Serine proteases

Abstract

The present disclosure relates to serine proteases and variants thereof. Compositions containing the serine proteases are suitable for use in cleaning fabrics and hard surfaces, as well as in a variety of industrial applications.

We claim: 
     
         1 . A BspAL03279-clade of subtilisins comprising a subtilisin or recombinant polypeptide or active fragment thereof comprising a DLGDXXRFGX a GLLXXXXAVX (SEQ ID NO:26) motif, wherein X is any amino acid and X a  is N or S. 
     
     
         2 . The BspAL03279-clade of subtilisins according to  claim 1 , wherein the subtilisin or recombinant polypeptide or active fragment thereof further comprises an amino acid sequence having at least 70% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 12, and 15. 
     
     
         3 . The BspAL03279-clade of subtilisins according to  claim 1  or  2 , wherein X a  is N. 
     
     
         4 . The BspAL03279-clade of subtilisins according to  claim 1  or  2 , with proviso that the subtilisin or recombinant polypeptide or active fragment thereof does not comprise ADK62564. 
     
     
         5 . The BspAL03279-clade of subtilisins of any of the above claims, wherein the subtilisin and/or recombinant polypeptide or active fragment has protease activity. 
     
     
         6 . A recombinant polypeptide or active fragment thereof comprising an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, and 15, with the proviso that the recombinant polypeptide or active fragment thereof does not comprise ADK62564 or optionally, WP_035392836, WP_038476582, WP_035392836, or WP_047989534. 
     
     
         7 . A recombinant polypeptide or active fragment thereof of the BspAL03279-clade. 
     
     
         8 . The recombinant polypeptide or active fragment thereof of  claim 7 , wherein the recombinant polypeptide or active fragment thereof comprises a DLGDXXRFGX a GLLNXXXAVX (SEQ ID NO:26) motif, wherein X is any amino acid and X a  is N or S. 
     
     
         9 . The recombinant polypeptide or active fragment thereof according to  claim 7  or  8 , wherein the recombinant polypeptide or an active fragment thereof comprises an amino acid sequence having at least 70% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 12, and 15. 
     
     
         10 . The recombinant polypeptide or active fragment thereof of any one of  claims 7  to  9 , wherein X a  is N. 
     
     
         11 . The recombinant polypeptide or an active fragment thereof of any one of  claims 7  to  9 , with proviso that the subtilisin or recombinant polypeptide or active fragment thereof does not comprise ADK62564. 
     
     
         12 . The recombinant polypeptide or an active fragment thereof of any one of  claims 6 - 11 , wherein the polypeptide has protease activity. 
     
     
         13 . The recombinant polypeptide or an active fragment thereof of  claim 12 , wherein the protease activity is subtilisin protease activity on a dimethylcasein substrate. 
     
     
         14 . A composition comprising a surfactant and a subtilisin or recombinant polypeptide or active fragment thereof of any one of  claims 1 - 5 , or the recombinant polypeptide or active fragment thereof of any one of  claims 6 - 13 . 
     
     
         15 . The composition of  claim 14 , wherein the surfactant is selected from the group consisting of an anionic surfactant, a cationic surfactant, a zwitterionic surfactant, an ampholytic surfactant, a semi-polar non-ionic surfactant, and a combination thereof. 
     
     
         16 . The composition of  claim 14  or  15 , wherein the composition is a detergent composition. 
     
     
         17 . The composition of  claim 16 , wherein the detergent composition is selected from the group consisting of a laundry detergent, a fabric softening detergent, a dishwashing detergent, and a hard-surface cleaning detergent. 
     
     
         18 . The composition of any one of  claims 14 - 17 , wherein said composition further comprises at least one calcium ion and/or zinc ion; at least one stabilizer; from about 0.001% to about 1.0 weight % of said subtilisin or recombinant polypeptide or active fragment thereof of any one of  claims 1 - 5 , or the recombinant polypeptide or active fragment thereof of any one of  claims 6 - 13 ; at least one bleaching agent; at least one adjunct ingredient; and/or one or more additional enzymes or enzyme derivatives selected from the group consisting of acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1,4-glucanases, endo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, oxidases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases, metalloproteases, additional serine proteases, and combinations thereof. 
     
     
         19 . The composition of any one of  claims 14 - 18 , wherein said composition contains phosphate or is phosphate-free and/or contains borate or is borate-free. 
     
     
         20 . The composition of any one of  claims 14 - 19 , wherein said composition is a granular, powder, solid, bar, liquid, tablet, gel, paste or unit dose composition. 
     
     
         21 . The composition of any one of  claims 14 - 20 , wherein said composition is formulated at a pH of from about 8 to about 12. 
     
     
         22 . A method of cleaning comprising contacting a surface or an item in need of cleaning with the subtilisin or recombinant polypeptide or active fragment thereof of any one of  claims 1 - 5 , the recombinant polypeptide or active fragment thereof of any one of  claims 6 - 13 , or the composition of any one of  claims 14 - 21 ; and optionally further comprising the step of rinsing said surface or item after contacting said surface or item with said subtilisin, recombinant polypeptide, or composition. 
     
     
         23 . The method of  claim 22 , wherein said item is dishware or fabric. 
     
     
         24 . A polynucleotide comprising a nucleic acid sequence encoding the subtilisin or recombinant polypeptide or active fragment thereof of any one of  claims 1 - 5 , or the recombinant polypeptide or active fragment thereof of any one of  claims 6 - 13 . 
     
     
         25 . The polynucleotide of  claim 24 , wherein said polynucleotide comprises a nucleic acid sequence having at least 70% identity to the nucleic acid sequence of SEQ ID NO:1, 4, 10, 13, 16, 18, 22, or 24. 
     
     
         26 . An expression vector comprising the polynucleotide of  claim 24  or  25 . 
     
     
         27 . A host cell comprising the vector of  claim 26 . 
     
     
         28 . The host cell of  claim 27 , wherein the host cell is of a species selected from  Bacillus  spp.,  Streptomyces  spp.,  Escherichia  spp.,  Aspergillus  spp.,  Trichoderma  spp.,  Pseudomonas  spp.,  Corynebacterium  spp.,  Saccharomyces  spp., or  Pichia  spp. 
     
     
         29 . The host cell of  claim 28 , wherein said  Bacillus  spp. is  Bacillus subtilis.    
     
     
         30 . A method for producing the subtilisin or recombinant polypeptide or active fragment thereof of any one of  claims 1 - 5 , or the recombinant polypeptide or active fragment thereof of any one of  claims 6 - 13  comprising:
 (a) stably transforming the host cell of any one of  claims 27 - 29  with the expression vector of  claim 26 ; 
 (b) cultivating said transformed host cell under conditions suitable for said host cell to produce said subtilisin polypeptide; and 
 (c) recovering said subtilisin or polypeptide. 
 
     
     
         31 . The method of  claim 30 , wherein said expression vector comprises a heterologous polynucleotide sequence encoding a heterologous pro-peptide. 
     
     
         32 . The method of  claim 30  or  31 , wherein said expression vector comprises one or both of a heterologous promoter and a polynucleotide sequence encoding a heterologous signal peptide. 
     
     
         33 . A textile, leather, or feather processing composition comprising the subtilisin or recombinant polypeptide or active fragment thereof of any one of  claims 1 - 5 , or the recombinant polypeptide or active fragment thereof of any one of  claims 6 - 13 . 
     
     
         34 . A composition comprising the subtilisin or recombinant polypeptide or active fragment thereof of any one of  claims 1 - 5 , or the recombinant polypeptide or active fragment thereof of any one of  claims 6 - 13 , wherein said composition is an animal feed, contact lens cleaning, or wound cleaning composition.