HYDROLASES, NUCLEIC ACIDS ENCODING THEM AND METHODS TO PRODUCE TRIGLYCERIDES

• US 20110287495A1
• Filed: 04/08/2011
• Published: 11/24/2011
• Est. Priority Date: 08/29/2008
• Status: Active Grant

• Current Assignee: Christopher L.G. Dayton, Jonathan Lyon, Katie Kline, Mark A. Wall, Nelson R. Barton, Tim Hitchman
• Original Assignee: Christopher L.G. Dayton, Jonathan Lyon, Katie Kline, Mark A. Wall, Nelson R. Barton, Tim Hitchman
• Inventors: Dayton, Christopher L.G., Hitchman, Tim, Kline, Katie, Lyon, Jonathan, Wall, Mark A., Barton, Nelson R.
• Granted Patent: US 8,313,918 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• 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 of catalyzing an interesterification reaction to produce new triacylglycerides comprising the following steps:

(a) providing a composition comprising a polypeptide having a 1,3-specific lipase activity, said polypeptide being selected from the group consisting of isolated, synthetic or recombinant polypeptides having a hydrolase activity and said polypeptide either

i. being encoded by either

(1) a nucleic acid comprising a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO;

1 and having one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more, or all the base residue changes set forth in Table 3 or Table 4, wherein the nucleic acid encodes at least one polypeptide having a hydrolase activity, or

(2) a nucleic acid comprising a sequence that hybridizes under stringent conditions to a nucleic acid comprising SEQ ID NO;

1 and having one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more or all the base residue changes set forth in Table 3 or Table 4, wherein the nucleic acid encodes a polypeptide having a hydrolase activity;

or

ii. having at least 50% sequence identity to SEQ ID NO;

2 over a region of at least about 100 residues, and having one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more or all the amino acid residue changes set forth in Table 3 or Table 4, or

iii. comprising an amino acid sequence as set forth in SEQ ID NO;

2 but also comprising at least one of amino acid residue modification D61A;

D61E;

R72E;

R72K;

E116A;

E116Q;

E116R;

E116T;

E116V;

S133A;

I151G;

I151A;

V163R;

D164R, or a combination thereof, or

iv. comprising an amino acid sequence as set forth in SEQ ID NO;

2 but also comprising at least one of amino acid residue modification 120L;

V62S;

G77P;

V83C;

D88H;

Y113G;

E116T;

E116G;

H140K;

K146S;

I167S;

L180E;

E194M;

A211Q;

S212Y;

G215C;

G215V;

G215W;

A218H;

A218S;

V223A;

A225M;

A225Q, or a combination thereof;

(b) providing a mixture of triacylglycerides and free fatty acids;

(c) treating the mixture of step (b) with the polypeptide under conditions wherein the polypeptide can catalyze exchange of free fatty acids with the acyl groups of triacylglycerides, thereby producing new triacylglycerides enriched in said fatty acids.

2. The method of claim 1, wherein said mixture of step (b) comprises 1,3-dipalmitoyl-2-monoleine (POP) and the new triacylglycerides of step (c) comprise one or both of 1-palmitoyl-3-stearoyl-2-monoleine (POSt) and 1,3-distearoyl-2-monoleine (StOSt).

3. The method of claim 1, wherein said new triacylglycerides of step (c) have a lower saturated fat content than said triacylglycerides of step (b).

4. The method of claim 1, wherein said new triacylglycerides of step (c) have a lower trans fat content than said triacylglycerides of step (b).

5. The method of claim 1, wherein the free fatty acid comprises stearic acid.

6. The method of claim 1, wherein the free fatty acid comprises an omega-3 unsaturated fatty acid.

7. The method of claim 6, wherein the omega-3 unsaturated fatty acid is α

-linolenic acid, stearidonic acid, eicosapentaenoic acid, or docosahexaenoic acid.

8. The method of claim 1, wherein the free fatty acid comprises a polyunsaturated fatty acid.

9. The method of claim 1, wherein the free fatty acid comprises a fish oil fatty acid.

10. The method of claim 1, wherein the free fatty acid comprises an omega-6 unsaturated fatty acid.

11. The method of claim 10, wherein the omega-6 unsaturated fatty acid is linoleic acid, gamma-linoleic acid, eicosadienoic acid, dihomo-gamma-linoleic acid, arachidonic acid, docoasdienoic acid, adrenic acid, or docosapentaenoic acid.

12. The method of claim 1, wherein the free fatty acid comprises an omega-9 unsaturated fatty acid.

13. The method of claim 12, wherein the omega-9 unsaturated fatty acid is oleic acid, eicosaenoic acid, mead acid, erucic acid, nervonic acid, or palmitoleic acid.

14. The method of claim 1, wherein the polypeptide is encoded by a nucleic acid comprising a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO:

1 and having one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more, or all the base residue changes set forth in Table 3 or Table 4.

15. The method of claim 1, wherein the polypeptide is encoded by a nucleic acid comprising a sequence that hybridizes under stringent conditions to a nucleic acid comprising SEQ ID NO:

1 and having one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more or all the base residue changes set forth in Table 3 or Table 4.

16. The method of claim 1, wherein the polypeptide has at least 50% sequence identity to SEQ ID NO:

2 over a region of at least about 100 residues, and has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more or all the amino acid residue changes set forth in Table 3 or Table 4.

17. The method of claim 1, wherein the polypeptide comprises an amino acid sequence as set forth in 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;

D164R, or a combination thereof.

18. The method of claim 1, wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:

2 and further comprises at least one of amino acid residue modification 120L;

V62S;

G77P;

V83C;

D88H;

Y113G;

E116T;

E116G;

H140K;

K146S;

I167S;

L180E;

E194M;

A211Q;

S212Y;

G215C;

G215V;

G215W;

A218H;

A218S;

V223A;

A225M;

A225Q, or a combination thereof.