# US 8,541,191 B2 · Hydrolases, nucleic acids encoding them and methods for biocatalytic synthesis of structured lipids

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- Number: US 8,541,191 B2
- Document ID: US8541191B2
- Status: Granted
- Kind: B2
- Application number: 13/083,480
- Filed: 2011-04-08
- Published: 2012-11-01
- Granted: 2013-09-24
- Earliest claimed: 2008-08-29
- Assignees: Bunge Oils, Inc.
- Applicants: Christopher L. G. Dayton; Tim Hitchman; Katie Kline; Jonathan Lyon; Mark A. Wall; Nelson R. Barton
- Inventors: Christopher L. G. Dayton; Tim Hitchman; Katie Kline; Jonathan Lyon; Mark A. Wall; Nelson R. Barton
- Web page: https://patentgrep.com/patent/US8541191B2

## CPC

- C12P 7/6472: containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
- A23D 7/04: Working-up
- A23D 9/04: Working-up
- C11B 3/003: {by enzymes or microorganisms, living or dead}
- C11C 1/045: {using enzymes or microorganisms, living or dead}
- C11C 3/08: with fatty acids
- C11C 3/12: by hydrogenation
- C12N 9/20: Triglyceride splitting, e.g. by means of lipase
- C12P 7/6418: by hydrolysis of fatty acid esters
- C12P 7/6427: Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
- C12P 7/6431: Linoleic acids [18:2[n-6]]
- C12P 7/6432: Eicosapentaenoic acids [EPA]
- C12P 7/6434: Docosahexenoic acids [DHA]
- C12P 7/6445: Glycerides
- C12P 7/6454: by esterification
- C12P 7/6458: by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- A23D: EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS (preservation thereof A23B20/00; production, refinement or preservation of animal or vegetable fats or oils C11B, C11C)
- C11B: PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES (drying-oils C09F)
- C11C: FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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

### Claim 1

A method of producing a diacylglyceride, the method comprising the steps of:
(a) providing an oil composition comprising a quantity of triacylglyceride,
(b) 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,
Table A
Amino
Original
New
Acid
Amino
Amino
Residue
Acid
Acid
27
G
Q
45
G
L
54
S
H
61
D
A
E
62
V
A
G
72
R
E
K
S
T
74
F
I
83
V
M
96
A
I
98
G
L
116
E
A
F
H
L
Q
Q
R, H140R
R
S
S
T
V
117
L
R, I161L
120
K
L
133
S
A
147
I
F
151
I
A
G
H
160
P
T
172
R
Q
212
S
R
213
K
T
214
T
V
N
R
Y
215
G
A
H
216
A
V
C
L
or
iii. having at least 85% sequence identity to full length of SEQ ID NO:2 and comprising at least one amino acid residue modification D61A; D61E; R72E; R72K; E116A; E116Q; E116R; E116T; E116V; S133A; I151G; I151A; V163R; or D164R
(c) contacting said oil composition of step (a) with said polypeptide of step (b) under conditions sufficient for the polypeptide to hydrolyze a palmitase residue on the triacylglyceride to form a diacylglyceride.

### Claim 2 (depends on 1)

The method of claim 1, wherein said polypeptide is Sn2 specific and said diacylglyceride is a 1,3-diacylglyceride.

### Claim 3 (depends on 1)

The method of claim 1, wherein said polypeptide is Sn1 or Sn3 specific and said diacylglyceride is a 1,2-diacylglyceride or a 2,3-diacylglyceride.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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; 1151A; V163R; or D164R.

### Claim 6 (depends on 1)

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.