Beta-glucosidase variants

• US 9,150,843 B2
• Filed: 11/13/2014
• Issued: 10/06/2015
• 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 R., Andor, Attila, Clark, Louis A., Newman, Lisa Marie
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US14/540,422
• Publication Number:
  US 20150064766A1
• Time in Patent Office: 327 Days
• Field of Search: None
• US Class Current: 1/1
• 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 recombinant β

-glucosidase protein variant that comprises a first segment with at least 90% sequence identity to the GH3 Domain Consensus Sequence as set forth in SEQ ID NO;

53 and comprises a second segment with at least 90% sequence identity to the GH3-C Domain Consensus Sequence as set forth in SEQ ID NO;

54, wherein the segments are in the order N-first segment-second segment-C;

wherein the β

-glucosidase comprises no more than one of the following residues;

a) alanine at a position corresponding to position 104 of SEQ ID NO;

1;

b) leucine at a position corresponding to position 157 of SEQ ID NO;

1;

c) isoleucine at a position corresponding to position 210 of SEQ ID NO;

1;

d) alanine at a position corresponding to position 485 of SEQ ID NO;

1;

e) alanine at a position corresponding to position 572 of SEQ ID NO;

1; and

f) tyrosine at a position corresponding to position 649 of SEQ ID NO;

1;

wherein the β

-glucosidase protein variant is catalytically active.

2. The recombinant β

-glucosidase protein of claim 1, that has greater thermoactivity than a reference β

-glucosidase protein that differs only by having alanine at the position corresponding to position 104 of SEQ ID NO;

1, leucine at the position corresponding to position 157 of SEQ ID NO;

1, isoleucine at the position corresponding to position 210 of SEQ ID NO;

1, alanine at the position corresponding to position 485 of SEQ ID NO;

1, alanine at the position corresponding to position 572 of SEQ ID NO;

1, and tyrosine at the position corresponding to position 649 of SEQ ID NO;

1.

3. The recombinant β

-glucosidase protein of claim 1, comprising none of residues (a)-(f).

4. A catalytically active recombinant β

-glucosidase variant protein that comprises a first segment with at least 90% sequence identity to the GH3 Domain Consensus Sequence as set forth in SEQ ID NO;

53 and comprises a second segment with at least 90% sequence identity to the GH3-C Domain Consensus Sequence as set forth in SEQ ID NO;

54, wherein the segments are in the order N-first segment-second segment-C; and

wherein the β

-glucosidase protein comprises amino acid substitutions, relative to a naturally occurring β

-glucosidase protein, at one or more performance sensitive positions, wherein the performance sensitive position(s) corresponds to one or more positions in SEQ ID NO;

1 selected from residues 60, 87, 104, 116, 122, 123, 130, 160, 163, 164, 210, 484, 521, 572, 211, 338, 339, 295, 299, 350, 415, 463, 485, 108, 157, and 649 of SEQ ID NO;

1, and wherein the β

-glucosidase variant is thermostable and retains 80% or more β

-glucosidase activity after incubation at pH 5.0, at 65°

C., for 6 hours.

5. The recombinant β

-glucosidase variant protein of claim 4, wherein the naturally occurring β

-glucosidase protein is selected from SEQ ID NOS;

4-52.

6. A recombinant β

-glucosidase variant protein that has β

-glucosidase activity, wherein said recombinant β

-glucosidase variant protein comprises a first segment with at least 90% sequence identity to the GH3 Domain Consensus Sequence as set forth in SEQ ID NO;

53 and comprises a second segment with at least 90% sequence identity to the GH3-C Domain Consensus Sequence as set forth in SEQ ID NO;

54, wherein the segments are in the order N-first segment-second segment-C;

has a sequence in the first segment that differs from SEQ ID NO;

53 at one or more performance sensitive positions selected from positions 39, 43, 51, 57, 58, 65, 91, 94, 97, 98, 133 and 134 of SEQ ID NO;

53, and differs from SEQ ID NO;

54 at one or more performance sensitive positions selected from positions 61, 82, 83, 115 and 163 of SEQ ID NO;

54, wherein the number of said positions at which the variant protein differs from SEQ ID NOs;

53 and 54 is 9 or more.