# US 2014/0154752 A1 · Glycoside hydrolases having multiple hydrolase activities

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- Number: US 2014/0154752 A1
- Document ID: US20140154752A1
- Status: Published application
- Kind: A1
- Application number: 14/114,990
- Filed: 2012-05-01
- Published: 2014-06-05
- Earliest claimed: 2011-05-02
- Assignees: Sandia Corporation; The Regents of the University of California
- Inventors: Zhiwei Chen; Gregory D. Friedland; Swapnil R. Chhabra; Dylan C. Chivian; Blake A. Simmons
- Web page: https://patentgrep.com/patent/US20140154752A1

## CPC

- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C12N 9/2434: {acting on beta-1,4-glucosidic bonds}
- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- 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

## Abstract

Glycoside hydrolases having at least two different hydrolytic activities are provided. In one embodiment, an isolated recombinant hydrolase having at least two activities selected from a group including asparagine derivatives, glutamine derivatives, and histidine derivatives is provided. Further, a method of generating free sugars from a mixture comprising asparagine derivatives, glutamine derivatives, and histidine derivatives is provided.

## Claims

### Claim 1

An isolated recombinant glycoside hydrolase having at least two activities selected from the group consisting of cellulase activity, xylanase activity, and mannanase activity, wherein the glycoside hydrolase comprises the following amino acids (corresponding to positions in SEQ ID NO:1): asparagine (N), aspartic acid (D), glutamine (Q) or glutamic acid (E) at position 20, aspartic acid (D) or glutamic acid (E) at position 23, proline (P) at position 53, histidine (H) at position 95, histidine (H) at position 96 and aspartic acid (D) or glutamic acid (E) at position 287, wherein the recombinant glycoside hydrolase has an increased cellulase activity, xylanase activity, and/or mannanase activity compared to the glycoside hydrolase of SEQ ID NO:1.

### Claim 2 (depends on 1)

The recombinant glycoside hydrolase of claim 1, wherein the recombinant glycoside hydrolase is a GH5 glycoside hydrolase.

### Claim 3 (depends on 1)

The recombinant glycoside hydrolase of claim 1, wherein the recombinant glycoside hydrolase has a cellulase activity, xylanase activity, and/or mannanase activity at least 10% higher than the activity of glycoside hydrolase of SEQ ID NO:1.

### Claim 4 (depends on 1)

The recombinant glycoside hydrolase of claim 1, wherein the hydrolase has cellulase activity, xylanase activity, and mannanase activity.

### Claim 5 (depends on 1)

The recombinant glycoside hydrolase of claim 1, wherein the recombinant glycoside hydrolase comprises at least one amino acid substitution compared to a naturally-occurring glycoside hydrolase at at least one amino acid position corresponding to positions 20, 23, 53, 95, 96, or 287 of SEQ ID NO:1.

### Claim 6 (depends on 1)

The recombinant glycoside hydrolase of claim 1, wherein the recombinant glycoside hydrolase comprises an amino acid sequence substantially (e.g., at least 60%, 70%, 80%, 85%, 90%, or 95%) identical to any of SEQ ID NOs: 3, 5, 7, 9, or 11.

### Claim 7 (depends on 1)

An isolated nucleic acid comprising a polynucleotide encoding the glycoside hydrolase of claim 1.

### Claim 8 (depends on 7)

An expression vector comprising an expression cassette, the expression cassette comprising a heterologous promoter operably linked to the polynucleotide of claim 7.

### Claim 9 (depends on 8)

A cell culture comprising cells that comprise the expression cassette of claim 8.

### Claim 10

A method of generating free sugars from a mixture comprising at least two polymeric substrates selected from the group consisting of cellulose, xylan and mannan, the method comprising contacting the mixture with a glycoside hydrolase under conditions such that the glycoside hydrolase hydrolyzes the at least two polymeric substrates, wherein the glycoside hydrolase comprises the following amino acids (corresponding to positions in SEQ ID NO:1): asparagine (N), aspartic acid (D), glutamine (Q) or glutamic acid (E) at position 20, aspartic acid (D) or glutamic acid (E) at position 23, proline (P) at position 53, histidine (H) at position 95, histidine (H) and position 96 and aspartic acid (D) or glutamic acid (E) at position 287, thereby generating free sugars.

### Claim 11 (depends on 10)

The method of claim 10, wherein the glycoside hydrolase is a recombinant glycoside hydrolase.

### Claim 12 (depends on 10)

The method of claim 10, wherein the glycoside hydrolase is a GH5 glycoside hydrolase.

### Claim 13 (depends on 10)

The method of claim 10, wherein the glycoside hydrolase has an increased cellulase activity, xylanase activity, and/or mannanase activity compared to the glycoside hydrolase of SEQ ID NO:1.

### Claim 14 (depends on 10)

The method of claim 10, wherein the mixture comprises cellulose, xylan and mannan and the glycoside hydrolase hydrolyzes each of the cellulose, xylan and mannan.

### Claim 15 (depends on 10)

The method of claim 10, wherein the glycoside hydrolase comprises at least one amino acid substitution compared to a naturally-occurring glycoside hydrolase at at least one amino acid position corresponding to positions 20, 23, 53, 95, 96, or 287 of SEQ ID NO:1.

### Claim 16 (depends on 10)

The method of claim 10, wherein the glycoside hydrolase comprises an amino acid sequence substantially (e.g., at least 60%, 70%, 80%, 85%, 90%, or 95%) identical to any of SEQ ID NOs: 3, 5, 7, 9, or 11.

### Claim 17

A method of generating a polynucleotide encoding a mutated glycoside hydrolase with activity to hydrolyze at least two polymeric substrates selected from the group consisting of cellulose, xylan and mannan, the method comprising:
introducing at least one nucleotide change to a coding region of a polynucleotide encoding a native glycoside hydrolase, resulting in at least one amino acid change in the glycoside hydrolase to generate a mutated polynucleotide encoding a mutated glycoside hydrolase, wherein the introducing changes at least one amino acid at positions 20, 23, 53, 95, 96, or 287 (corresponding to positions in SEQ ID NO:1) of the native glycoside hydrolase such that the mutated glycoside hydrolase comprises each of the following amino acids (corresponding to positions in SEQ ID NO:1): asparagine or aspartic acid at position 20, aspartic acid or glutamate at position 23, proline at position 53, histidine at position 95, histidine and position 96 and aspartate or glutamate at position 287, wherein the mutated glycoside hydrolase has an increased cellulase activity, xylanase activity, and/or mannanase activity compared to the native glycoside hydrolase.

### Claim 18 (depends on 10)

The method of claim 10, wherein glycoside hydrolase is a GH5 glycoside hydrolase.

### Claim 19 (depends on 17)

The method of claim 17, further comprising translating and purifying the mutated glycoside hydrolase.

### Claim 20 (depends on 17)

The method of claim 17, wherein the mutated glycoside hydrolase has an increased cellulase activity, xylanase activity, and mannanase activity compared to the native glycoside hydrolase.

### Claim 21 (depends on 17)

The method of claim 17, wherein the mutated glycoside hydrolase comprises an amino acid sequence substantially (e.g., at least 60%, 70%, 80%, 85%, 90%, or 95%) identical to any of SEQ ID NOs: 3, 5, 7, 9, or 11.