# US 8,465,949 B2 · Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

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- Number: US 8,465,949 B2
- Document ID: US8465949B2
- Status: Granted
- Kind: B2
- Application number: 13/548,718
- Filed: 2012-07-13
- Published: 2013-02-21
- Granted: 2013-06-18
- Earliest claimed: 2009-07-07
- Assignees: Novozymes, Inc.; Novozymes A/S
- Applicants: Junxin Duan; Lan Tang; Ye Liu; Wenping Wu; Jason Quinlan; Randall Kramer
- Inventors: Junxin Duan; Lan Tang; Ye Liu; Wenping Wu; Jason Quinlan; Randall Kramer
- Web page: https://patentgrep.com/patent/US8465949B2

## CPC

- C12N 15/8246: {Non-starch polysaccharides, e.g. cellulose, fructans, levans}
- C07K 14/4705: {stimulating, promoting or activating activity}
- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12N 9/86: acting on amide bonds in cyclic amides, e.g. penicillinase {(3.5.2)}
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- C12Y 305/02006: Beta-lactamase (3.5.2.6)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## Abstract

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

## Claims

### Claim 1

A method of producing a mutant cell, comprising disrupting or deleting a nucleotide sequence encoding a polypeptide having cellulolytic enhancing activity in a parent Penicillium cell, which results in the mutant producing less of the polypeptide than the parent Penicillium cell, wherein the polypeptide having cellulolytic enhancing activity is selected from the group consisting of: (a) a polypeptide having at least 95% sequence identity to amino acids 22 to 332 of SEQ ID NO: 2; (b) a polypeptide encoded by the polynucleotide having nucleotides 64 to 1018 of SEQ ID NO: 1; and (c) a fragment of a polypeptide of (a) or (b) that has cellulolytic enhancing activity.

### Claim 2 (depends on 1)

The method of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 97% identity to amino acids 22 to 332 of SEQ ID NO: 2.

### Claim 3 (depends on 1)

The method of claim 1, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 2.

### Claim 4 (depends on 1)

The method of claim 1, wherein the polypeptide comprises or consists of amino acids 22 to 332 of SEQ ID NO: 2.

### Claim 5 (depends on 1)

The method of claim 1, wherein the polypeptide is encoded by a polynucleotide comprising or consisting of nucleotides 64 to 1018 of SEQ ID NO: 1.

### Claim 6 (depends on 1)

The method of claim 1, wherein the polypeptide is encoded by the polynucleotide contained in plasmid pGEM-T-Ppin7 which is contained in E. coli DSM 22711.

### Claim 7 (depends on 1)

The method of claim 1, wherein the mutant cell is a Penicillium pinophilum cell.

### Claim 8

A mutant cell, comprising a disruption or deletion of a nucleotide sequence encoding a polypeptide having cellulolytic enhancing activity in a parent Penicillium cell, wherein the mutant produces less of the polypeptide than the parent Penicillium cell, and wherein the polypeptide having cellulolytic enhancing activity is selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 95% sequence identity to amino acids 22 to 332 of SEQ NO: 2; (b) a polypeptide encoded by a polynucleotide comprising SEQ ID NO: 1; and (c) a fragment of SEQ ID NO: 2 having cellulolytic enhancing activity.

### Claim 9 (depends on 8)

The mutant cell of claim 8, wherein the polypeptide comprises an amino acid sequence having at least 97% identity to amino acids 22 to 332 of SEQ ID NO: 2.

### Claim 10 (depends on 8)

The mutant cell of claim 8, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 2.

### Claim 11 (depends on 8)

The mutant cell of claim 8, wherein the polypeptide comprises or consists of amino acids 22 to 332 of SEQ ID NO: 2.

### Claim 12 (depends on 8)

The mutant cell of claim 8, wherein the polypeptide is encoded by a polynucleotide comprising or consisting of nucleotides 64 to 1018 of SEQ ID NO: 1.

### Claim 13 (depends on 8)

The mutant cell of claim 8, wherein the polypeptide is encoded by the polynucleotide contained in plasmid pGEM-T-Ppin7 which is contained in E. coli DSM 22711.

### Claim 14 (depends on 8)

The mutant cell of claim 8, further comprising an expression vector for a protein.

### Claim 15 (depends on 14)

The mutant cell of claim 14, wherein the protein is a native protein.

### Claim 16 (depends on 14)

The mutant cell of claim 14, wherein the protein is a heterologous protein.

### Claim 17 (depends on 8)

The mutant cell of claim 8, wherein the mutant cell is a Penicillium pinophilum cell.

### Claim 18 (depends on 14)

A method of producing a protein, comprising: (a) cultivating the mutant cell of claim 14 under conditions conducive for production of the protein; and (b) recovering the protein.

### Claim 19 (depends on 18)

The method of claim 18, wherein the mutant cell is a Penicillium pinophilum cell.