Pullulanase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-10030237-B2
Application number	US-201515112096-A
Country	US
Kind code	B2
Filing date	Jan 21, 2015
Priority date	Jan 22, 2014
Publication date	Jul 24, 2018
Grant date	Jul 24, 2018

Abstract

The present invention relates to pullulanase variants having increased thermoactivity and comprising substitutions of the parent pullulanase at one or more positions corresponding to positions 393, 143, 150, 243, 244, 345, 346, 368, 370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3. 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. The variants are mainly derived from pullulanases from Bacillus deramificans, Bacillus acidopullulyticus or hybrid pullulanases.

First claim

The invention claimed is:

1. A pullulanase variant comprising a substitution at one or more positions corresponding to positions 393, 143, 150, 243, 244, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 456, 486, 492, 610, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3 wherein the variant has pullulanase activity, and;
a) wherein the variant has at least 85%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO: 3 and wherein the variant has increased thermoactivity compared to the pullulanase of SEQ ID NO: 3; or
b) wherein the variant has at least 85%, sequence identity to the polypeptide of SEQ ID NO: 21.

2. The pullulanase variant according to claim 1 , comprising a substitution at two or more positions corresponding to positions 393, 143, 150, 243, 244, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 456, 486, 492, 610, 624, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3.

3. The pullulanase variant according to claim 1 , comprising a substitution at three or more positions corresponding to positions 393, 143, 150, 243, 244, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 431, 456, 486, 492, 610, 624, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3.

4. The pullulanase variant according to claim 1 , comprising a substitution at four or more positions corresponding to positions 393, 143, 150, 243, 244, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3.

5. The pullulanase variant according to claim 1 , comprising a substitution at five or more positions corresponding to positions 393, 143, 150, 243, 244, 345, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3.

6. The pullulanase variant according to claim 1 , comprising a substitution at six or more positions corresponding to positions 393, 143, 150, 243, 244, 345, 346, 368, 370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3.

7. The pullulanase variant according to claim 1 , comprising a substitution at one position corresponding to positions 393, 143, 150, 243, 244, 345, 346, 368, 456, 492, 610 or 624 of the polypeptide of SEQ ID NO: 3 wherein the variant has pullulanase activity.

8. The variant according to claim 1 , wherein the variant comprises or consists of one or more substitutions selected from the group consisting of 393A, 143G, 150R, 243E, 244K, 345P, 346S, 368G, 370S, 373L, 381V, 382T, 385F, 387L, 402T, 429V, 430R, 431E, 432F, 456A, 486C, 492S,A, 610R,L, 624S, 631S, 632C and 665I.

9. The variant of claim 1 , wherein the variant comprises at least one of the following substitutions or combinations of substitutions:
N368G;
E150R;
N346S;
N243E;
S244K;
V143G;
N393A;
N610R;
N610L;
G624S;
F456A;
T492S,A;
V486C+T492S,A;
N368G+M402T;
T631S+S632C;
V486C+T492S,A+T631S+S632C;
N393A+T631S+S632C;
N393A+V486C+T492S,A+T631S+S632C;
N393A+G624S+S632C;
N393A+N610R+T631S+S632C;
N393A+G624S+T631S+S632C;
N393A+N610R+G624S+T631S+S632C;
N393A+V486C+T492S,A+G624S+T631S+S632C;
N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;
N368G+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;
N346S+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;
N393A+F456A+V486C+T492S,A+N610R+G624S+T631S+S632C;
N393A+T492S,A+N610R+G624S+T631S+S632C;
N368G+N393A+T492S,A+N610R+G624S+T631S+S632C;
A345P+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;
N368G+K370S+I373L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;
I381V+Q385E+Q387L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;
I381V+N382T+Q385E+Q387L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;
A345P+N368G+N393A+T492S,A+N610R+G624S+T631S+S632C;
N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C;
A345P+N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C;
A345P+N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C+V665I;
N393A+T430R+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;
N393A+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;
N393A+I429V+Q431E+V486C+T492S,A+N610R+G624S+T631S+S632C;
N393A+I429V+T430R+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;
N368G+N393A+A492S,A;
N368G+N393A;
N393A+N610R;
N368G+N393A+N610R;
N368G+N393A+T492S,A+N610R+G624S;
N368G+N393A+T492S,A+N610R+G624S+T631S+S632C+Q431E+L432F;
N368G+N393A+N610R+G624S+T631S+S632C;
N368G+N393A+T492S,A+N610R+G624S+T631S+S632C; or
N368G+N393A+N610R+G624S+T631S+S632C+Q431E+L432F.

10. A variant catalytic domain, wherein the variant catalytic domain comprises a substitution at one or more positions corresponding to positions 393, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 456, 486, 492, 531, 532, 610, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3; and;
a) wherein the variant catalytic domain has at least 85%, but less than 100% sequence identity to amino acids 330 to 828 of SEQ ID NO: 3; or
b) wherein the variant catalytic domain has at least 85%, SEQ ID NO: 21.

11. The variant catalytic domain according to claim 10 comprising a substitution at two or more positions corresponding to positions 393, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 456, 486, 492, 610, 624, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3.

12. The variant catalytic domain according to claim 10 comprising a substitution at three or more positions corresponding to positions 393, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 431, 456, 486, 492, 610, 624, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3.

13. The variant catalytic domain according to claim 10 comprising a substitution at four or more positions corresponding to positions 393, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3.

14. The variant catalytic domain according to claim 10 comprising a substitution at five or more positions corresponding to positions 393, 345, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3.

15. The variant catalytic domain according to claim 10 comprising a substitution at six or more positions corresponding to positions 393, 345, 346, 368, 370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632 and 665 of the polypeptide of SEQ ID NO: 3.

16. A pullulanase variant comprising the variant catalytic domain according to claim 10 , wherein the pullulanase variant has pullulanase activity and increased thermoactivity compared to the parent pullulanase, and the variant has at least 60% relative activity when measured at 70° C. relative to activity at 65° C.

17. The pullulanase variant according to claim 16 , wherein the variant comprises or consists of one or more substitutions selected from the group consisting of 393A, 345P, 346S, 368G, 370S, 373L, 381V, 382T, 385F, 387L, 402T, 429V, 430R, 431E, 432F, 456A, 486C, 492S,A, 610R,L, 624S, 631S, 632C and 665I.

18. The pullulanase variant according to claim 16 , further comprising an N-terminal part comprising at least one of the domains selected from a CBM41 domain, an X45 domain and a CBM48 domain.

19. The pullulanase variant according to claim 18 , comprising an N-terminal part comprising a CBM41 domain, an X45 domain and a CBM48 domain.

20. The pullulanase variant according to claim 19 , further comprising an X25 domain.

21. The pullulanase variants according to

Assignees

• Novozymes As

Inventors

• Matsui Tomoko
• Yamagishi Akihiko

Classifications

• C12N9/2457 Primary

  Pullulanase (3.2.1.41) · CPC title

• C12P7/06

  Ethanol, i.e. non-beverage · CPC title

• C12P7/02

  containing a hydroxy group · CPC title

• C12Y302/01041

  Pullulanase (3.2.1.41) · CPC title

• C12P2203/00

  Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source · CPC title