Enzymes For Starch Processing

Abstract

The present invention relates to a hybrid enzyme comprising carbohydrate-binding module amino acid sequence and a fungal alpha-amylase amino acid sequence and to a variant of a fungal wild type enzyme comprising a carbohydrate-binding module and an alpha-amylase catalytic module. The invention also relates to the use of the hybrid enzyme or the variant in starch liquefaction.

1 . A hybrid enzyme which comprises an amino acid sequence of a catalytic module and an amino acid sequence of a carbohydrate binding module, wherein
 (a) the catalytic module is an amino acid sequence which has at least 70% homology to the amino acid sequence of SEQ ID NO:8 or an amino acid sequence which has at least 70% homology to the amino acid sequence of SEQ ID NO:30; and   (b) the carbohydrate binding module is an amino acid sequence having at least 50% homology to an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25.   
     
     
         2 . The hybrid enzyme of  claim 1 , wherein the hybrid enzyme further comprises a linker amino acid sequence which connects the amino acid sequence of the catalytic module and the amino acid sequence of the carbohydrate-binding module. 
     
     
         3 . The hybrid enzyme of  claim 2 , wherein the linker sequence is a linker sequence selected from the group consisting of the linker sequence of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 or a linker sequence which differs from the amino acid sequence shown in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29 by no more than 10 positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions, no more than 2 positions, or no more than 1 position. 
     
     
         4 - 16 . (canceled) 
     
     
         17 . A variant alpha-amylase having at least 80% homology to SEQ ID NO:41, which variant comprises an alteration at one or more of the positions selected from the group consisting of: 13, 15, 18, 31, 32, 33, 34, 35, 36, 61, 63, 64, 68, 69, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 89, 117, 118, 119, 120, 121, 122, 123, 124, 125, 152, 153, 154, 155, 156, 157, 158, 161, 162, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 204, 205, 206, 207, 208, 209, 210, 211, 216, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 242, 245, 250, 252, 253, 255, 256, 257, 259, 260, 275, 292, 295, 296, 297, 298, 299, 304, 328, 339, 344, 348, 378, 383, 386, 387, 405, 448 and 480, wherein
 (a) the alteration(s) are independently,
 (i) an insertion of an amino acid downstream of the amino acid which occupies the position, 
 (ii) a deletion of the amino acid which occupies the position, or 
 (iii) a substitution of the amino acid which occupies the position with a different amino acid, 
   (b) the variant has alpha-amylase activity.   
     
     
         18 . The variant according to  claim 17 , comprising one or more of the following substitutions: G33A, 136K, S74A, D75Y, E77D, P120A, 1153D, D154N, W155Y, D156E, N157D, L158Q, Q162E, E166L, T169N, 1170T, E199K, E199L, D232L, N233D, N235D, L238Y, D239T, W256Y, Q257P, E331Q, S336A, D339K, D339N, V340D, and Y342A. 
     
     
         19 . The variant according to  claim 17 , comprising an alteration at one or more of the following positions: 31, 74, 89, 209, 245, 348, 378, 383, 386, 387, 405, 448 and 480. 
     
     
         20 . The variant according to  claim 17 , comprising one or more of the following substitutions: N31D, S74A, Y89D, E209L, Y245V, D348K, D378A, K383A, P386A, 1387F, 1405V, N448S, N480R. 
     
     
         21 . The variant according to  claim 17 , comprising the following substitutions: N31D, S74A, Y89D, E209L, Y245V, D348K, D378A, K383A, P386A, 1387F, 1405V, N448S, N480R. 
     
     
         22 - 26 . (canceled) 
     
     
         27 . A method for liquefying starch, comprising treating a gelatinized or granular starch substrate with the hybrid enzyme or the variant according to  claim 1  in an aqueous medium. 
     
     
         28 - 30 . (canceled) 
     
     
         31 . A process for preparing a dough-based product, comprising adding the hybrid enzyme or the variant of  claim 1  to a dough. 
     
     
         32 . A hybrid enzyme comprising the catalytic domain of the  A. oryzae  alpha amylase shown as amino acids 21-498 of the sequence SEQ ID NO:40 or a sequence having at least 50% homology amino acid sequence shown as amino acids 21-498 of the sequence SEQ ID NO:40, and a carbohydrate binding domain. 
     
     
         33 - 36 . (canceled)