Pullulanase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-10640755-B2
Application number	US-201816016950-A
Country	US
Kind code	B2
Filing date	Jun 25, 2018
Priority date	Jan 22, 2014
Publication date	May 5, 2020
Grant date	May 5, 2020

Abstract

The present invention relates to pullulanase variants 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.

First claim

The invention claimed is:

1. A pullulanase variant comprising a substitution at one or more positions corresponding to positions 368, 393, 492, 610, 631, and 632, of the polypeptide of SEQ ID NO: 3, wherein the pullulanase variant has:
(a) pullulanase activity,
(b) at least 85%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3,
(c) has increased thermoactivity compared to the pullulanase of SEQ ID NO: 3; and
(d) the substitution is selected from the group consisting of:
(i) a substitution at a position corresponding to position 368 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(ii) a substitution at a position corresponding to position 393 with Ala, Arg, Asp, Cyt, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(iii) a substitution at a position corresponding to position 492 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(iv) a substitution at a position corresponding to position 610 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(v) a substitution at a position corresponding to position 631 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(vi) a substitution at a position corresponding to position 632 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val.

2. The pullulanase variant of claim 1 , comprising at least two of the following substitutions:
(a) a substitution at a position corresponding to position 368 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(b) a substitution at a position corresponding to position 393 with Ala, Arg, Asp, Cyt, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(c) a substitution at a position corresponding to position 492 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(d) a substitution at a position corresponding to position 610 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(e) a substitution at a position corresponding to position 631 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(f) a substitution at a position corresponding to position 632 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val.

3. The pullulanase variant of claim 1 , comprising at least three of the following substitutions:
(a) a substitution at a position corresponding to position 368 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(b) a substitution at a position corresponding to position 393 with Ala, Arg, Asp, Cyt, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(c) a substitution at a position corresponding to position 492 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(d) a substitution at a position corresponding to position 610 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(e) a substitution at a position corresponding to position 631 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(f) a substitution at a position corresponding to position 632 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val.

4. The pullulanase variant of claim 1 , comprising at least four of the following substitutions:
(a) a substitution at a position corresponding to position 368 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(b) a substitution at a position corresponding to position 393 with Ala, Arg, Asp, Cyt, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(c) a substitution at a position corresponding to position 492 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(d) a substitution at a position corresponding to position 610 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(e) a substitution at a position corresponding to position 631 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(f) a substitution at a position corresponding to position 632 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val.

5. The pullulanase variant of claim 1 , comprising at least five of the following substitutions:
(a) a substitution at a position corresponding to position 368 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(b) a substitution at a position corresponding to position 393 with Ala, Arg, Asp, Cyt, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(c) a substitution at a position corresponding to position 492 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(d) a substitution at a position corresponding to position 610 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(e) a substitution at a position corresponding to position 631 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(f) a substitution at a position corresponding to position 632 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val.

6. The pullulanase variant of claim 1 , comprising each of the following substitutions:
(a) a substitution at a position corresponding to position 368 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(b) a substitution at a position corresponding to position 393 with Ala, Arg, Asp, Cyt, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(c) a substitution at a position corresponding to position 492 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(d) a substitution at a position corresponding to position 610 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,
(e) a substitution at a position corresponding to position 631 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,
(f) a substitution at a position corresponding to position 632 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val.

7. The pullulanase variant of claim 1 , which has at least 90% sequence identity to the polypeptide of SEQ ID NO: 3.

8. The pullulanase variant of claim 1 , which has at least 95% sequence identity to the polypeptide of SEQ ID NO: 3.

9. A composition comprising a pullulanase variant of claim 1 and one or more enzymes selected from the group consisting of: alpha-amylase, beta-amylase, glucoamylase, and protease.

10. A process of producing a fermentation product, comprising:
(a) liquefying a 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 pullulanase variant of claim 1 .

Assignees

• Novozymes As

Inventors

• Matsui Tomoko
• Yamagishi Akihiko

Classifications

• C12P7/02

  containing a hydroxy group · CPC title

• C12P19/02

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title

• C12P7/06

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

• C12P2203/00

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

• C12Y302/01041

  Pullulanase (3.2.1.41) · CPC title