β-Glucosidase variants

• US 8,772,010 B2
• Filed: 06/16/2010
• Issued: 07/08/2014
• Est. Priority Date: 06/16/2009
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Zhang, Xiyun, Yang, Jie, Baidyaroy, Dipnath, Postlethwaite, Sally Rhiannon, Andor, Attila, Clark, Louis, Newman, Lisa M.
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US13/375,189
• Publication Number:
  US 20120077216A1
• Time in Patent Office: 1,483 Days
• Field of Search: None
• US Class Current: 435/209
• CPC Class Codes: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12N 9/2482 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

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

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.

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.