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US 2011/0033575 A1Published application
Pseudomonas saccharophila g4-amylase variants and uses thereof
OwnerKarsten Matthias Kragh, Anja Hemmingsen Kellet-Smith +2inventors
InventorsKarsten Matthias Kragh, Anja Hemmingsen Kellet-Smith, Andrew Shaw, Regina Chin
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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.
Claims18
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.
8 more elements
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.
57 more elements
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.
Dependencies are inferred from the claim text.