Thermolysin variants

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.

We claim: 
     
         1 . A composition comprising an isolated thermolysin and a neutral metalloprotease inhibitor, wherein said thermolysin is a  Geobacillus  thermolysin or a  Bacillus  thermolysin. 
     
     
         2 . The composition of  claim 1 , wherein said neutral metalloprotease inhibitor is phosphoramidon or galardin. 
     
     
         3 . The composition of  claim 1 , wherein said  Geobacillus  is  G. caldoproteolyticus  or  G. stearothermophilus.    
     
     
         4 . The composition of  claim 1 , wherein said  Bacillus  is  B. thermoproteolyticus.    
     
     
         5 . The composition of  claim 1 , wherein said thermolysin has at least 50% amino acid identity with the amino acid sequence set forth in SEQ ID NO:3. 
     
     
         6 . The composition of  claim 1 , wherein said thermolysin comprises the amino acid sequence set forth in SEQ ID NO:3. 
     
     
         7 . An isolated thermolysin variant having improved stability and/or performance. 
     
     
         8 . The isolated thermolysin variant of  claim 7 , 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 . The isolated thermolysin variant of  claim 8 , wherein said one or more substitutions comprise one, two, three, four or five 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. 
     
     
         10 . The isolated thermolysin variant of  claim 7 , 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 . The isolated thermolysin variant of  claim 10 , wherein said one or more substitutions comprise one, two, three, four or five 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. 
     
     
         12 . The isolated thermolysin variant of  claim 7 , wherein said thermolysin variant comprises one or more substitutions chosen from substitutions T006G, T006H, T006I, 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, F063I, 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, Q273I, Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651, 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, N280I, 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 . The isolated thermolysin variant of  claim 12 , wherein said one or more substitutions comprise one, two, three, four or five substitutions chosen from substitutions T006G, T006H, T006I, 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, F063I, 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, Q273I, Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651, 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, N280I, 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. 
     
     
         14 . An isolated thermolysin variant having an improvement in stability and/or performance as compared to wild-type  Geobacillus  sp. thermolysin (e.g., thermolysin comprising the amino acid sequence set forth as SEQ ID NO:3). 
     
     
         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 . A method for producing an enzyme having thermolysin activity, comprising:
 i) transforming a host cell with an expression vector comprising a polynucleotide encoding a thermolysin variant having 50 to 99% amino acid identity with the amino acid sequence set forth in SEQ ID NO:3; and   ii) cultivating said transformed host cell under conditions suitable for the production of said thermolysin.   
     
     
         17 . The method of  claim 16 , wherein said method further comprises the step of harvesting the produced thermolysin. 
     
     
         18 . The method of  claim 16 , wherein said host cell is a  Bacillus  species (e.g.,  B subtilis ). 
     
     
         19 . A composition comprising at least one thermolysin variant obtained from the recombinant  Bacillus  sp. host cell of  claim 18 . 
     
     
         20 . The composition of  claim 7 , wherein said composition further comprises at least one calcium ion and/or zinc ion. 
     
     
         21 . The composition of  claim 7 , 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 . The composition of  claim 7 , wherein said composition is a cleaning composition. 
     
     
         25 . The composition of  claim 24 , wherein said cleaning composition is a detergent. 
     
     
         26 . The composition of  claim 7 , 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 7 , wherein said composition comprises at least about 0.0001 weight percent of said thermolysin variant. 
     
     
         28 . The composition of  claim 7 , wherein said composition comprises from about 0.001 to about 0.5 weight percent of said thermolysin variant. 
     
     
         29 . The composition of  claim 7 , further comprising at least one adjunct ingredient. 
     
     
         30 . The composition of  claim 7 , further comprising a sufficient amount of a pH modifier to provide the composition with a neat pH of from about 3 to about 5, the composition being essentially free of materials that hydrolyze at a pH of from about pH 3 to about pH 5. 
     
     
         31 . The composition of  claim 30 , wherein said materials that hydrolyze at a pH of from about pH 3 to about pH 5 comprise at least one surfactant. 
     
     
         32 . The composition of  claim 31 , wherein said surfactant is a sodium alkyl sulfate surfactant comprising an ethylene oxide moiety. 
     
     
         33 . The composition of  claim 7 , wherein said composition is a liquid. 
     
     
         34 . An animal feed composition comprising the isolated thermolysin variant of  claim 7 . 
     
     
         35 . A textile processing composition comprising the isolated thermolysin variant of  claim 7 . 
     
     
         36 . A leather processing composition comprising the isolated thermolysin variant of  claim 7 . 
     
     
         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 7 . 
     
     
         38 . The method of  claim 37 , further comprising the step of rinsing the surface and/or material after contacting the surface or material with the cleaning composition.