Multiple Mutation Variants of Serine Protease

Abstract

The present invention provides novel Micrococcineae spp serine proteases having multiple substitutions. In particular, the present invention provides serine proteases having multiple substitutions, DNA encoding these proteases, vectors comprising the DNA encoding the proteases, host cells transformed with the vector DNA, and enzymes produced by the host cells. The present invention also provides cleaning compositions (e.g., detergent compositions), animal feed compositions, and textile and leather processing compositions comprising these serine protease variants. In particularly preferred embodiments, the present invention provides mutant (i.e., variant) proteases derived from the wild-type proteases described herein. These variant proteases also find use in numerous applications.

1 - 76 . (canceled) 
     
     
         77 . An isolated serine protease variant of the  Cellulomonas  69B4 protease set forth in SEQ ID NO: 8 wherein the variant consists of at least 90% amino acid identity to SEQ ID NO: 8, comprises substitution at N024, and has the serine protease substrate activity of the  Cellulomonas  69B4 protease set forth by SEQ ID NO: 8. 
     
     
         78 . A cleaning composition comprising the isolated serine protease variant of  claim 77 . 
     
     
         79 . A cleaning composition comprising the isolated serine protease variant of  claim 77 , wherein said isolated serine protease variant has immunological cross-reactivity with an antibody to the serine  Cellulomonas  69B4 protease set forth by the amino acid sequence of SEQ ID NO: 8. 
     
     
         80 . The variant of  claim 77 , wherein said variant has improved thermostability as compared to the  Cellulomonas  69B4 protease set forth by the amino acid sequence of SEQ ID NO: 8. 
     
     
         81 . The variant of  claim 77 , wherein said variant has improved linear alkylbenzene sulfonate (LAS) stability as compared to the  Cellulomonas  69B4 protease set forth by the amino acid sequence of SEQ ID NO: 8. 
     
     
         82 . The variant of  claim 77  wherein said variant has enhanced protease activity as measured by Performance Index (PI), as compared to the  Cellulomonas  69B4 protease set forth by the amino acid sequence of SEQ ID NO: 8. 
     
     
         83 . The variant of  claim 82 , wherein said variant has improved caseinolytic or keratinolytic activity as compared to the  Cellulomonas  69B4 protease set forth by the amino acid sequence of SEQ ID NO: 8. 
     
     
         84 . The variant of  claim 77 , wherein said variant has improved wash performance activity as compared to the  Cellulomonas  69B4 protease set forth by the amino acid sequence of SEQ ID NO: 8. 
     
     
         85 . The variant of  claim 84 , wherein said variant has improved dishwashing performance activity as compared to the  Cellulomonas  69B4 protease set forth by the amino acid sequence of SEQ ID NO: 8. 
     
     
         86 . The variant of  claim 84 , wherein said variant has improved stain removal activity as compared to the  Cellulomonas  69B4 protease set forth by the amino acid sequence of SEQ ID NO: 8. 
     
     
         87 . The cleaning composition of  claim 78 , further comprising one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. 
     
     
         88 . The cleaning composition of  claim 87 , further comprising a stabilizing agent selected from the group consisting of borax, glycerol, and competitive inhibitors. 
     
     
         89 . The cleaning composition of  claim 78 , wherein said serine protease variant is an autolytically stable variant. 
     
     
         90 . A cleaning composition comprising at least 0.0001 weight percent of the serine protease variant of  claim 77 . 
     
     
         91 . The cleaning composition of  claim 90 , wherein said composition comprises from about 0.001 to about 0.5 weight percent of the serine protease variant. 
     
     
         92 . The cleaning composition of  claim 78 , wherein said cleaning composition is selected from liquid, powder, granular, and tablet compositions. 
     
     
         93 . The cleaning composition of  claim 78 , wherein said composition further comprises a hydrogen peroxide source. 
     
     
         94 . The cleaning composition of  claim 93 , wherein said hydrogen peroxide source comprises at least one persalt, wherein said persalt is alkalimetal perborate, alkalimetal percarbonate, alkalimetal perphosphate, alkalimetal persulfate, or a mixture thereof. 
     
     
         95 . The cleaning composition of  claim 94 , wherein said composition further comprises a bleach catalyst, bleach activator and/or mixtures thereof.