Thermolysin variants and detergent compositions therewith

Abstract

The present invention provides methods and compositions comprising at least one thermolysin-like neutral protease enzyme with improved storage stability and/or catalytic activity. In some embodiments, the thermolysin finds use in cleaning and other applications comprising detergent. In some particularly preferred embodiments, the present invention provides methods and compositions comprising thermolysin formulated and/or engineered to resist detergent-induced inactivation.

1 - 4 . (canceled) 
     
     
         5 . An isolated thermolysin variant wherein said thermolysin has at least 95% amino acid identity with the amino acid sequence set forth in SEQ ID NO: 3. 
     
     
         6 . The thermolysin variant of  claim 5 , wherein said thermolysin variant comprises the amino acid sequence set forth in SEQ ID NO: 3. 
     
     
         7 . (canceled) 
     
     
         8 . The isolated thermolysin variant of  claim 5 , wherein said thermolysin variant is a  Geobacillus  thermolysin variant having an amino acid sequence comprising one or more substitutions at positions chosen from positions equivalent to positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196, 273, 278, and 280 of the amino acid sequence set forth as SEQ ID NO: 3. 
     
     
         9 . (canceled) 
     
     
         10 . The isolated thermolysin variant of  claim 5 , wherein said thermolysin variant is a  Geobacillus  thermolysin variant having an amino acid sequence comprising one or more substitutions at positions chosen from positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58, 61, 63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261, 265, 273, 278, 280 and 297 of the amino acid sequence set forth as SEQ ID NO: 3. 
     
     
         11 . (canceled) 
     
     
         12 . The isolated thermolysin variant of  claim 5 , wherein said thermolysin variant comprises one or more substitutions chosen from substitutions T006G, T006H, T0061, T006K, T006M, T006N, T006P, T006Q, T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L, V007M, V007P, V007Q, V007R, V007T, V007Y, T049G, T049H, T049I, T049K, T049L, T049N, T049P, T049Q, T049W, A058I, A058P, A058R, F0631, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V, Y151W, I156M, I156R, I156T, I156W, G196R, Q2731, Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S065I, S065T, S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W, I156E, I156H, I156K, I156M, I156R, I156T, I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N, T278S, T278Y, N280E, N2801, N280L, N280M, N280S, T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D, S065E, S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, and I156E. 
     
     
         13 . (canceled) 
     
     
         14 . An isolated thermolysin variant having an improvement in stability and/or performance as compared to wild-type  Geobacillus  sp. thermolysin. 
     
     
         15 . The isolated thermolysin variant of  claim 14 , wherein said improvement comprises one or more of improved thermostability, improved performance under lower pH conditions, improved performance under higher pH conditions, and improved autolytic stability. 
     
     
         16 - 19 . (canceled) 
     
     
         20 . The composition of  claim 39 , wherein said composition further comprises at least one calcium ion and/or zinc ion. 
     
     
         21 . The composition of  claim 39 , wherein said composition further comprises at least one stabilizer. 
     
     
         22 . The composition of  claim 21 , wherein said stabilizer is chosen from borax, glycerol, zinc ions, calcium ions, and calcium formate. 
     
     
         23 . The composition of  claim 21 , wherein said stabilizer is at least one competitive inhibitor that stabilizes the at least one thermolysin in the presence of an anionic surfactant. 
     
     
         24 - 25 . (canceled) 
     
     
         26 . The composition of  claim 39 , further comprising at least one additional enzyme or enzyme derivative chosen from proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. 
     
     
         27 . The composition of  claim 39 , wherein said composition comprises at least about 0.0001 weight percent of said thermolysin variant. 
     
     
         28 . The composition of  claim 39 , wherein said composition comprises from about 0.001 to about 0.5 weight percent of said thermolysin variant. 
     
     
         29 - 32 . (canceled) 
     
     
         33 . The composition of  claim 39 , wherein said composition is a liquid. 
     
     
         34 . An animal feed composition comprising the isolated thermolysin variant of  claim 39 . 
     
     
         35 . A textile processing composition comprising the isolated thermolysin variant of  claim 39 . 
     
     
         36 . A leather processing composition comprising the isolated thermolysin variant of  claim 39 . 
     
     
         37 . A method of cleaning, comprising the step of contacting a surface and/or an article comprising a fabric with a cleaning composition comprising the isolated thermolysin variant of  claim 39 . 
     
     
         38 . (canceled) 
     
     
         39 . A cleaning composition comprising the isolated thermolysin variant of  claim 8  and a detergent.