US 12,509,667 B2Granted

Cytochrome P450 enzyme mutant and application thereof

AssigneeAsymchem Life Science (Tianjin) Co., Ltd

InventorsHao Hong, Gage James, Na Zhang, Xuecheng Jiao, Yulei Ma, Shan Cao, Yibing Cheng, Kaihua Sun

Life of this patent

Estimating the term…

Abstract

Provided are a cytochrome P450 enzyme mutant and an application thereof. The enzyme activity and anti-Markov oxidation selectivity of P450 derived from a wild-type strain of Bacillus megaterium undergo protein modification by means of directed evolution, thus improving enzyme activity and selectivity, and developing a series of P450 enzyme mutants that may be used for industrial production.

Claims15

Dependent claims

A cytochrome P450 enzyme mutant, wherein the cytochrome P450 enzyme obtained from Bacillus megaterium;

wherein the cytochrome P450 enzyme mutant has the amino acid sequence of SEQ ID NO:
1 with a mutation of one or more amino acids:
V79A + F332A
V79A + F332A + A75V
V79A + F332A + A75F
V79A + F332A + L76A
V79A + F332A + A83F
V79A + F332A + A83V
V79A + F332A + F88A
V79A + F332A + F88V
V79A + F332A + T89A
V79A + F332A + T89V
V79A + F332A + A265V
V79A + F332A + A265F
V79A + F332A + T269V
V79A + F332A + T269A
V79A + F332A + T269F
V79A + F332A + A329V
V79A + F332A + A329F
V79A + F332A + A331F
V79A + F332A + A331V
V79A + F332A + L438A
V79A + F332A + L438F
V79A + F332A + C63Y
V79A + F332A + C63Y + S107C
V79A + F332A + C63Y + S107C + Q129L
V79A + F332A + C63Y + S107C + Q129L + E229K
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
N320I
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
S165G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A331P
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A331G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A331D
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329L
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A75R
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329Y + A331P
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329L + A331P
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331R
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331D
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + R48H
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + Y52G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + Q74L
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + T269P
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333M
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + M355F
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + M355K
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + L182I
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + L182M
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + S333Q
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + S333V
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + W91M
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + S90C
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + L18V
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + S90V
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + S90G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182A
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182M
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + A331P
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + A331S
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333M + M355W
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333M + M355G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333M + A331D
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + S73G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + W91F
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + P173Q
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + P173R
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + L72F
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + S73A.

120 more elements

Dependencies are inferred from the claim text.

Facts

Application: 18/267,326Filed 25 Apr 2021
Priority: CN 202110039458.813 Jan 2021
Published: US 2024/0010997 A111 Jan 2024
Granted: 30 Dec 2025
Term ends: Not estimatedThe source records don’t support an estimate.

Classification12 codes

C07K 14/415
Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof: from plants
C12N 9/0081
With a reduced iron-sulfur protein as one donor: cholesterol monooxygenase
C12N 9/0071
Oxidoreductases: acting on paired donors with incorporation of molecular oxygen
C12N 15/70
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: vectors or expression systems…

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US 2024/0010997 A1Pre-grant publication · published 11 Jan 2024
US 12,509,667 B2This patent

From the US filing history. Foreign family members aren’t listed.

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Abstract

A cytochrome P450 enzyme mutant, wherein the cytochrome P450 enzyme obtained from Bacillus megaterium;

wherein the cytochrome P450 enzyme mutant has the amino acid sequence of SEQ ID NO:
1 with a mutation of one or more amino acids:
V79A + F332A
V79A + F332A + A75V
V79A + F332A + A75F
V79A + F332A + L76A
V79A + F332A + A83F
V79A + F332A + A83V
V79A + F332A + F88A
V79A + F332A + F88V
V79A + F332A + T89A
V79A + F332A + T89V
V79A + F332A + A265V
V79A + F332A + A265F
V79A + F332A + T269V
V79A + F332A + T269A
V79A + F332A + T269F
V79A + F332A + A329V
V79A + F332A + A329F
V79A + F332A + A331F
V79A + F332A + A331V
V79A + F332A + L438A
V79A + F332A + L438F
V79A + F332A + C63Y
V79A + F332A + C63Y + S107C
V79A + F332A + C63Y + S107C + Q129L
V79A + F332A + C63Y + S107C + Q129L + E229K
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
N320I
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
S165G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A331P
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A331G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A331D
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329L
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A75R
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329Y + A331P
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329L + A331P
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331R
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331D
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + R48H
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + Y52G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + Q74L
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + T269P
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333M
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + M355F
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + M355K
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + L182I
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + L182M
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + S333Q
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + S333V
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + W91M
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + S90C
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + L18V
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + S90V
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + A331F + S90G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182A
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182M
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + A331P
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + A331S
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333M + M355W
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333M + M355G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333M + A331D
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + S73G
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + W91F
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + P173Q
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + P173R
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + L72F
V79A + F332A + C63Y + S107C + Q129L + E229K + Q388L + T428I +
F78L + I175S + A329K + S333H + L182F + S73A.

120 more elements

Facts

Application

18/267,326Filed 25 Apr 2021

Priority

CN 202110039458.813 Jan 2021

Published

US 2024/0010997 A111 Jan 2024

Granted

30 Dec 2025

Term ends

Not estimatedThe source records don’t support an estimate.

Classification12 codes

C07K 14/415
Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof: from plants
C12N 9/0081
With a reduced iron-sulfur protein as one donor: cholesterol monooxygenase
C12N 9/0071
Oxidoreductases: acting on paired donors with incorporation of molecular oxygen
C12N 15/70
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: vectors or expression systems…