# US 8,465,958 B2 · Polypeptides having endoglucanase activity and polynucleotides encoding same

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 8,465,958 B2
- Document ID: US8465958B2
- Status: Granted
- Kind: B2
- Application number: 13/600,071
- Filed: 2012-08-30
- Published: 2012-12-27
- Granted: 2013-06-18
- Earliest claimed: 2007-12-05
- Assignees: Novozymes A/S
- Applicants: Alfredo Lopez de Leon; Michael Rey
- Inventors: Alfredo Lopez de Leon; Michael Rey
- Web page: https://patentgrep.com/patent/US8465958B2

## CPC

- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12N 9/2434: {acting on beta-1,4-glucosidic bonds}
- C12N 15/8246: {Non-starch polysaccharides, e.g. cellulose, fructans, levans}
- 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
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present invention relates to isolated polypeptides having endoglucanase 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

An isolated polynucleotide encoding a polypeptide having endoglucanase activity, selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 2;
(b) a polynucleotide encoding a polypeptide 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 of the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) the 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.; and
(c) a polynucleotide encoding a polypeptide comprising a nucleotide sequence having at least 90% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 1.

### Claim 2 (depends on 1)

The polynucleotide of claim 1, wherein the encoded polypeptide having endoglucanase activity comprises an amino acid sequence having at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 2.

### Claim 3 (depends on 2)

The polynucleotide of claim 2, wherein the encoded polypeptide having endoglucanase activity comprises an amino acid sequence having at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 2.

### Claim 4 (depends on 3)

The polynucleotide of claim 3, wherein the encoded polypeptide having endoglucanase activity comprises an amino acid sequence having at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 2.

### Claim 5 (depends on 1)

The polynucleotide of claim 1, which hybridizes under at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 1 or the genomic DNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 3, or (iii) the 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 6 (depends on 5)

The polynucleotide of claim 5, which hybridizes under at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 1 or the genomic DNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 3, or (iii) the full-length complementary strand of (i) or (ii), wherein very 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 70° C.

### Claim 7 (depends on 1)

The polynucleotide of claim 1, which comprises a nucleotide sequence having at least 90% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 1.

### Claim 8 (depends on 7)

The polynucleotide of claim 7, which comprises a nucleotide sequence having at least 95% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 1.

### Claim 9 (depends on 8)

The polynucleotide of claim 8, which comprises a nucleotide sequence having at least 97% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 1.

### Claim 10 (depends on 1)

The polynucleotide of claim 1, which encodes a polypeptide having endoglucanase activity comprising or consisting of the amino acid sequence of SEQ ID NO: 2, or a fragment thereof having endoglucanase activity.

### Claim 11 (depends on 1)

The polynucleotide of claim 1, which encodes a polypeptide having endoglucanase activity comprising or consisting of the mature polypeptide of SEQ ID NO: 2.

### Claim 12 (depends on 1)

The polynucleotide of claim 1, which comprises or consists of the nucleotide sequence of SEQ ID NO: 1; or a subsequence thereof encoding a polypeptide fragment having endoglucanase activity.

### Claim 13 (depends on 1)

The polynucleotide of claim 1, which is contained in plasmid pTter16A which is contained in E. coli NRRL B-50081.

### Claim 14 (depends on 1)

A nucleic acid construct comprising the polynucleotide of claim 1 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

### Claim 15 (depends on 14)

A recombinant expression vector comprising the nucleic acid construct of claim 14.

### Claim 16 (depends on 14)

An isolated recombinant host cell comprising the nucleic acid construct of claim 14.

### Claim 17 (depends on 16)

A method for producing a polypeptide having endoglucanase activity, comprising: (a) cultivating the recombinant host cell of claim 16 under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

### Claim 18 (depends on 1)

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

### Claim 19 (depends on 1)

A transgenic plant, plant part or plant cell, which has been transformed with the polynucleotide of claim 1.