# US 7,608,689 B2 · Methods for enhancing the degradation or conversion of cellulosic material

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- Number: US 7,608,689 B2
- Document ID: US7608689B2
- Status: Granted
- Kind: B2
- Application number: 11/541,099
- Filed: 2006-09-29
- Published: 2007-04-05
- Granted: 2009-10-27
- Earliest claimed: 2005-09-30
- Assignees: Novozymes, Inc.
- Inventors: Paul Harris; Michael Rey; Hanshu Ding
- Web page: https://patentgrep.com/patent/US7608689B2

## CPC

- C12P 7/10: substrate containing cellulosic material
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12P 7/26: Ketones
- C12P 7/40: containing a carboxyl group {including Peroxycarboxylic acids}
- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- C12Y 302/01021: Beta-glucosidase (3.2.1.21)
- C13K 1/02: obtained by saccharification of cellulosic materials (manufacture of fodder A23K10/32)
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- C13K: SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES (chemically synthesised sugars or sugar derivatives C07H; polysaccharides, e.g. starch, derivatives thereof C08B; malt C12C; fermentation or enzyme-using processes for preparing compounds containing saccharide radicals C12P19/00; production of sucrose C13B)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present invention relates to methods for degrading or converting a cellulosic material and for producing a substance from a cellulosic material.

## Claims

### Claim 1

A method for degrading or converting a cellulosic material, comprising: treating the cellulosic material with an effective amount of one or more cellulolytic proteins in the presence of an effective amount of a polypeptide having cellulolytic enhancing activity, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulosic material compared to the absence of the polypeptide having cellulolytic enhancing activity and wherein the polypeptide having cellulolytic enhancing activity does not itself degrade or convert the cellulosic material in the absence of one or more cellulolytic protein and wherein the polypeptide having cellulolytic enhancing activity is selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence which has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 2;
(b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which has at least 95% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 1; and
(c) a polypeptide encoded by a polynucleotide which hybridizes under at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii), wherein high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

### Claim 2 (depends on 1)

The method of claim 1, further comprising treating the cellulosic material with an effective amount of one or more enzymes selected from the group consisting of a hemicellulase, esterase, protease, laccase, peroxidase, or a mixture thereof.

### Claim 3 (depends on 1)

The method of claim 1, further comprising recovering the degraded cellulosic material.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cellulolytic protein and/or the polypeptide having cellulolytic enhancing activity are in the form of a fermentation broth with or without cells.

### Claim 5 (depends on 1)

The method of claim 1, wherein the polypeptide having cellulolytic enhancing activity comprises or consists of the amino acid, sequence of SEQ ID NO: 2 or the mature polypeptide thereof; or a fragment thereof having cellulolytic enhancing activity.

### Claim 6 (depends on 1)

The method of claim 1, wherein the polypeptide having cellulolytic enhancing activity is encoded by the polynucleotide contained in plasmid pTr3337 which is contained in E. coli NRRL B-30878.

### Claim 7 (depends on 1)

The method of claim 1, wherein the mature polypeptide coding sequence of SEQ ID NO: 1 is nucleotides 77 to 766 of SEQ ID NO: 1.

### Claim 8 (depends on 1)

The method of claims 1, wherein the mature polypeptide of SEQ ID NO: 2 is amino acids 20 to 249 of SEQ ID NO: 2.

### Claim 9 (depends on 1)

The method of claim 1, wherein the polypeptide having cellulolytic enhancing activity comprises an amino acid sequence which has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 2.

### Claim 10 (depends on 9)

The method of claim 9, wherein the polypeptide having cellulolytic enhancing activity comprises an amino acid sequence which has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 2.

### Claim 11 (depends on 1)

The method of claim 1, wherein the polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide comprising a nucleotide sequence which has at least 95% a sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 1.

### Claim 12 (depends on 11)

The method of claim 11, wherein the polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide comprising a nucleotide sequence which has at least 97% a sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 1.

### Claim 13 (depends on 1)

The method of claim 1, wherein the polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide which hybridizes under high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii), wherein high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.30% SDS, 200 μg/ml sheared and denatured salmon sperm DNA and 50% a formamide and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.