# US 8,785,170 B2 · Variant CBH2 cellulases and related polynucleotides

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

- Number: US 8,785,170 B2
- Document ID: US8785170B2
- Status: Granted
- Kind: B2
- Application number: 13/393,495
- Filed: 2010-08-31
- Published: 2012-06-21
- Granted: 2014-07-22
- Earliest claimed: 2009-09-04
- Assignees: Codexis, Inc.
- Applicants: Ish K. Dhawan; Erika N. Segraves
- Inventors: Ish K. Dhawan; Erika N. Segraves
- Web page: https://patentgrep.com/patent/US8785170B2

## CPC

- C12N 9/2408: {acting on alpha -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 7/10: substrate containing cellulosic material
- C12P 7/14: Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
- 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 302/01091: Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
- 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 invention provides variants of a Streptomyces sp. CBH2 that have improved properties compared to the wild type enzyme and methods of using the variants in the hydrolysis of substrates comprising cellulose.

## Claims

### Claim 1

An isolated Streptomyces sp cellobiohydrolase II (CBH2) polypeptide variant comprising:
an amino acid sequence that is at least 90% identical to the wild type Streptomyces sp CBH2 of SEQ ID NO:-4 and comprises the substitution L201F, wherein the amino acid position is determined by optimal alignment with SEQ ID NO:-4, and wherein said CBH2 variant exhibits at least 2-fold improvement in cellulase activity compared to the wild-type Streptomyces sp. CBH2 set forth in SEQ ID NO:4.

### Claim 2 (depends on 1)

The isolated CBH2 polypeptide variant according to claim 1, wherein the variant amino acid sequence is at least 95% identical to SEQ ID NO: 4.

### Claim 3 (depends on 1)

The isolated CBH2 polypeptide variant according to claim 1, wherein the variant amino acid sequence is at least 97% identical to SEQ ID NO: 4.

### Claim 4 (depends on 1)

The isolated CBH2 polypeptide variant according to claim 1, wherein the variant amino acid sequence is at least 99% identical to SEQ ID NO: 4.

### Claim 5 (depends on 1)

The isolated CBH2 polypeptide variant of claim 1 further comprising a substitution at a position selected from A30, A118, S122, S175, I180, V183, G202, Q206, G216, V219, D221, S233, P234, T241, Q253, N274, V324, and/or S395.

### Claim 6 (depends on 5)

The isolated CBH2 polypeptide variant according to claim 5, wherein the further substitution in the variant CBH2 polypeptide is selected from A30T, A118R, S122V/H, S175Q/L, I180K/C, V183G, G202F/Y, Q206L, G216K, V219E/R, D221L, S233C, P234S/A, T241R/K, Q253M/A/S, N274K/P, V324H/F and/or S395T.

### Claim 7 (depends on 1)

The isolated CBH2 polypeptide variant of claim 1 further comprising a substitution at positions P234, S299 and Q378.

### Claim 8 (depends on 7)

The isolated CBH7 polypeptide variant according to claim 7 wherein the substitution is selected from P234A, S299P, and Q378R.

### Claim 9 (depends on 1)

The isolated CBH2 polypeptide variant of claim 1, wherein the variant amino acid sequence comprises at least 95% sequence identity to SEQ ID NO: 6 and optionally at least 1 further amino acid substitution[s].

### Claim 10 (depends on 1)

The isolated CBH2 polypeptide variant of claim 1, wherein the variant exhibits at least 3-fold improvement in cellulase activity compared to wild type Streptomyces sp CBH2 set forth in SEQ ID NO: 4.

### Claim 11 (depends on 1)

An isolated polynucleotide encoding the CBH2 polypeptide variant of claim 1.

### Claim 12 (depends on 11)

The polynucleotide of claim 11 which has been codon optimized.

### Claim 13 (depends on 11)

A vector comprising a polynucleotide according to claim 11.

### Claim 14 (depends on 13)

A host cell transformed with vector of claim 13.

### Claim 15 (depends on 14)

The host cell of claim 14, wherein the host cell is selected from a bacterial cell, a filamentous fungal cell or a yeast cell.

### Claim 16 (depends on 15)

The host cell of claim 15, wherein the host cell is a bacterial cell.

### Claim 17 (depends on 16)

The host cell of claim 16, wherein the host cell is a Streptomyces cell.

### Claim 18 (depends on 16)

The host cell of claim 16, wherein the host cell is a Bacillus cell.

### Claim 19 (depends on 15)

The host cell of claim 15, wherein the host cell is a filamentous fungal cell.

### Claim 20 (depends on 19)

The host cell of claim 19, wherein the host cell is an Aspergillus, a Chrysosporium, a Humicola, a Myceliopthora, a Neurospora, a Piromyces, a Thielavia, or a Trichoderma host cell.

### Claim 21 (depends on 1)

An enzyme composition comprising the CBH2 polypeptide variant of claim 1.

### Claim 22 (depends on 21)

The enzyme composition of claim 21 further comprising additional cellulase enzymes.

### Claim 23 (depends on 21)

The enzyme composition of claim 21 further comprising one or more additional enzymes selected from amylases, glucoamylases, esterases, cutinases, phytases, xylanases, lipases, and proteases.