Pullulanase variants and polynucleotides encoding same

• US 10,030,237 B2
• Filed: 01/21/2015
• Issued: 07/24/2018
• Est. Priority Date: 01/22/2014
• Status: Active Grant

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: Matsui, Tomoko, Yamagishi, Akihiko
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US15/112,096
• Publication Number:
  US 20170044511A1
• Time in Patent Office: 1,280 Days
• Field of Search: 435210, 435209, 435200, 435 98, 435 691, 435 911, 4353201, 4352523, 43525411, 536 231, 536 232, 530350
• CPC Class Codes: C12N 9/2457 Pullulanase (3.2.1.41)
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/16 produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin non-biological hydrolysis of starch C08B30/00
• CPC Class Codes: C12P 2203/00 Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source ethanol C12P7/10
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12Y 302/01041 Pullulanase (3.2.1.41)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

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.

Claims

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.

24. The variant according to claim 1, wherein the variant has increased thermoactivity.

25. The variant according to claim 1, wherein the variant has increased specific activity toward starch or maltodextrin compared to any of SEQ ID NO:

3.

28. A composition comprising the variant pullulanase of claim 1.

29. The composition according to claim 28, further comprising one or more enzymes selected from the group consisting of:

glucoamylase, alpha-amylase, beta-amylase, and protease.

30. The composition according to claim 28, comprising a variant pullulanase of claim 1 and:

i) a glucoamylase, an alpha-amylase and a protease;

ii) an alpha-amylase and a protease;

iii) a glucoamylase and an alpha-amylase;

iv) a beta-amylase;

or v) a glucoamylase.

31. A process of producing a fermentation product from starch-containing material comprising the steps of:

(a) liquefying starch-containing material in the presence of an alpha amylase;

(b) saccharifying the liquefied material in the presence of a glucoamylase; and

(c) fermenting with a fermenting organism;

wherein step (a) and/or step (b) is carried out in the presence of a variant pullulanase of claim 1.

32. A process of producing a fermentation product from starch-containing material, comprising the steps of:

(a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material; and

(b) fermenting with a fermenting organism

wherein step (a) is carried out using at least a glucoamylase, and a variant pullulanase of claim 1.

33. The process according to claim 32, wherein an alpha amylase is added in step (a).

34. The process according to claim 33, wherein saccharification and fermentation is carried out simultaneously.

35. The process according to claim 31, wherein the fermentation product is ethanol.

36. A polynucleotide encoding the variant pullulanase of claim 1.

37. A nucleic acid construct comprising the polynucleotide of claim 36.

38. An expression vector comprising the polynucleotide of claim 36.

39. An isolated host cell comprising the polynucleotide of claim 36.

40. A whole broth formulation or cell culture composition comprising a variant pullulanase of claim 1.

41. The variant of claim 1, which has at least 90% , but less that 100%, sequence identity to the polypeptide of SEQ ID NO:

3.

42. The variant of claim 1, which has at least 95%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:

3.

43. The variant of claim 1, which has at least 97%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:

3.

44. The variant of claim 1, which has at least 98%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:

3.

45. The variant of claim 1, which has at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:

3.

46. The variant of claim 1, which has at least 90% sequence identity to the polypeptide of SEQ ID NO:

21.

47. The variant of claim 1, which has at least 95% sequence identity to the polypeptide of SEQ ID NO:

21.

48. The variant of claim 1, which has at least 96% sequence identity to the polypeptide of SEQ ID NO:

21.

49. The variant of claim 1, which has at least 98% sequence identity to the polypeptide of SEQ ID NO:

21.

50. The variant of claim 1, which has at least 99% sequence identity to the polypeptide of SEQ ID NO:

21.

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 claim 18, wherein the N-terminal parts are selected from CBM41, X45, X25, and CBM48 obtainable from a Bacillus bacterium.

22. The pullulanase variant according to claim 17, wherein the variant comprises one or more substitutions selected from the group consisting of 393A, 368G, 492S,A, 610R,L, 624S, 631S, 632C, 431E, 432F.

23. The pullulanase variant according to claim 22, wherein the variant comprises at least one of the following substitutions or combinations of substitutions:

368G+393A+492S,A;

368G+393A+T492S,A+610R+624S;

393A+492S,A+610R+624S+631S+632C;

368G+393A+492S,A+610R+624S+631S+632C;

368G+393A+492S,A+610R+624S+631S+632C+431E+432F;

368G+393A+610R+624S+631S+632C;

368G+393A+610R+624S+631S+632C+431E+432F.

26. A method for producing a variant pullulanase of a parent pullulanase comprising substitution 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, 531, 532, 610, 624, 631, 632 and 665 of the polypeptide of SEQ ID NO:

3 wherein the variant has pullulanase activity and increased thermoactivity compared to the parent; and

;

a) wherein the variant has at least 85%, but less than 100% sequence identity to the polypeptide of SEQ ID NO;

3;

or

b) wherein the variant has at least 85%, sequence identity to the polypeptide of SEQ ID NO;

21.

27. A variant pullulanase produced by the method of claim 26.