# US 7,465,571 B1 · Endoglucanases

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- Number: US 7,465,571 B1
- Document ID: US7465571B1
- Status: Granted
- Kind: B1
- Application number: 09/914,543
- Filed: 1997-05-22
- Granted: 2008-12-16
- Earliest claimed: 1996-05-22
- Assignees: Verenium Corporation
- Inventors: David Lam; Eric J. Mathur
- Web page: https://patentgrep.com/patent/US7465571B1

## CPC

- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- 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
- 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

## Abstract

In one aspect, the invention provides a purified thermostable enzyme derived from the archael bacterium AEPII 1a. In one aspect, the enzyme has a molecular weight of about 60.9 kilodaltons and has a cellulase activity. The enzyme can be produced from native or recombinant host cells, and can be used to aid in the digestion of cellulose. The invention also provides polypeptides having endoglucanase activity having homology to SEQ ID NO:2.

## Claims

### Claim 1

An isolated, synthetic or recombinant polypeptide having endoglucanase or cellulase activity comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:46, or encoded by a nucleic acid encoding a polypeptide having endoglucanase or cellulase activity and having at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:45.

### Claim 2 (depends on 1)

An isolated, synthetic or recombinant polynucleotide comprising a sequence encoding the endoglucanase or cellulase of claim 1, or comprising a nucleic acid sequence encoding a polypeptide having endoglucanase or cellulase activity and having at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:45.

### Claim 3 (depends on 2)

The isolated, synthetic or recombinant polynucleotide of claim 2, wherein the polynucleotide is isolated from a prokaryote.

### Claim 4 (depends on 2)

A vector comprising a nucleic acid having a sequence as set forth in claim 2.

### Claim 5 (depends on 4)

The vector of claim 4, wherein the vector comprises a plasmid.

### Claim 6 (depends on 4)

The vector of claim 4, wherein the vector comprises virus-derived sequences.

### Claim 7 (depends on 4)

An isolated host cell comprising the vector of claim 4.

### Claim 8 (depends on 7)

A method for producing an enzyme comprising (a) (i) growing a host cell of claim 7 under conditions which allow the expression of the enzyme-encoding nucleic acid and (ii) isolating the enzyme encoded by the nucleic acid, or (b) the method of (a), wherein the cell is a plant cell, a yeast cell, a bacterial cell, a fungal cell, an insect cell or an animal cell.

### Claim 9 (depends on 7)

The host cell of claim 7, wherein the cell is a plant cell.

### Claim 10 (depends on 7)

The host cell of claims 7, wherein the cell is a yeast cell, a bacterial cell, a fungal cell, an insect cell or an animal cell.

### Claim 11 (depends on 7)

The host cell of claim 7, wherein the cell is prokaryotic.

### Claim 12 (depends on 1)

The isolated, synthetic or recombinant polypeptide of claim 1, wherein the polypeptide has the amino acid sequence of SEQ ID NO:46.

### Claim 13 (depends on 1)

The isolated, synthetic or recombinant polypeptide of claim 1, wherein the polypeptide has endoglucanase activity.

### Claim 14 (depends on 1)

The isolated, synthetic or recombinant polypeptide of claim 1, wherein the polypeptide has cellulase activity.

### Claim 15 (depends on 14)

The isolated, synthetic or recombinant polypeptide of claim 14, wherein the cellulase activity comprises a carboxymethyl cellulase activity.

### Claim 16 (depends on 1)

The isolated, synthetic or recombinant polypeptide of claim 1, wherein the cellulase activity comprises a carboxymethylcellulase.

### Claim 17

An isolated, synthetic or recombinant polypeptide having endoglucanase or cellulase activity comprising (a) a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:46, (b) a polypeptide encoded by a nucleic acid encoding a polypeptide having endoglucanase or cellulase activity and having at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:45, or (c) enzymatically active fragments of (a) or (b).

### Claim 18 (depends on 17)

A method for degrading carboxymethylcellulose comprising contacting a carboxymethylcellulose with an effective amount of the polypeptide of claim 17.

### Claim 19 (depends on 17)

A method for hydrolyzing a beta 1,4 glycosidic bond in a cellulose comprising contacting an effective amount of the polypeptide of claim 17 with the cellulose to hydrolyze the glycosidic bond.

### Claim 20 (depends on 19)

The method of claim 19, wherein the method comprises contacting the cellulose with a detergent comprising an effective amount of the polypeptide.

### Claim 21 (depends on 19)

The method of claim 19, wherein the method comprises contacting a textile comprising the cellulose with an effective amount of the polypeptide in.

### Claim 22 (depends on 19)

The method of claim 19, wherein the method comprises contacting an animal feed comprising the cellulose with an effective amount of the polypeptide in.

### Claim 23 (depends on 19)

The method of claim 19, wherein the method comprises contacting a waste comprising the cellulose with an effective amount of the polypeptide.

### Claim 24 (depends on 19)

The method of claim 19, wherein the method comprises contacting a juice or a brew comprising the cellulose with an effective amount of the polypeptide for the clarification or extraction of juices or brews.

### Claim 25 (depends on 17)

A method for converting plant biomass into fuels or chemicals comprising contacting a plant biomass comprising cellulose with an effective amount of a polypeptide of claim 17, thereby hydrolyzing the cellulose for converting the plant biomass into a fuel or a chemical.

### Claim 26 (depends on 17)

A composition comprising the isolated, synthetic or recombinant polypeptide of claim 17, and a textile.

### Claim 27 (depends on 17)

A composition comprising the isolated, synthetic or recombinant polypeptide of claim 17, and a feed.

### Claim 28 (depends on 17)

A composition comprising the isolated, synthetic or recombinant polypeptide of claim 17, and a detergent.

### Claim 29 (depends on 17)

A composition comprising the isolated, synthetic or recombinant polypeptide of claim 17, and a juice or a brew.

### Claim 30 (depends on 17)

An isolated, synthetic or recombinant polynucleotide sequence encoding an endoglucanase or cellulase of claim 17.

### Claim 31 (depends on 17)

A composition comprising the isolated, synthetic or recombinant polypeptide of claim 17, and a plant biomass.

### Claim 32

An isolated, synthetic or recombinant polypeptide having endoglucanase or cellulase activity comprising a polypeptide having the amino acid sequence of SEQ ID NO:46, or enzymatically active fragments thereof.

### Claim 33 (depends on 32)

An isolated, synthetic or recombinant nucleic acid encoding the polypeptide of claim 32.

### Claim 34 (depends on 33)

A vector comprising a nucleic acid having the nucleic acid sequence of claim 33.

### Claim 35 (depends on 33)

An isolated host cell comprising (a) the nucleic acid of claim 33, or (b) the host cell of (a), wherein the cell is a plant cell, a yeast cell, a bacterial cell, a fungal cell, an insect cell or an animal cell.

### Claim 36

An isolated, synthetic or recombinant polypeptide having endoglucanase or cellulase activity comprising the amino acid sequence of SEQ ID NO:46 and having at least one conservative amino acid substitution, wherein the conservative amino acid substitution comprises: a replacement, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile; or an interchange of the hydroxyl residues Ser and Thr; or an exchange of the acidic residues Asp and Glu; or a substitution between the amide residues Asn and Gln; or an exchange of the basic residues Lys and Arg; or a replacement among the aromatic residues Phe, Tyr,
wherein the polypeptide has an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:46, or the polypeptide is encoded by a nucleic acid having at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:45.

### Claim 37

An isolated, synthetic or recombinant nucleic acid encoding a polypeptide having endoglucanase or cellulase activity, wherein the nucleic acid hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:45, and the stringent conditions comprise a wash step comprising a wash for 30 minutes at room temperature in a solution comprising 150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA, 0.5% SDS, followed by 30 minute wash in fresh solution at Tm-10° C.

### Claim 38 (depends on 37)

The isolated, synthetic or recombinant nucleic acid of claim 37, wherein the nucleic acid that hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:45 has at least 97% sequence identity to the polynucleotide of SEQ ID NO:45.

### Claim 39 (depends on 38)

The isolated, synthetic or recombinant nucleic acid of claim 38, wherein the nucleic acid has the polynucleotide sequence of SEQ ID NO:45.

### Claim 40 (depends on 37)

An isolated host cell comprising (a) a heterologous nucleic acid having the sequence of claim 37, or (b) the host cell of (a), wherein the cell is a plant cell, a yeast cell, a bacterial cell, a fungal cell, an insect cell or an animal cell.

### Claim 41 (depends on 40)

A method for producing an enzyme comprising growing a host cell of claim 40 under conditions which allow the expression of the nucleic acid and isolating the enzyme encoded by the nucleic acid.

### Claim 42 (depends on 37)

The isolated, synthetic or recombinant nucleic acid claim 37, wherein the nucleic acid that hybridizes under stringent conditions to the polynucleotide sequence SEQ ID NO:45 has at least 95% sequence identity to the polynucleotide of SEQ ID NO:45.

### Claim 43 (depends on 37)

A method for degrading carboxymethylcellulose comprising (i) expressing the nucleic acid of claim 37 to generate a recombinant enzyme, and (ii) contacting a carboxymethylcellulose with an effective amount of the recombinant enzyme of (i).

### Claim 44 (depends on 37)

A method for hydrolyzing a beta 1,4 glycosidic bond in a cellulose comprising (i) expressing the nucleic acid of claim 37 to generate a recombinant enzyme, and (ii) contacting an effective amount of the recombinant enzyme of (i) with the cellulose to hydrolyze the glycosidic bond.

### Claim 45 (depends on 37)

A composition comprising the isolated, synthetic or recombinant polypeptide encoded by the nucleic acid of claim 37, and a textile.

### Claim 46 (depends on 37)

A composition comprising the isolated, synthetic or recombinant polypeptide encoded by the nucleic acid of claim 37, and a feed.

### Claim 47 (depends on 37)

A composition comprising the isolated, synthetic or recombinant polypeptide encoded by the nucleic acid of claim 37, and a detergent.

### Claim 48 (depends on 37)

A vector comprising a nucleic acid having the nucleic acid sequence of claim 37.

### Claim 49 (depends on 37)

A composition comprising the isolated, synthetic or recombinant polypeptide encoded by the nucleic acid of claim 37, and a juice or a brew.

### Claim 50 (depends on 37)

A composition comprising the isolated, synthetic or recombinant polypeptide encoded by the nucleic acid of claim 37, and a plant biomass.