# US 11,441,099 B2 · Partial enzymatic hydrolysis of triacylglycerols

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- Number: US 11,441,099 B2
- Document ID: US11441099B2
- Status: Granted
- Kind: B2
- Application number: 16/066,603
- Filed: 2016-12-29
- Published: 2021-07-08
- Granted: 2022-09-13
- Earliest claimed: 2015-12-30
- Assignees: Dsm IP Assets B.V.
- Applicants: DSM IP Assets B.V.
- Inventors: Paul Mugford; Monika Mueller; Martin Schurmann
- Web page: https://patentgrep.com/patent/US11441099B2

## CPC

- C11C 1/045: {using enzymes or microorganisms, living or dead}
- C11B 3/00: Refining fats or fatty oils
- C11B 3/003: {by enzymes or microorganisms, living or dead}
- C12N 9/20: Triglyceride splitting, e.g. by means of lipase
- C12P 7/6432: Eicosapentaenoic acids [EPA]
- C12P 7/6434: Docosahexenoic acids [DHA]
- C12Y 301/01003: Triacylglycerol lipase (3.1.1.3)
- 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
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}
- C12Y: ENZYMES

## Abstract

Disclosed herein are host cells transformed with a nucleic acid molecule comprising a polynucleotide sequence, wherein the polynucleotide sequence encodes a polypeptide that hydrolyzes an ester linkage of a triacylglycerol in an oil comprising at least one long-chain polyunsaturated fatty acid, methods for using such host cells, and processes for production of a lipase using such host cells.

## Claims

### Claim 1

A host cell transformed with a nucleic acid molecule comprising a polynucleotide sequence having at least 90% identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, wherein the polynucleotide sequence encodes a polypeptide that hydrolyzes an ester linkage of a triacylglycerol in an oil comprising at least one long-chain polyunsaturated fatty acid (LC-PUFA).

### Claim 2 (depends on 1)

The host cell of claim 1 wherein the host cell is Pichia pastoris.

### Claim 3 (depends on 1, 2)

The host cell of claim 1 or claim 2, wherein the polynucleotide sequence has a 90% to 99% identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

### Claim 4 (depends on 1)

The host cell of claim 1, wherein the polynucleotide sequence encodes a lipase gene.

### Claim 5 (depends on 1)

The host cell of claim 1, wherein the polynucleotide sequence encodes a Candida rugosa lipase 1 gene.

### Claim 6 (depends on 1)

The host cell of claim 1, wherein the polynucleotide sequence encodes a Candida rugosa lipase 3 gene.

### Claim 7 (depends on 1)

The host cell of claim 1, wherein the polynucleotide sequence encodes a Geotrichum candidum lipase 2 gene.

### Claim 8 (depends on 1)

The host cell of claim 1, wherein the long-chain polyunsaturated fatty acid comprises eicosopentaenoic acid (EPA), docosahexaneoic acid (DHA), and combinations thereof.

### Claim 9 (depends on 1)

The host cell of claim 1, wherein the oil is a crude or unrefined oil.

### Claim 10 (depends on 1)

The host cell of claim 1, wherein the oil is a marine oil.

### Claim 11

A method of using a host cell transformed with a nucleic acid molecule comprising a polynucleotide sequence having at least 90% identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, wherein the polynucleotide sequence encodes a polypeptide that hydrolyzes an ester linkage of a triacylglycerol in an oil comprising at least one long-chain polyunsaturated fatty acid.

### Claim 12 (depends on 11)

The method of claim 11, wherein the host cell is Pichia pastoris.

### Claim 13 (depends on 11, 12)

The method of claim 11 or claim 12, wherein the polynucleotide sequence has a 90% to 99% identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

### Claim 14 (depends on 11)

The method of claim 11 wherein the polynucleotide sequence encodes a lipase gene.

### Claim 15 (depends on 11)

The method of claim 11, wherein the polynucleotide sequence encodes a Candida rugosa lipase 1 gene.

### Claim 16 (depends on 11)

The method of claim 11, wherein the polynucleotide sequence encodes a Candida rugosa lipase 3 gene.

### Claim 17 (depends on 11)

The method of claim 11, wherein the polynucleotide sequence encodes a Geotrichum candidum lipase 2 gene.

### Claim 18 (depends on 11)

The method of claim 11, wherein the long-chain polyunsaturated fatty acid comprises eicosopentaenoic acid (EPA), docosahexaneoic acid (DHA), and combinations thereof.

### Claim 19 (depends on 11)

The method of claim 11, wherein the oil is a crude or unrefined oil.

### Claim 20 (depends on 11)

The method of claim 11, wherein the oil is a marine oil.

### Claim 21 (depends on 1, 2)

The host cell of claim 1 or claim 2, wherein the polynucleotide sequence has at least 95% identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

### Claim 22 (depends on 1, 2)

The host cell of claim 1 or claim 2, wherein the polynucleotide sequence has 100% identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.