# US 12,767,966 B2 · Lipase mutant and application thereof

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- Number: US 12,767,966 B2
- Document ID: US12767966B2
- Status: Granted
- Kind: B2
- Application number: 18/691,605
- Filed: 2021-10-13
- Published: 2024-12-05
- Granted: 2026-10-20
- Earliest claimed: 2021-09-13
- Assignees: Asymchem Life Science (Tianjin) Co., Ltd.
- Applicants: Asymchem Life Science (Tianjin) Co., Ltd.
- Inventors: Hao Hong; Gage James; Na Zhang; Xuecheng Jiao; Fang Liu; Ru Jia; Wenjing Liu
- Web page: https://patentgrep.com/patent/US12767966B2

## CPC

- C12P 7/62: Carboxylic acid esters
- C12N 9/18: Carboxylic ester hydrolases {(3.1.1)}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12R 2001/19: Escherichia coli
- C12Y 301/01003: Triacylglycerol lipase (3.1.1.3)
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12Y: ENZYMES

## Abstract

Provided are a lipase mutant and an application thereof. Specifically, one or more mutations selected from A262H, A338V, V364I, A158P/V, and 1159N are generated on the basis of an amino acid sequence as shown in SEQ ID NO: 1; and compared with a parental lipase, a change in the structure and function of a protein occurs in the lipase mutant, and the stereoselectivity is improved, such that a usage amount of an enzyme is decreased to a certain extent, the post-treatment difficulty is reduced, and the lipase mutant is suitable for industrial production.

## Claims

### Claim 1

A lipase mutant, wherein the lipase mutant comprises:
1) A protein having the amino acid sequence of SEQ ID NO: 1 with a mutation of one or more amino acids selected from the group consisting of:
A262H
A338V
V364I
A262H + A338V + V364I + L65I
A158P
A262H + A338V + V364I + P243K
A158V
A262H + A338V + V364I + F66Y
I159N
A262H + A338V + V364I + A158P + L365Q
A262H + A338V
A262H + A338V + V364I + A158P + N263G
A262H + A338V +
A262H + A338V + V364I + A158P + C68S
V364I
A262H + A338V +
A262H + A338V + V364I + A158P + C68Y
V364I + A158P
A262H + A338V +
A262H + A338V + V364I + A158P + L365V
V364I + A158V
A262H + A338V +
A262H + A338V + V364I + A158P + Y363M
V364I + A158P +
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + P336G
V364I + A158V +
I159N
A262H + A338F
A262H + A338V + V364I + A158P + S67P
A262H + A338L
A262H + A338V + V364I + A158P + V226I
A262H + I245W
A262H + A338V + V364I + A158P + V226W
A262H + I245H
A262H + A338V + V364I + A158P + T236R
A262H + I245S
A262H + A338V + V364I + A158P + L365T
A262H + I245C
A262H + A338V + V364I + A158V + L365Q
A262H + S67R
A262H + A338V + V364I + A158V + N263G
A262H + A338V +
A262H + A338V + V364I + A158V + C68S
A158P
A262H + A338V +
A262H + A338V + V364I + A158V + C68Y
A158V
A262H + A338V +
A262H + A338V + V364I + A158V + L365V
R123K
A262H + A338V +
A262H + A338V + V364I + A158V + Y363M
H262E
A262H + A338V +
A262H + A338V + V364I + A158V + P336G
V364H
A262H + A338V +
A262H + A338V + V364I + A158V + S67P
R237F
A262H + A338V +
A262H + A338V + V364I + A158P + L365Q +
G337A
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + N263G +
V364L
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + C68S +
T236N
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + C68Y +
V364F
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + L365V +
H262Y
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + Y363M +
V364I + I245W
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + P336G +
V364I + I159Y
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + S67P +
V364I + I159F
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + V226I +
V364I + V364L
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + V226W +
V364I + A244I
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + T236R +
V364I + I245D
I159N
A262H + A338V +
A262H + A338V + V364I + A158P + L365T +
V364I + L65T
I159N
A262H + A338V +
A262H + A338F + V364I
V364I + T160L
A262H + A338V +
A262H + A338L + V364I
V364I + P243M
and the protein has an amino acid sequence with at least 98% identity to the amino acid sequence of SEQ ID NO:1.

### Claim 2 (depends on 1)

The lipase mutant according to claim 1, the protein in 2) has an amino acid sequence with more than 99% identity to the amino acid sequence of the protein in 1), and the lipase mutant is obtained from Pseudomonas putida and has the lipase activity.

### Claim 3 (depends on 1)

The lipase mutant according to claim 1, the protein in 2) has an amino acid sequence with more than 99.5% identity to the amino acid sequence of the protein in 1), and the lipase mutant is obtained from Pseudomonas putida and has the lipase activity.

### Claim 4 (depends on 1)

A DNA molecule, wherein the DNA molecule encodes the lipase mutant according to claim 1.

### Claim 5 (depends on 4)

A recombinant plasmid, wherein the recombinant plasmid comprises the DNA molecule according to claim 4.

### Claim 6 (depends on 5)

The recombinant plasmid according to claim 5, wherein the recombinant plasmid is selected from any one of the following: pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18 and pUC-19.

### Claim 7 (depends on 5)

A non-plant host cell, wherein the host cell comprises the recombinant plasmid according to claim 5.

### Claim 8 (depends on 7)

The host cell according to claim 7, wherein the host cell is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a yeast cell.

### Claim 9 (depends on 7)

The host cell according to claim 7, wherein the host cell is a competent cell.

### Claim 10 (depends on 9)

The host cell according to claim 9, wherein the competent cell is an Escherichia coli BL21 cell or an Escherichia coli W3110 cell.

### Claim 11 (depends on 1)

A method for preparing a chiral compound, wherein the method comprises:
using the lipase mutant according to claim 1 to catalyze hydrolysis of an ester compound shown in Formula I into an acid compound shown in Formula II and an alcohol compound shown in Formula III:
wherein, R1 is selected from any one of the following: CH3, CH2CH3, CH2—CH2CH3, or CHCH3CH3;
R2, R3, R4, and R5 are each independently selected from any one of the following: H, F, Cl, Br, I, OH, CH3, or CH2CH3; and
the presence or absence of a double bond in a cyclohexane ring, when it is present, the double bond forms at any one or more of the following sites: between R3 and R4, between R5 and R6, between R7 and R5, and between R9 and R10.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the ester compound is selected from any one of the following:

### Claim 13 (depends on 11)

The method according to claim 11, wherein the lipase mutant catalyzes a hydrolysis reaction of the ester compound shown in Formula I at a temperature of 15° C.~30° C.

### Claim 14 (depends on 11)

The method according to claim 11, wherein a mass ratio of a bacterial sludge amount of the lipase mutant to the ester compound is 1:10~1:1.

### Claim 15 (depends on 11)

The method according to claim 11, wherein a reaction system comprises an organic solvent, and the organic solvent is selected from any one of the following: dimethylsulfoxide (DMSO), acetone, dimethyltetrahydrofuran, isopropanol, and n-propanol.

### Claim 16 (depends on 15)

The method according to claim 15, wherein a volume percentage content of the organic solvent in the reaction system is 5%~10%.