Pseudomonas saccharophila g4-amylase variants and uses thereof

Abstract

Pseudomonas saccharophila G4-forming amylase (PS4) variants, and nucleic acids encoding these, and their uses in producing food products and in an enzyme-catalyzed high temperature liquefaction step to produce ethanol from starch, e.g., cornstarch are provided.

1 . A  Pseudomonas saccharophila  amylase (PS4) variant, wherein the variant comprises a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2, and wherein the PS4 variant comprises one or more amino acid substitutions at the following positions: 7, 8, 32, 38, 49, 62, 63, 64, 67, 72, 73, 74, 75, 76, 104, 106, 107, 110, 112, 116, 119, 122, 123, 124, 125, 126, 128, 130, 137, 138, 140, 142, 143, 144, 148, 149, 150, 151, 154, 156, 163, 164, 168, 169, 182, 183, 192, 195, 196, 200, 202, 208, 213, 220, 222, 225, 226, 227, 232, 233, 234, 236, 237, 239, 253, 255, 257, 260, 264, 267, 269, 271, 276, 282, 285, 295, 297, 300, 302, 305, 308, 312, 323, 324, 325, 341, 358, 367, 379, and/or 390 of SEQ ID NO: 1, 2, 3, 4, 5, or 6; and/or one or more of the following amino acid substitutions: A3T, G9A, H13R, I46F, D68E, G69A/E/H/I/K/M/R/T, G70A/E/L/P/Q/S/V, K71M, G100A/S, G121I/P/R, A131T, G134C, A141S, N145S, Y146D/E, G153A/D, G158C/F/I/L/P/Q/V, S161G/H/K/P/R/TN, G166N, I170E/K/L/M/N, L178N/Q/W, A179E/N/P/R/S, A179S, G184Q, G188A, A199P, G223C/F/H/M/N/Q/W/Y, S229N, W238E/G/K/P/Q/R, G303L, H307D/E/F/G/K/M/P/Q/R/S/W/Y, A309E/I/M/T/V, S334A/H/K/L/M/Q/R/T, and/or H335M of SEQ ID NO: 1, 2, 3, 4, 5, or 6; and/or one or more amino acid substitutions at the following positions: 420, 422, and/or 424 of SEQ ID NO: 1. 
     
     
         2 . The PS4 variant of  claim 1 ,
 comprising a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2 or   comprising one or more of the following amino acid substitutions: A3T, P7S, A8N, G9A, H13R, P32S, I38M, I46F, D49V, D62N, F63A/D/E/L/V, S64N/T, T67G/H/K/N/Q/R/V, D68E, G69A/E/H/I/K/M/R/T, G70A/E/L/P/Q/S/V, K71M, S72E/K/N/T, G73D/E/L/M/N/S/T, G74S, G75C/E/F/R/S/W/Y, E76V, G100A/S, G104N/R, G106K, V107M, L110F, D112E, N116D, N119E/G/S/Y, G121I/P/R, Y122A/E/Q/W, P123S, D124S, K125A/D/E/G/P/Q/W, E126D/N, N128E, P130S, A131T, G134C, R137C, N138D/E/S, C140A/R, A141S, D142E/G/N, P143T, G144E, N145S, Y146D/E, N148K/S, D149H/L/V, C150A, D151A/V/W, G153A/D, D154E/G/Y, F156Y, G158C/F/I/L/P/Q/V, S161G/H/K/P/R/TN, L163M, N164R, G166N, P168L, Q169D/E/G/K/N/R/V, I170E/K/L/M/N, L178N/Q/W, A179E/N/P/R/S, R182D/G/H/M/S, S183G, G184Q, G188A, F192M/Y, V195D, R196A/G/K/P/Q/S/T/V/Y, A199P, P200A/G, R202K, S208T, S213N, L220A/T, K222M/Y, G223C/F/H/M/N/Q/W/Y, S225E/G/V, E226C/D/G/W, Y227C/D/G/K/T, S229N, W232F/G/H/I/K/L/N/P/Q/R/S/T/Y, R233H, N234R, A236E, S237D/G, W238E/G/K/P/Q/R, Q239L, V253G, D255V, A257V, E260K/R, N264D, V267I, D269N/S/V, K271A/L/Q, G276R, W282S, V285A, T295C, Y297H, G300E, N302K, G303L, Q305E/L/T, H307D/E/F/G/K/M/P/Q/R/S/W/Y, W308A/C/G/K/N/Q/R/S/T, A309E/I/M/T/V, D312E, W323M, T324A/L/M, S325G, S334A/H/K/L/M/Q/R/T, H335M, Y341C/E, R358A/E/G/L/N/Q/T/V, S367Q/R, S379G, and/or D390E of SEQ ID NO: 1, 2, 3, 4, 5, or 6, and/or one or more substitutions of S420G, D422N/P/Q, and/or G424D/S of SEQ ID NO: 1 or   comprising one or more amino acid substitutions at the following positions: 7, 32, 49, 62, 63, 64, 72, 73, 74, 75, 76, 107, 110, 112, 116, 119, 122, 123, 125, 128, 130, 137, 138, 140, 142, 143, 144, 148, 149, 150, 151, 154, 156, 163, 164, 168, 169, 182, 183, 192, 195, 196, 202, 220, 222, 226, 227, 232, 233, 234, 236, 237, 239, 253, 255, 257, 260, 264, 269, 271, 276, 282, 285, 297, 300, 302, 305, 308, 312, 323, 324, 325, 341, 358, 367, and/or 379 of SEQ ID NO: 1, 2, 3, 4, 5, or 6; and/or one or more of the following amino acid substitutions: A3T, H13R, I38M, I46F, T67G/H/K/N/Q/R/V, G69A/E/H/I/K/M/R/T, G70E/L/P/Q/V, K71M, G100A/S, G104R, G106K, G121I/P/R, D124S, E126D/N, A131T, G134C, A141S, N145S, Y146D/E, G153A/D, G158C/F/I/L/P/Q/V, S161G/H/K/P/R/TN, G166N, I170E/K/L/M/N, L178N/Q/W, A179E/N/P/R/S, G188A, A199P, P200A, G223C/F/H/M/N/Q/W/Y, S225E/G/V, W238E/G/K/P/Q/R, T295C, G303L, H307D/G/M/P/S, A309E/I/M/T/V, S334A/H/K/L/M/Q/R/T, H335M, and/or D390E of SEQ ID NO: 1, 2, 3, 4, 5, or 6; and/or one or more of the amino acid substitutions S420G and/or D422/N/P/Q of SEQ ID NO: 1; and/or an amino acid substitution at position 424 of SEQ ID NO: 1 or   comprising one or more of the following amino acid substitutions: A3T, P7S, H13R, P32S, I38M, I46F, D49V, D62N, F63A/D/E/L/V, S64N/T, T67G/H/K/N/Q/R/V, G69A/E/H/I/K/M/R/T, G70E/L/P/Q/V, K71M, S72E/K/N/T, G73D/E/L/M/N/S/T, G74S, G75C/E/F/R/S/W/Y, E76V, G100A/S, G104R, G106K, V107M, L110F, D112E, N116D, N119E/G/S/Y, G121I/P/R, Y122A/E/Q/W, P123S, D124S, K125A/D/E/G/P/Q/W, E126D/N, N128E, P130S, A131T, G134C, R137C, N138D/E/S, C140A/R, A141S, D142E/G/N, P143T, G144E, N145S, Y146D/E, N148K/S, D149H/L/V, C150A, D151A/V/W, G153A/D, D154E/G/Y, F156Y, G158C/F/I/L/P/Q/V, S161G/H/K/P/R/TN, L163M, N164R, G166N, P168L, Q169D/E/G/K/N/R/V, I170E/K/L/M/N, L178N/Q/W, A179E/N/P/R/S, R182D/G/H/M/S, S183G, G188A, F192M/Y, V195D, R196A/G/K/P/Q/S/T/V/Y, A199P, P200A, R202K, L220A/T, K222M/Y, G223C/F/H/M/N/Q/W/Y, S225E/G/V, E226C/D/G/W, Y227C/D/G/K/T, W232F/G/H/I/K/L/N/P/Q/R/S/T/Y, R233H, N234R, A236E, S237D/G, W238E/G/K/P/Q/R, Q239L, V253G, D255V, A257V, E260K/R, N264D, D269N/S/V, K271A/L/Q, G276R, W282S, V285A, T295C, Y297H, G300E, N302K, G303L, Q305E/L/T, H307D/G/M/P/S, W308A/C/G/K/N/Q/R/S/T, A309E/I/M/T/V, D312E, W323M, T324A/L/M, S325G, S334A/H/K/L/M/Q/R/T, H335M, Y341C/E, R358A/E/G/L/N/Q/TN, S367Q/R, S379G, and/or D390E of SEQ ID NO: 1, 2, 3, 4, 5, or 6, and/or one or more of the following amino acid substitutions: S420G, D422N/P/Q, and/or G424D/S of SEQ ID NO: 1 or   comprising one or more of the amino acid substitutions at the following positions: 49, 62, 63, 64, 72, 73, 74, 75, 76, 107, 112, 116, 119, 122, 123, 125, 128, 130, 137, 140, 143, 144, 148, 149, 150, 151, 154, 156, 163, 164, 168, 169, 182, 183, 192, 195, 196, 202, 257, 282, 285, 297, 300, 305, 308, 312, 323, and/or 325 of SEQ ID NO: 1, 2, 3, 4, 5, or 6; and/or one or more of the following amino acid substitutions: A3T, P7S, H13R, I38M, I46F, T67G/H/K/N/Q/R/V, G69A/E/H/I/K/M/R/T, G70E/L/P/Q/V, K71M, G100A/S, G104R, G106K, L110F, G121I/P/R, D124S, E126D/N, A131T, G134C, N138D/E, D142/E/G/N, N145S, Y146D/E, G153A/D, G158C/F/I/L/P/Q/V, S161G/H/K/P/R/TN, G166N, I170E/K/L/M, L178N/Q/W, A179E/N/P/R/S, G188A, A199P, P200A, L220T, K222M/Y, G223C/F/H/M/N/Q/W/Y, S225EN, E226C/D/G/W, Y227C/D/G/K/T, W232F/G/H/I/K/N/P/Q/R/S/T/Y, R233H, N234R, A236E, S237D/G, W238E/G/K/P/Q/R, Q239L, V253G, D255V, E260K/R, N264D, D269N/S/V, K271A/L/Q, G276R, T295C, N302K, G303L, H307D/G/M/P/S, A309E/I/M/T/V, T324L/M, S334A/H/K/L/M/Q/R/T, H335M, Y341C/E, R358A/E/G/L/N/Q/T/V, S367Q/R, S379G, and/or D390E of SEQ ID NO: 1, 2, 3, 4, 5, or 6; and/or one or more of the following amino acid substitutions: S420G, D422/N/P/Q, and/or G424S of SEQ ID NO: 1 or   comprising one or more of the following amino acid substitutions: A3T, P7S, H13R, I38M, I46F, D49V, D62N, F63A/D/E/L/V, S64N/T, T67G/H/K/N/Q/R/V, G69A/E/H/I/K/M/R/T, G70E/L/P/Q/V, K71M, S72E/K/N/T, G73D/E/L/M/N/S/T, G74S, G75C/E/F/R/S/W/Y, E76V, G100A/S, G104R, G106K, V107M, L110F, D112E, N116D, N119E/G/S/Y, G121I/P/R, Y122A/E/Q/W, P123S, D124S, K125A/D/E/G/P/Q/W, E126D/N, N128E, P130S, A131T, G134C, R137C, N138D/E, C140A/R, D142E/G/N, P143T, G144E, N145S, Y146D/E, N148K/S, D149H/L/V, C150A, D151A/V/W, G153A/D, D154E/G/Y, F156Y, G158C/F/I/L/P/Q/V, S161G/H/K/P/R/T/V, L163M, N164R, G166N, P168L, Q169E/G/K/N/R/V, I170E/K/L/M, L178N/Q/W, A179E/N/P/R/S, R182D/G/H/M/S, S183G, G188A, F192M/Y, V195D, R196A/G/K/P/Q/S/T/V/Y, A199P, P200A, R202K, L220T, K222M/Y, G223C/F/H/M/N/Q/W/Y, S225E/V, E226C/D/G/W, Y227C/D/G/K/T, W232F/G/H/I/K/N/P/Q/R/S/T/Y, R233H, N234R, A236E, S237D/G, W238E/G/K/P/Q/R, Q239L, V253G, D255V, A257V, E260K/R, N264D, D269N/S/V, K271A/L/Q, G276R, W282S, V285A, T295C, Y297H, G300E, N302K, G303L, Q305E/L/T, H307D/G/M/P/S, W308A/C/G/K/N/Q/R/S/T, A309E/I/M/T/V, D312E, W323M, T324L/M, S325G, S334A/H/K/L/M/Q/R/T, H335M, Y341C/E, R358A/E/G/L/N/Q/T/V, S367Q/R, S379G, and/or D390E of SEQ ID NO: 1, 2, 3, 4, 5, or 6, and/or one or more of the following amino acid substitutions: S420G, D422N/P/Q, and/or G424S of SEQ ID NO: 1 or   comprising additional one or more amino acid substitutions at the following positions: N33, D34, G70, G121, G134, A141, Y146, I157, S161, L178, A179, G223, S229, L307, A309, and/or 5334 of SEQ ID NO: 1 or 2 or   comprising one or more of the following amino acid substitutions: N33Y, D34N, G70D, G121F, G134R, A141P, Y146G, I157L, S161A, L178F, A179T, G223E, S229P, L307K, A309P, and/or S334P of SEQ ID NO: 1 or 2 or   comprising up to 25 amino acid deletions, additions, insertions, or substitutions compared to the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or   comprising an altered thermostability compared to the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2 or   comprising more thermostability than the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2.   
     
     
         3 . The PS4 variant according to  claim 2 ,
 wherein the PS4 variant comprises one or more of the following amino acid substitutions: A3T, I38M, G70L, Q169K/R, R182G/H, P200G, G223N, S237D, D269V, K271A/Q, S367Q/R, S379G, and/or S420G of SEQ ID NO: 1 or 2 or   wherein the PS4 variant comprises additional one or more amino acid substitutions at the following positions: G134, A141, I157, G223, H307, 5334, and/or D343 of SEQ ID NO: 1 or 2 or   wherein the PS4 variant comprises one or more of the following amino acid substitutions: G134R, A141P, I157L, G223A, H307L, S334P, and/or D343E of SEQ ID NO: 1 or 2 or   wherein the PS4 variant further comprises one or more amino acid substitutions at the following positions: N33, D34, K71, L178, and/or A179 of SEQ ID NO: 1 or 2 or   wherein the PS4 variant comprises one or more following amino acid substitutions: N33Y, D34N, K71R, L178F, and/or A179T of SEQ ID NO: 1 or 2 or   wherein the PS4 variant has an altered endo-amylase activity, an altered exo-amylase activity, and/or an altered ratio of exo- to endo-amylase activity compared to the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2 or   wherein the PS4 variant comprises one or more of the following amino acid substitutions: A3T, G69K, G70E, K71M, G73D/E, G75C/E, Y122A, C140A, G144E, Y146D/146E, N148K, C150A, D151A/V/W, G153A, G158I/P, S161G/H/K/P/R, Q169D/E/G/N/R, R196Q/S/T, R202K, 5208T, S213N, K222M, G223C/F/H/M/Q/W/Y, E226D, Y227D/G/K/T, S229N, W232Q/S/T, T295C, Q305T, W308A/C/G/Q/R/S/T, A3091N, W323M, T324L/M, S334A/H/M/Q, and/or R358E/L/N/Q/T/V of SEQ ID NO: 1 or 2 or   wherein the PS4 variant has an increased endo-amylase activity or a decreased ratio of exo- to endo-amylase activity compared to the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2 or   wherein the PS4 variant comprises one or more following amino acid substitutions: G69K, G73D/E, Y122A, C140A, C150A, G153A, G158I/P, S161G/H/K/P/R, Q169R, S208T, S229N, T295C, Q305T, and/or R358E/L/Q/T/V of SEQ ID NO: 1 or 2 or   wherein the PS4 variant has an increased exo-amylase activity or an increased ratio of exo- to endo-amylase activity compared to the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2 or   wherein the PS4 variant comprises one or more of the following amino acid substitutions: A3T, G70E, K71M, G75C/E, G144E, Y146D/E, N148K, D151A/V/W, Q169D/E/G/N, R196Q/S/T, R202K, S213N, K222M, G223C/F/H/M/Q/W/Y, E226D, Y227D/G/K/T, W232Q/S/T, W308A/C/G/Q/R/S/T, A309I/V, W323M, T324L/M, S334A/H/M/Q, and/or R358N of SEQ ID NO: 1 or 2 or   wherein the PS4 variant comprises additional one or more amino acid substitutions at the following positions: W66, I157, E160, S161, R196, W221, K222, E226, D254, Q305, H307, and/or W308 of SEQ ID NO: 1 or 2 or   wherein the PS4 variant comprises one or more of the following amino acid substitutions: W66S, E160F/G/L/P/R/S, S161A, R196H/P/V, W221A, K222T, Q305T/L, H307L, and/or W308A/L/S of SEQ ID NO: 1 or 2 or   wherein the PS4 variant comprises additional one or more of the following amino acid substitutions: W66S, R196H/P/V, W221A, K222T, H307L, and/or W308 of SEQ ID NO: 1 or 2 or   wherein the PS4 variant comprises additional one or more of the following amino acid substitutions: E160F/G/L/P/R/S, S161A, and/or Q305T/L of SEQ ID NO: 1 or 2 or   wherein the PS4 variant has an additional amino acid substitution in position 141 or   wherein the PS4 variant has the additional amino acid substitution A141P or   wherein the PS4 variant has an additional amino acid substitution in position 223 or   wherein the PS4 variant has the additional amino acid substitution G223A or   wherein the PS4 variant has further amino acid substitution(s) in one or more of the following position(s) selected from the group consisting of 134, 141, 223, 157, 307, and 334 or   wherein the PS4 variant further has one or more of the amino acid substitution(s) selected from the group consisting of G134R, A141P, G223A, I157L, H307L, S334P, and D343E or   wherein the PS4 variant further has the following amino acid substitution(s) G134R, A141P, G223A, I157L, H307L, S334P, and D343E or   wherein the PS4 variant according to any one of  claims 1 - 34  additional having amino acid substitution(s) in one or more of the following position(s) selected from the group consisting of 157, 158, 160, 161, and 307 or   wherein the PS4 variant in which the half life (t½), preferably at 60 degrees C., is increased by 15% or more, preferably 50% or more, most preferably 100% or more, compared to the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2.   
     
     
         4 . The PS4 variant according to  claim 3 ,
 wherein a food product treated with the PS4 variant has any one or more, preferably all of the following properties: (a) lower firmness; (b) higher resilience; (c) higher cohesiveness; (d) lower crumbliness; and (e) higher foldability compared to a food product which has been treated with the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2 or   wherein the resilience, cohesiveness or foldability of the food product is independently increased by 15% or more, preferably 50% or more, most preferably 100% or more, compared to a food product which has been treated with the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2 or   wherein each of resilience cohesiveness and foldability of a food product treated with the PS4 variant is increased compared to a food product which has been treated with the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2 or   wherein the firmness or the crumbliness of the food product is independently decreased by 15% or more, preferably 50% or more, most preferably 100% or more, relative to a food product which has been treated with the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2 or   wherein each of the firmness and crumblines of a food product treated with the polypeptide is decreased compared to a food product which has been treated with the amino acid sequence of SEQ ID NO: 1, residues 1 to 429 of SEQ ID NO: 1, or SEQ ID NO: 2 or   wherein the PS4 variant comprises a polypeptide fragment of at least 20 residues and has non-maltogenic exoamylase activity.   
     
     
         5 . A process for treating a starch,
 wherein the process comprises contacting the starch with a PS4 variant according to  claim 4  and allowing the polypeptide to generate from the starch one or more linear products or   wherein the process comprises admixing a PS4 variant as set out in any of  claims 1  to  42  with a food or feed ingredient.   
     
     
         6 . A process according to  claim 5 , wherein the food product comprises a dough or a dough product, preferably a processed dough product or a bakery product. 
     
     
         7 . A process for making a bakery product comprising: (a) providing a starch medium; (b) adding to the starch medium a PS4 variant as set out in  claim 1 ; and (c) applying heat to the starch medium during or after step (b) to produce a bakery product. 
     
     
         8 . A food product, feed product, dough product or a bakery product obtained by a process according to  claim 7 . 
     
     
         9 . An improver composition for a dough,
 wherein the improver composition comprises a PS4 variant according to  claim 4 , and at least one further dough ingredient or dough additive or   wherein the improver composition comprises a flour and a PS4 variant according to  claim 4 .   
     
     
         10 . A combination of a PS4 variant according to  claim 4 , together with any one or more of the following:
 (a) maltogenic alpha-amylase also called glucan 1,4-α-maltohydrolase (EC 3.2.1.133) from  Bacillus stearothermophilus , or a variant, homologue, or mutants thereof which have maltogenic alpha-amylase activity;   (b) a bakery xylanase (EC 3.2.1.8) from e.g.  Bacillus  sp.,  Aspergillus  sp.,  Thermomyces  sp. or  Trichoderma  sp.;   (c) α-amylase (EC 3.2.1.1) from  Bacillus amyloliquefaciens  or from  Aspergillus  sp. or a variant, homologue, or mutants thereof which have alpha-amylase activity; and   (d) a lipase such as glycolipase from  Fusarium heterosporum.      
     
     
         11 . A method of desizing textiles, comprising contacting the PS4 variant of  claim 1  with a textile for a time sufficient to desize the textile. 
     
     
         12 . A polynucleotide that encodes a polypeptide according to  claim 1 . 
     
     
         13 . A vector comprising the polynucleotide of  claim 12 . 
     
     
         14 . A bacterial cell comprising the vector of  claim 13 . 
     
     
         15 . A host cell that expresses the polynucleotide of  claim 12 . 
     
     
         16 . The host cell of  claim 15 , wherein the host cell is a  B. subtilis  or  B. licheniformis.    
     
     
         17 . A starch processing composition comprising the PS4 variant of  claim 1 . 
     
     
         18 . A method of baking, comprising adding a PS4 variant of  claim 1  to a substance to be baked, and baking the substance.