# US 8,772,010 B2 · β-Glucosidase variants

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- Number: US 8,772,010 B2
- Document ID: US8772010B2
- Status: Granted
- Kind: B2
- Application number: 13/375,189
- Filed: 2010-06-16
- Published: 2012-03-29
- Granted: 2014-07-08
- Earliest claimed: 2009-06-16
- Assignees: Codexis, Inc.
- Applicants: Xiyun Zhang; Jie Yang; Dipnath Baidyaroy; Sally Rhiannon Postlethwaite; Attila Andor; Louis Clark; Lisa M. Newman
- Inventors: Xiyun Zhang; Jie Yang; Dipnath Baidyaroy; Sally Rhiannon Postlethwaite; Attila Andor; Louis Clark; Lisa M. Newman
- Web page: https://patentgrep.com/patent/US8772010B2

## CPC

- C12N 9/2445: {Beta-glucosidase (3.2.1.21)}
- C12N 9/2482: {Endo-1,4-beta-xylanase (3.2.1.8)}
- C12Y 302/01021: Beta-glucosidase (3.2.1.21)
- 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)
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The invention relates to recombinantly produced β-Glucosidase Variants with enhanced thermoactivity compared to naturally occurring proteins. The invention also provides methods for producing a variant β-glucosidase polypeptide with improved thermoactivity by identifying performance sensitive positions in a target β-glucosidase polypeptide and substituting the residue at that position with a thermoactivity enhancing residue.

## Claims

### Claim 1

A method of producing a variant Myceliophthora thermophila β-glucosidase polypeptide with improved thermoactivity, comprising:
a) identifying a first performance sensitive position (PSP) in a target Myceliophthora thermophila β-glucosidase polypeptide, comprising the steps of:
i) aligning the primary sequence of the target Myceliophthora thermophila β-glucosidase polypeptide with one or both of SEQ ID NO:53 or portion thereof, and SEQ ID NO:54, or portion thereof, and
ii) identifying a position in the target Myceliophthora thermophila β-glucosidase polypeptide that corresponds in the alignment to a PSP in the one or more β-glucosidase polypeptides, wherein the position so identified is a PSP; and
b) expressing a variant Myceliophthora thermophila β-glucosidase polypeptide in which the residue at the first performance sensitive position is replaced with a thermoactivity enhancing residue,
wherein the variant Myceliophthora thermophila β-glucosidase polypeptide has greater thermoactivity than the target Myceliophthora thermophila β-glucosidase polypeptide.

### Claim 2

A method of producing a variant Myceliophthora thermophila β-glucosidase polypeptide with improved thermoactivity, comprising
a) identifying a first performance sensitive position (PSP) in a target Myceliophthora thermophila β-glucosidase polypeptide, comprising the steps of:
i) aligning the primary sequence of the target Myceliophthora thermophila β-glucosidase polypeptide with one or more Myceliophthora thermophila β-glucosidase polypeptides in which performance sensitive positions have been defined, and
ii) identifying a position in the target Myceliophthora thermophila β-glucosidase polypeptide that corresponds in the alignment to a PSP in the one or more Myceliophthora thermophila β-glucosidase polypeptides, wherein the position so identified is a PSP; and
b) expressing a variant Myceliophthora thermophila β-glucosidase polypeptide in which the residue at the first performance sensitive position is replaced with a thermoactivity enhancing residue,
wherein the variant Myceliophthora thermophila β-glucosidase polypeptide has greater thermoactivity than the target Myceliophthora thermophila β-glucosidase polypeptide, wherein the step of identifying a PSP comprises aligning the primary sequence of the target Myceliophthora thermophila β-glucosidase polypeptide with at least a portion of SEQ ID NO: 1, and wherein the PSP corresponds to a Myceliophthora thermophila Bgl1 PSP at position 350, wherein position 350 is determined relative to SEQ ID NO: 1.