Hydrolases, nucleic acids encoding them and methods for making and using them

• US 8,153,391 B2
• Filed: 08/29/2008
• Issued: 04/10/2012
• 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:
  US12/202,204
• Publication Number:
  US 20100055234A1
• Time in Patent Office: 1,320 Days
• Field of Search: None
• US Class Current: 435/18
• 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 hydrolyzing an oil or fat comprising the steps of:

(a) obtaining a composition comprising an oil or fat, wherein the oil or fat can be hydrolyzed by a polypeptide having a palmitase activity, wherein the palmitase activity comprises hydrolyzing at least 45% palmitic acid from the oil or fat, 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 a palmitase activity and the polypeptide having at least one base residue change described 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 described in Table A,

2. The method of claim 1, wherein said hydrolyzed oil or fat has a lower saturated fat content than said oil or fat prior to hydrolysis.

3. The method of claim 1, wherein said oil or fat comprises an algal oil, an animal oil, a vegetable oil, an oil having an altered fatty acid composition, a low saturated oil, fish oil or a combination thereof.

4. The method of claim 3, wherein said oil comprises Neochloris oleoabundans oil, Scenedesmus dimorphus oil, Euglena gracilis oil, Phaeodactylum tricornutum oil, Pleurochrysis carterae oil, Prymnesium parvum oil, Tetraselmis chui oil, Tetraselmis suecica oil, Isochrysis galbana oil, Nannochloropsis salina oil, Botryococcus braunii oil, Dunaliella tertiolecta oil, Nannochloris species oil, Spirulina species oil, Chlorophycease oil, Bacilliarophy oil, canola oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, other nut oils, hempseed oil, linseed oil, meadowfoam oil, olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, sasanqua oil, sesame oil, soybean oil, sunflower seed oil, tall oil, tsubaki oil, tallow, lard, butter fat, chicken fat, or a blend of any of said fats or oils.

5. The method of claim 3, wherein the altered fatty acid comprises high oleic oil, low linolenic oil or a combination thereof.

6. The method of claim 3, wherein the low saturated oil comprises high oleic canola oil, low linolenic soybean oil, high stearic sunflower oil or a combination thereof.

7. The method of claim 3, wherein the fish oil comprises candlefish oil, cod-liver oil, orange roughy oil, sardine oil, herring oil, menhaden oil or a combination thereof.

8. The method of claim 1, wherein said fats or oils comprise molecules having a triacylglyceride backbone and wherein during said hydrolysis palmitic acid is removed from at least some of the triacylglyceride backbones.

9. The method of claim 8, wherein said palmitic acid is selectively removed from an Sn1 or Sn3 position on the triacylglyceride backbone.

10. The method of claim 1, wherein said oil or fat is present in a feed or a food and said hydrolysis is accomplished prior to the consumption of said feed or food by an animal or an individual.

11. The method of claim 1, wherein said oil or fat comprises one or more of a triacylglycerol, a diacylglycerol, or a monoacylglycerol, and wherein said polypeptide of step (a) is contacted with said oil or fat under conditions wherein said polypeptide hydrolyzes one or more of said triacylglycerol, diacylglycerol or monoacylglycerol.

12. The method of claim 11 wherein said hydrolysis causes the amount of triacylglycerol, diacylglycerol or monoacylglycerol in the composition to decrease.

13. The method of claim 1, wherein the oil or fat comprises a glycerol ester of a polyunsaturated fatty acid.

14. The method of claim 1, wherein said hydrolyzed oil or fat comprises any one or more of a low-saturate oil or fat, a no-trans oil or fat, a lipid containing essential fatty acids, a lipid containing monounsaturated fatty acids, a lipid containing phospho-choline, a lipid containing phospho-serine, a lipid containing a phytosterol, a 1,3-diacylglyceride, a 2-monoacylglyceride, and a triacylglyceride.

15. The method of claim 1, wherein said composition comprises a milk or vegetable-based dietary composition, wherein the polypeptide can hydrolyze the oil or fat in the composition, thereby reducing its fat content.

16. The method of claim 1, wherein polypeptide comprises at least one of amino acid residue modification D61A;

D61E;

R72E;

R72K;

E116A;

E116Q;

E116R;

E116T;

E116V;

S133A;

1151G;

1151A;

V163R;

D164R.