Hydrolases, nucleic acids encoding them and methods for making and using them for biocatalytic synthesis of a structured lipid

• US 8,227,215 B2
• Filed: 04/08/2011
• Issued: 07/24/2012
• Est. Priority Date: 08/29/2008
• Status: Active Grant

• Current Assignee: Bunge Oils, Inc. (Bunge Limited)
• Original Assignee: Bunge Oils, Inc. (Bunge Limited)
• Inventors: Dayton, Christopher L. G., Hitchman, Tim, Kline, Katie, Lyon, Jonathan, Wall, Mark A., Barton, Nelson R.
• Primary Examiner(s): PAK, YONG D
• Application Number:
  US13/083,455
• Publication Number:
  US 20110281312A1
• Time in Patent Office: 473 Days
• Field of Search: None
• US Class Current: 435/132
• CPC Class Codes: A23D 7/04 Working-up
• CPC Class Codes: A23D 9/04 Working-up
• CPC Class Codes: C11B 3/003 by enzymes or microorganisms, living or dead
• CPC Class Codes: C11C 1/045 using enzymes or microorganisms, living or dead
• CPC Class Codes: C11C 3/08 with fatty acids
• CPC Class Codes: C11C 3/12 by hydrogenation
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12P 7/6418 by hydrolysis of fatty acid esters
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6431 Linoleic acids [18:2[n-6]]
• CPC Class Codes: C12P 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6434 Docosahexenoic acids [DHA]
• CPC Class Codes: C12P 7/6445 Glycerides
• CPC Class Codes: C12P 7/6454 by esterification
• CPC Class Codes: C12P 7/6458 by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

Provided are hydrolases, including lipases, saturases, palmitases and/or stearatases, and polynucleotides encoding them, and methods of making and using these polynucleotides and polypeptides. Further provided are polypeptides, e.g., enzymes, having a hydrolase activity, e.g., lipases, saturases, palmitases and/or stearatases and methods for preparing low saturate or low trans fat oils, such as low saturate or low trans fat animal or vegetable oils, e.g., soy or canola oils.

Claims

1. A method for biocatalytic synthesis of a structured lipid comprising the following steps:

(a) providing a polypeptide having a palmitase activity, said polypeptide being selected from the group consisting of isolated, synthetic and recombinant polypeptides having a palmitase activity and said polypeptide either

i. being encoded by

a nucleic acid comprising a nucleic acid sequence having at least 85% sequence identity to full length SEQ ID NO;

1, and wherein the nucleic acid encodes at least one polypeptide having palmitase activity and the polypeptide having at least one amino acid residue change recited below in Table A, or

ii. having at least 85% sequence identity to full length SEQ ID NO;

2, and having at least one amino acid residue change recited in Table A,

2. The method of claim 1, wherein the R1-specific hydrolase is an Sn2-specific lipase.

3. The method of claim 1, wherein the structured lipid is selected from the group consisting of a cocoa butter alternative, a synthetic cocoa butter, a natural cocoa butter, 1,3-dipalmitoyl-2-oleoylglycerol, 1,3-distearoyl-2-oleoylglycerol, 1-palmitoyl-2-oleoyl-3-stearoylglycerol and 1-oleoyl-2,3-dimyristoylglycerol.

4. The method of claim 1, wherein said R1 ester comprises one or more of an omega-3 fatty acid, an omega-6 fatty acid, an omega-9 fatty acid, or a mono-unsaturated fatty acid.

5. The method of claim 1, wherein said R1 ester comprises a moiety selected from the group consisting of alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, oleic acid, and palmitoleic acid.

6. The method of claim 1, wherein said synthesized structured lipid has a lower saturated fat content than said triacylglyceride.

7. The method of claim 1, wherein said synthesized structured lipid has a lower trans fat content than said triacylglyceride.

8. The method of claim 1, wherein the polypeptide has at least 85% sequence identity to full length SEQ ID NO:

2, and has at least one amino acid residue change recited in Table A.

9. The method of claim 1, wherein the polypeptide has at least 85% sequence identity to full length SEQ ID NO:

2 and further comprises at least one of amino acid residue modification D61A;

D61E;

R72E;

R72K;

E116A;

E116Q;

E116R;

E116T;

E116V;

S133A;

I151G;

I151A;

V163R;

or D164R.

10. The method of claim 1, wherein the polypeptide has at least 85% sequence identity to full length SEQ ID NO:

2 and further comprises at least one of amino acid residue modification I20L;

V62S;

G77P;

V83C;

D88H;

Y113G;

E116T;

E116G;

H140K;

K146S;

I167S;

L180E;

E194M;

A211Q;

S212Y;

G215C;

G215V;

G215W;

A218H;

A218S;

V223A;

A225M;

or A225Q.