Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof

Abstract

This invention relates to amylase polypeptides, and nucleic acids encoding the polpypeptides and uses thereof. The amylases of the present invention have been engineered to have more beneficial qualities. Specifically, the amylases of the current invention show an altered exospecifity.

1 . An exo-specific non-maltogenic exoamylase comprising an amino acid sequence at least 75% identical with SEQ ID Nos. 1, 5, 7 or 11 and at least one substitution selected from the group consisting of 4, 33, 34, 70, 71, 87, 99, 108, 113, 121, 134, 141, 157, 158, 171, 178, 179, 188, 198, 199, 223, 290, 307, 315, 334, 343, 399, and 405.  
     
     
         2 . The exoamylase of  claim 1 , wherein said amino acid sequence is at least 75% identical with SEQ ID NO.:1.  
     
     
         3 . The exoamylase of  claim 1 , wherein said amino acid sequence is at least 75% identical with SEQ ID NO.:5.  
     
     
         4 . The exoamylase of  claim 1 , wherein said amino acid sequence is at least 75% identical with SEQ ID NO.:7.  
     
     
         5 . The exoamylase of  claim 1 , wherein said amino acid sequence is at least 75% identical with SEQ ID NO.:11.  
     
     
         6 . The exoamylase of  claim 1 , wherein said amino acid sequence comprises an amino acid sequence at least 75% identical with SEQ ID Nos. 2-4b or 8-10.  
     
     
         7 . The exoamylase of  claim 1 , wherein said at least one substitution is selected from G4D, N33Y, D34N, G70D, K71R, G87S, A99V, K108R, V113I, G121D, G134R, A141P, I157L, G158D, Y171S, L178F, A179T, G188A, Y198F, Y198F, Y198L, A199v, G223A, V290I, H307L, I315V, S334P, D343E, S399P, A405F and A405E.  
     
     
         8 . The exoamylase of  claim 1  wherein said at least one position is selected from 33, 34, 71, 87, 121, 134, 141, 157, 178, 179, 223, 207, 334 and 343.  
     
     
         9 . The exo amylase of  claim 8 , wherein said at least one substitution is selected from N33Y, D34N, K71R, G87S, G121D G134R, A141P, I157L, L178F, A179T, G223A, H307L, S334P, and D343E.  
     
     
         10 . The exo amylase of  claim 8 , further comprising at least one additional substitution at a position selected from 108, 158, 171 and 188.  
     
     
         11 . The exo-amylase of  claim 10 , wherein said at least one additional substitution is selected from K108R, G158D, Y171S, and G188A.  
     
     
         12 . The exo-amylase of  claim 1 , wherein said at least one substitution comprises a combination selected from the following: 
 G134R, A141P I157L G223A H307L S334P D343E G121D;    G134R A141P I157L G223A H307L S334P D343E N33Y G121D;    G134R A141P I157L G223A H307L S334P D343E N33Y;    G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G87S G121D S214N T375A;    G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D Y171S G188A N138D;    G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D;    G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T;    G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G188A;    G134R, A141P I157L G223A H307L S334P K71R L178F A179T;    G134R, A141P I157L G223A H307L S334P L178F A179T;    G134R, A141P I157L G223A H307L S334P N33Y D34N L178F A179T;    G134R, A141P I157L G223A H307L S334P L178F A179T G87S G121D;    G134R, A141P I157L G223A H307L S334P L178F A179T G121D;    G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D E343D;    G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T;    G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T Y33N N34D E343D;    G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D;    G134R, A141P I157L G223A H307L S334P K71R L178F A179T G121D;    G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    113F, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    A99V, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    V113I, I157L, Y198F, G223A, V290I, H307L, S334P, D343E    V113I, G134R, A141 P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    A199V, D343E, V131I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P;    V113I, A141 P, I157L, Y198F, G223A, V290I, S334P, D343E;    V113I, G121D, G134R, A141 P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    V113I, G121D, G134R, A141 P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    V113I, A141P, Y198F, G223A, V290I, H307L;    V113I, A141P, Y198F, G223A, V290I, S334P, D343E;    V113I, A141P, Y198F, G223A, A268P, V290I, S399P V113I, A141 P, Y198F, G223A, V290I, S399P;    V113I, A141P, Y198W, G223A, V290I;    V113I, A141P, Y198F, G223A, V290I;    Y198F, G223A, V290I;    Y198W, G223A, V290I;    V113, A141P, I157L, Y198F, G223A, V290I;    V113M;    V113A;    V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, I315V, S334P, D343E;    D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    V113I, G134R, A141P, I157L, G188S, Y198F, G223A, V290I, H307L, S334P, D343E;    K71R, V113I, G134R, A141P, I157L, L178L, Y198F, G223A, V290I, H307L, G313G, S334P, D343E;    D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, G313G, S334P, D343E; V113I,    G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L, S334P, D343E;    V113I, G134R, A141P, I157L, I170I, Y198F, G223A, V290I, H307L, G313G, S334P, D343E V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L, G313G, S334P, D343E;    G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405E;    V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405V;    A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    V113I, G134R, A141P, I157L, Y198L, G223A, V290I, H307L, S334P, D343E;    V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    K71R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    K108R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    D34G, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E;    G4D, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; and    A141P, G134R, G223A, H307L, I157L, V113I, V290I, Y198F, G188A;    
     
     
         12 . The exo amylase of  claim 3 , wherein said at least one substitution comprises a combination selected from the following: 
 G134R, A141P, I157L, G223A, H307L and S334P;    G121D, G134R, A141P, I157L, G223A, H307L and S334;    G87S, G121D, G134R, A141P, I157L, G223A, H307L and S334P;    G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;    G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;    G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;    N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;    N33Y, D34N, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; and    N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P.    
     
     
         13 . The exo amylase of  claim 3 , wherein said at least one substitution comprises a combination selected from the following: 
 N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;    N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; and    N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P    
     
     
         14 . The exo amylase of  claim 1 , wherein said amylase comprises an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:4a, SEQ ID NO:4b, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.  
     
     
         15 . The exo-amylase of  claim 1 , wherein said exo-amylase is derived from a  Pseudomonas  sp.  
     
     
         16 . The exo-amylase of  claim 15 , wherein said  Pseudomonas  sp. is selected from  Pseudomonas saccharophilia  and  Pseudomonas stutzeri.    
     
     
         17 . A nucleic acid sequence at least 75% identical to the nucleic acid sequence encoding the exo-amylase of  claim 1 .  
     
     
         18 . A vector comprising the nucleic acid sequence of  claim 17 .  
     
     
         19 . A host cell comprising the nucleic acid sequence of  claim 18.