Alpha-amylase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-12415972-B2
Application number	US-202418595600-A
Country	US
Kind code	B2
Filing date	Mar 5, 2024
Priority date	May 8, 2015
Publication date	Sep 16, 2025
Grant date	Sep 16, 2025

Abstract

The present invention relates to alpha-amylase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

First claim

The invention claimed is:

1. A variant of a parent alpha-amylase having alpha-amylase activity, which:
a) is a polypeptide having at least 85% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14, and
b) comprises a substitution or a deletion at a position corresponding to N195, N54, V56, G109, R116, or Q172 of the amino acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14, and comprises a substitution and/or deletion in two, three or four positions corresponding to positions R181, G182, D183, and G184 of the amino acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14,
c) has alpha-amylase activity, and
d) has an improved wash performance at a low temperature of 5° C. to 40° C., wherein the improved wash performance is an Improvement Factor (IF) of >1, when compared to the parent alpha-amylase.

2. The variant of claim 1 , which has a substitution at a position corresponding to N195 of the amino acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14.

3. The variant of claim 1 , wherein the parent alpha-amylase comprises an A and a B domain having at least 85%, but less than 100% sequence identity to SEQ ID NO: 10.

4. The variant of claim 1 , wherein the parent alpha-amylase comprises a C domain having at least 85% sequence identity to SEQ ID NO: 11 or 12.

5. The variant of claim 1 , which has a substitution at a position corresponding to N195F of the amino acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14.

6. The variant of claim 1 wherein the parent alpha-amylase has at least 90% sequence identity to SEQ ID NO: 13 or 14.

7. The variant of claim 1 wherein the parent alpha-amylase has at least 95% sequence identity to SEQ ID NO: 13 or 14.

8. The variant of claim 3 , wherein the A and B domains further comprise a modification in one or more positions corresponding to positions: H1, T5, G7, Q11, N16, V17, Q32, A37, T40, P45, W48, G50, T51, A60, K72, R87, Q98, M105, F113, Q118, Q125, G133, T134, W140, G142, G149, T165, W167, Q169, R171, L173, A186, E190, T193, G196, A204, V206, P211, 1214, V215, L217, L219, A225, L228, L235, V238, K242, S244, M246, M248, L250, G255, Q256, N260, A263, V264, A265, Y267, K269, N270, G273, S280, W284, T285, M286, F289, V291, Y295, Q299, S304, R320, H321, S323, H324, V326, F328, T334, D337, Q345, G346, G348, T355, S376, D377, D379, Y382, S383, Q385, K391, K393, and Q395 of SEQ ID NO: 13 or 14.

9. The variant of claim 8 , wherein the modification is selected from the group consisting of H1*, T5K, G7K, G7A, Q11H, N16S, N16H, V17L, Q32S, A37H, A37M, A37V, A37S, A37Y, A37R, A37L, T40D, T40G, T40K, P45A, W48G, W48Y, W48F, G50A, T51K, T51E, T51G, T51A, T51S, T51G, T51D, N54S, V56T, A60V, K72R, K72H, K72S, K72Q, K72E, K72N, K72A, K72M, R87S, Q98S, Q98A, M105F, M105I, M105V, M105L, G109A, G109S, F113W, F113S, F113N, F113Y, F113R, F113L, F113Q, R116Q, R116V, R116K, R116W, R116L, R116A, R116H, R116M, R116E, R116S, R116I, R116G, Q118N, Q118K, Q118G, Q118S, Q118F, Q118R, Q125P, Q125K, Q125A, Q125T, N125D, G133S, F133Q, T134E, W140Y, G142T, G149Q, T165S, T165G, T165V, W167I, W167G, W167F, W167R, W167S, W167H, W167L, W167M, W167Y, Q169E, R171H, Q172G, Q172R, Q172N, Q172D, Q172Y, Q172M, Q172S, Q172T, Q172K, Q172H, Q172E, L173V, L173G, L173H, L173A, L173I, L173P, L173T, L173F, L173M, G184T, A186D, A186N, A186E, A186Q, A186H, E190P, T193K, G196R, A204T, A204V, A204S, A204G, V206L, V206S, V206Y, P211D, I214G, I214H, I214S, I214T, I214L, I214E, I214W, V215T, L217T, L217Q, L219V, L219H, A225V, L228I, L235V, L235A, V238A, V238T, V238G, K242Q, S244Q, M246L, M246A, M246I, M246F, M246V, M246S, M248T, L250I, L250V, L250T, L250A, L250F, L250M, G255N, G255A, G255S, Q256A, N260G, A263G, V264I, V264T, A265G, Y267I, Y267M, Y267H, Y267L, K269Q, N270G, N270T, G273R, S280W, S280L, S280T, S280A, S280K, S280Q, W284H, T285L, T285Q, M286F, M286L, A288L, A288V, F289I, F289L, V291G, V291A, V291T, Y295N, Y295F, Q299V, S304N, S304R, R320A, R320V, H321Y, S323N, H324L, V326L, F328L, F328M, F328V, F328I, T334S, D337H, Q345D, G346T, G346D, G346P, G348S, G348P, T355L, T355F, S376H, S376T, S376V, D377H, D377S, D377A, D377Q, D379S, D379G, D379R, D379A, Y382M, Y382I, Y382L, Y382F, S383G, S383A, Q385L, K391A, K391Y, K391V, K391M, K391E, K391D, K391R, K391H, K391W, K391I, K391Q, K391L, K393Y, K393R, K393Q, K393S, and Q395P.

10. The variant of claim 4 , wherein the C domain further comprises a modification in one or more positions corresponding to positions: T400, H402, A420, G423, T444, A445, Q449, T459, P473, C474, G476, G477, and K484 of SEQ ID NO: 13 or I405, A421, A422, A428, R439, G448, W467, D476, and G477 of SEQ ID NO: 14.

11. The variant of claim 10 , wherein the modification is selected from the group consisting of:
T400P, H402R, A420Q, A420S, A420K, A420L, G423H, T444Q, T444S, T444D, T444Y, T444H, T444V, T444R, T444A, A445Q, Q449T, T459N, P473R, P473A, P473G, P473T, P473K, C474V, G476K, G477A, G477Q, K484A, K484G, K484P, K484E, K484Q, and K484S, wherein the positions correspond to SEQ ID NO: 13; or
I405L, A421H, A422P, A428T, G448D, D476K, D476G, D476N, D476Y, G477S, G477A, G477T, and G477Q, wherein the positions correspond to SEQ ID NO: 14.

12. The variant of claim 1 , which comprises:
a) a deletion and/or a substitution at two or more positions corresponding to positions R181, G182, D183, and G184 of SEQ ID NO: 13 or 14, and
b) a further modification at one or more positions:
i) H1, T5, G7, Q11, N16, V17, Q32, A37, T40, P45, W48, G50, T51, K72, R87, Q98, M105, F113, Q118, Q125, G133, T134, W140, G142, G149, T165, W167, Q169, R171, L173, A186, E190, T193, A204, V206, P211, 1214, V215, L217, L219, A225, L235, V238, K242, S244, M246, M248, L250, G255, Q256, N260, A263, V264, Y267, K269, N270, G273, S280, W284, T285, M286, F289, V291, Y295, Q299, R320, H321, S323, H324, V326, F328, T334, D337, Q345, G346, G348, T355, S376, D377, D379, Y382, S383, Q385, K391, K393, Q395, A420, G423, T444, A445, Q449, T459, P473, C474, G476, G477, and K484, wherein the positions correspond to SEQ ID NO: 13; or
ii) A37, T40, A60, K72, F113, N125, F133, T134, T165, Q169, L173, A186, G196, A204, V206, A225, L228, K242, S244, G255, N260, A265, K269, S280, W284, Y295, S304, R320, H321, S323, V326, K391, 1405, A421, A422, A428, R439, G448, W467, D476, and G477, wherein the positions correspond to SEQ ID NO: 14.

13. The variant of claim 12 , wherein the deletion a) is selected from the group consisting of R181+G182, R181+D183, R181+G184, G182+D183, G182+G184, or D183+G184.

14. The variant of claim 12 , which comprises one or more of the following modifications:
a) H1*, T5K, G7K, G7A, Q11H, N16S, N16H, V17L, Q32S, A37H, A37M, A37V, A37S, A37Y, A37R, A37L, T40G, T40K, P45A, W48G, W48Y, W48F, G50A, T51K, T51E, T51G, T51A, T51S, T51G, T51D, N54S, V56T, K72R, K72H, K72S, K72Q, K72E, K72N, K72A, K72M, R87S, Q98S, Q98A, M105F, M105I, M105V, M105L, G109A, G109S, F113W, F113S, F113N, F113Y, F113R, F113L, F113Q, R116Q, R116V, R116K, R116W, R116L, R116A, R116H, R116M, R116E, R116S, R116I, R116G, Q118N, Q118K, Q118G, Q118S, Q118F, Q118R, 0125P, Q125K, Q125A, Q125T, G133S, T134E, W140Y, G142T, G149Q, T165S, T165G, T165V, W167I, W167G, W167F, W167R, W167S, W167H, W167L, W167M, W167Y, 0169E, R171H, 0172G, Q172R, Q172N, Q172D, Q172Y, Q172M, Q172S, Q172T, Q172K, Q172H, Q172E, L173V, L173G, L173H, L173A, L173I, L173P, L173T, L173F, L173M, G184T, A186D, A186N, A186E, A186Q, A186H, E190P, T193K, A204T, A204V, A204S, A204G, V206L, V206S, V206Y, P211D, I214G, I214H, I214S, I214T, I214L, I214E, I214W, V215T, L217T, L217Q, L219V, L219H, A225V, L235V, L235A, V238A, V238T, V238G, K242Q, S244Q, M246L, M246A, M246I, M246F, M246V, M246S, M248T, L250I, L250V, L250T, L250A, L250F, L250

Assignees

• Novozymes As

Inventors

• Andersen Carsten
• Ghadiyaram Chakshusmathi
• Sainathan Rajendra Kulothungan
• Balumuri Padmavathi
• Iyer Padma Venkatachalam
• Damager Iben
• Munch Astrid
• Dalal Sohel

Classifications

• C12Y302/01001

  Alpha-amylase (3.2.1.1) · CPC title

• C12N9/2414 Primary

  Alpha-amylase (3.2.1.1.) · CPC title

• C12N9/2417

  from microbiological source · CPC title

• C11D3/38681

  Chemically modified or immobilised enzymes · CPC title

• C11D3/386 Primary

  Preparations containing enzymes {, e.g. protease or amylase} · CPC title