N248D N252K 2.59 1.32 ND 1.61 SlOIG S103A V1041 S128G G159D A232V Q236H Q245R N248D N252K 1.30 1-.23 ND SlOIG S103A V1041 S128L G159D A232V Q236H Q245R N248D N252K 2.94 0.71 ND 1.08 N62D S1OG S103A V1041 G159 T213R A232V Q236H Q245R N248D N252K 3.17 0.83 ND 2.35 N62D S103A V1041 S128G G159D T213R A232V Q236H Q245R N248D N252K 2.15 1.38 ND 1.77 kinl V2A cmA A 'OnAl E T I of Irvrv I n v IU~fl 5-L '3 I ULJ I 4 I 3Ir mzazv WIZ.501 Q245R N48D N252K 3.07 t4fl4fl 1 tAfl* I ~AAAI I I~4%etI r t t I I 0.07 1.16
ND~
IUI"5 Q I 13 V I U'+I r1.1v k41 pU A,232V Q236H Q245R N248D N252K 2.26 A98V S1OIG S103A V1041 G159D A232V Q236H Q245R N248D N252K 1.82 1.34 ND S99G SlOIG S103A V1041 G159D A232V Q236H 0245R N248D N252K 2.16 1.47 ND SlOIG S103A V1041 G159D S212G A232V Q236H Q245R N248D N252K 1.79 1 .38 1 ND S1OG S103A V1041 G159D Y209W A232V Q236H Q245R N248D N252K[ 1.15 1.18 ND SlOIG S103A V1041 G159D P2101 A232V Q236H Q245R N248D N252K L 1.47 1.23 ND 1 cinitlP i zina IA Mnnl 4 can \olncl nnt_..
1.45 3.05 1.08 1.20 1.01 8.7 1.03 1.05 1.23 YIVIV VIVU~ I IV- l IVI~JV V~VJI I ~LJLV WZI+Z)KL4n N2,18D N252K 1.90 1.38 cininf A t IlflAI I %jc f 1 I I f-?4r I A 1 II I I~ V IV~I Ut JJU 4 VV I W3fOn WZdQrM 1.55 1.51
ND
NID
i 1. j I SlOIG S103A jV1041 G159D jA194P jA232V Q236H 0245R N248D N252K 1.96 11.30 1ND 11.10 N76D S101G S103A V1041 G159D Al194P A232V 0236H Q245R N248D N252K 2.49 10.80 1ND 1.2z51 00
LA
Claims (5)
1. An isolated protease variant comprising an amino acid substitution at a residue position corresponding to position 103 and an amino acid substitution at a residue corresponding to position 245 of Bacillus amyloliquefaciens subtilisin and one or more amino acid substitutions at residue positions selected from the group consisting of residue positions corresponding to positions 1, 3, 4, 8,10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61,62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101,102, 104,106, 107, 109, 111,114,116,117,119,121,123,126,128,130,131,133, 134,137, 140,141,142,146,147,158,159,160,166,167,170,173,174,177,181,182, 183, 184, 185, 188, 192,194,198,203,204,205,206,209,210,211,212,213, 214,215,216,217, 218,222,224,227,228,230,232,236,237, 238, 240,242, 243,244,246,247, 248, 249,251,252,253, 254, 255, 256,257, 258, 259,260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens subtilisin; wherein when a substitution at a position corresponding to residue position 103 is combined with a substitution at a position corresponding to residue position 76, there is also a substitution at one or more residue positions other than residue positions corresponding to positions 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265, or 274 of Bacillus amyloliquefaciens subtilisin.
2. The protease variant of claim 1 including substitution of the amino acid residues at position 236.
3. The protease variant of claim 1 including substitutions of the amino acid residues at positions 103 and 245 and at one or more of positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 170, 183, 185, 205, 209, 210, 211,212, 213, 215, 217,222, 230, 232,248, 252,257, 260,261,270 and 275.
4. The protease variant of claim 1 including substitutions of the amino acid residues at positions 103, 236 and 245 and at one or more of positions 1, 22 September 2005 -78- 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 243, 248, 252, 257, 260, 270 and
275. 5. The protease variant of any one of claims 1 to 4 including a substitution of the amino acid residue at position 159. 6. The protease variant according to any one of the preceding claims which is derived from a Bacillus subtilisin. 7. The protease variant according to claim 6 which is derived from Bacillus Lentus subtilisin. 8. An isolated DNA encoding a protease variant of any one of claims 1 to 7. 9. An expression vector encoding the DNA of claim 8. A host cell transformed with the expression vector of claim 9. 11. A cleaning composition comprising the protease variant of any one of claims 1 to 7. 12. An animal feed comprising the protease variant of any one of claims 1 to 7. 13. A composition for treating a textile comprising the protease variant of any one of claims 1 to 7. 14. The protease variant according to any one of claims 1 to 7 comprising a substitution set selected from the group consisting of residue positions corresponding to positions in Table 1 of Bacillus amyloliquefaciens subtilisin. 22 September 2005 -79- The protease variant according to claim 14 comprising a substitution set selected from the group consisting of residue positions corresponding to positions in Table 3 of Bacillus amyloliquefaciens subtilisin. 16. The protease variant according to claim 14 comprising a substitution set selected from the group consisting of residue positions corresponding to positions in Table 2 of Bacillus amyloliquefaciens subtilisin. 17. A cleaning composition as hereinbefore described with reference to Example 2 or 3. DATED: 23 September 2005 PHILLIPS ORMONDE FITZPATRICK Attorneys for: GENENCOR INTERNATIONAL, INC and THE PROCTER GAMBLE COMPANY 22 September 2005
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2002320741A AU2002320741B2 (en) | 1997-10-23 | 2002-12-20 | Multiply-substituted protease variants |
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08956324 | 1997-10-23 | ||
| US08956564 | 1997-10-23 | ||
| US08956323 | 1997-10-23 | ||
| AU12762/99A AU1276299A (en) | 1997-10-23 | 1998-10-23 | Multiply-substituted protease variants |
| PCT/US1998/022572 WO1999020770A2 (en) | 1997-10-23 | 1998-10-23 | Multiply-substituted protease variants |
| AU2002320741A AU2002320741B2 (en) | 1997-10-23 | 2002-12-20 | Multiply-substituted protease variants |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU12762/99A Division AU1276299A (en) | 1997-10-23 | 1998-10-23 | Multiply-substituted protease variants |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU2002320741A1 AU2002320741A1 (en) | 2003-04-03 |
| AU2002320741B2 true AU2002320741B2 (en) | 2005-10-06 |
Family
ID=39295841
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2002320741A Ceased AU2002320741B2 (en) | 1997-10-23 | 2002-12-20 | Multiply-substituted protease variants |
Country Status (1)
| Country | Link |
|---|---|
| AU (1) | AU2002320741B2 (en) |
-
2002
- 2002-12-20 AU AU2002320741A patent/AU2002320741B2/en not_active Ceased
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU758371B2 (en) | Multiply-substituted protease variants | |
| US7129076B2 (en) | Multiply-substituted protease variants with altered net charge for use in detergents | |
| EP2287321B1 (en) | Multiply-substituted protease variants | |
| AU2002320741B2 (en) | Multiply-substituted protease variants | |
| AU2002325461B2 (en) | Multiply-substituted protease variants with altered net charge for use in detergents | |
| CA2829859A1 (en) | Multiply-substituted protease variants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGA | Letters patent sealed or granted (standard patent) | ||
| MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |