# US 10,246,691 B2 · Variants of polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

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- Number: US 10,246,691 B2
- Document ID: US10246691B2
- Status: Granted
- Kind: B2
- Application number: 13/876,759
- Filed: 2011-09-29
- Published: 2013-08-22
- Granted: 2019-04-02
- Earliest claimed: 2010-09-30
- Assignees: Novozymes, Inc.
- Applicants: Matthew Sweeney; Mark Wogulis
- Inventors: Matthew Sweeney; Mark Wogulis
- Web page: https://patentgrep.com/patent/US10246691B2

## CPC

- C12N 9/18: Carboxylic ester hydrolases {(3.1.1)}
- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12N 9/2445: {Beta-glucosidase (3.2.1.21)}
- C12N 15/8257: {for the production of primary gene products, e.g. pharmaceutical products, interferon}
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- C12Y 302/01021: Beta-glucosidase (3.2.1.21)
- C12Y 302/01091: Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
- D21C 5/005: {Treatment of cellulose-containing material with microorganisms or enzymes}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- D21C: PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR

## Abstract

The present invention relates to variants of GH61 polypeptides having cellulolytic enhancing activity, wherein the variants comprise a substitution at one or more positions corresponding to positions 90, 131, 134, and 141 of the full-length polypeptide of SEQ ID NO: 2, and have cellulolytic enhancing activity. 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.

## Claims

### Claim 1

A variant of a parent GH61 polypeptide having cellulolytic enhancing activity, comprising a substitution at one or more positions corresponding to positions 90, 131, 134, and 141 of the full-length polypeptide of SEQ ID NO: 2, wherein the substitution at a position corresponding to position 90 is with Val, the substitution at a position corresponding to position 131 is with Ser, the substitution at a position corresponding to position 134 is with Leu, and the substitution at a position corresponding to position 141 is with Trp, wherein the variant has cellulolytic enhancing activity and increased thermal activity and/or increased thermostability, and wherein the variant has
at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2.

### Claim 2 (depends on 1)

The variant of claim 1, which comprises one or more substitutions selected from the group consisting of L90V, D131S, M134L, and A141W.

### Claim 3 (depends on 1)

The variant of claim 1, which comprises the substitutions L90V+D131S+M134L+A141W of the mature polypeptide of SEQ ID NO: 2.

### Claim 4 (depends on 1)

An isolated polynucleotide encoding the variant of claim 1.

### Claim 5 (depends on 4)

An isolated recombinant host cell transformed with the polynucleotide of claim 4.

### Claim 6 (depends on 5)

A method of producing a variant, comprising:
(a) cultivating the recombinant host cell of claim 5 under conditions suitable for the expression of the variant; and
(b) recovering the variant.

### Claim 7 (depends on 4)

A transgenic plant, plant part or plant cell transformed with the polynucleotide of claim 4.

### Claim 8 (depends on 1)

A method of producing the variant of claim 1, comprising:
(a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the variant under conditions conducive for production of the variant; and
(b) recovering the variant.

### Claim 9

A method for obtaining a variant of a parent GH61 polypeptide having cellulolytic enhancing activity, comprising introducing into the parent GH61 polypeptide a substitution at one or more positions corresponding to positions 90, 131, 134, and 141 of the full-length polypeptide of SEQ ID NO: 2, wherein the substitution at a position corresponding to position 90 is with Val, the substitution at a position corresponding to position 131 is with Ser, the substitution at a position corresponding to position 134 is with Leu, and the substitution at a position corresponding to position 141 is with Trp, wherein the variant has cellulolytic enhancing activity and increased thermal activity and/or increased thermostability, and wherein the variant has
at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2.

### Claim 10 (depends on 1)

A method for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition comprising the variant of claim 1.

### Claim 11 (depends on 10)

The method of claim 10, further comprising recovering the degraded cellulosic material.

### Claim 12 (depends on 1)

A method for producing a fermentation product, comprising:
(a) saccharifying a cellulosic material with an enzyme composition comprising the variant of claim 1;
(b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.

### Claim 13 (depends on 1)

A method of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition comprising the variant of claim 1.

### Claim 14 (depends on 13)

The method of claim 13, wherein the fermenting of the cellulosic material produces a fermentation product.

### Claim 15 (depends on 14)

The method of claim 14, further comprising recovering the fermentation product from the fermentation.

### Claim 16 (depends on 1)

A detergent composition comprising the variant of claim 1 and a surfactant.

### Claim 17 (depends on 1)

A whole broth formulation or cell culture composition comprising the variant of claim 1.

### Claim 18 (depends on 1)

The variant of claim 1, wherein the variant has at least 96% sequence identity to the mature polypeptide of SEQ ID NO: 2.

### Claim 19 (depends on 1)

The variant of claim 1, wherein the variant has at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 2.

### Claim 20 (depends on 1)

The variant of claim 1, which comprises a substitution at two positions corresponding to any of positions 90, 131, 134, and 141.

### Claim 21 (depends on 1)

The variant of claim 1, which comprises a substitution at three positions corresponding to any of positions 90, 131, 134, and 141.

### Claim 22 (depends on 1)

The variant of claim 1, which comprises a substitution at each position corresponding to positions 90, 131, 134, and 141.

### Claim 23 (depends on 10)

The method of claim 10, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

### Claim 24 (depends on 12)

The method of claim 12, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

### Claim 25 (depends on 13)

The method of claim 13, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

### Claim 26 (depends on 1)

The variant of claim 1, wherein the variant has at least 98% sequence identity to the mature polypeptide of SEQ ID NO: 2.

### Claim 27 (depends on 1)

The variant of claim 1, wherein the variant has at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 2.