Monooxygenase and use in preparation of optically pure sulfoxide

Patent metadata

Field	Value
Publication number	US-11345900-B2
Application number	US-201917294424-A
Country	US
Kind code	B2
Filing date	Nov 26, 2019
Priority date	Nov 26, 2018
Publication date	May 31, 2022
Grant date	May 31, 2022

Abstract

A monooxygenase having an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO:2 is disclosed. The use of the monooxygenase of the present invention in production of chiral sulfoxide-based drugs has advantages including mild reaction conditions, environmental friendliness, high yield, high optical purity of products, less peroxide products, and the like, and therefore the monooxygenase in the present invention has a good industrial application prospect in the production of proton pump inhibitors for the treatment of gastric ulcers.

First claim

What is claimed is:

1. A monooxygenase, comprising a mutant polypeptide of a parental polypeptide having the amino acid sequence shown in SEQ ID NO:2, wherein
the mutant polypeptide has an oxidation activity,
mutations of the mutant polypeptide comprise replacements of amino acid residues at specified positions of Xaa143, Xaa326, Xaa426, Xaa432, Xaa433, Xaa435, Xaa438, and Xaa505 and optionally at least one position selected from the group consisting of Xaa21; Xaa40; Xaa55; Xaa70; Xaa145; Xaa156; Xaa185; Xaa220; Xaa244; Xaa246; Xaa248; Xaa249; Xaa277; Xaa281; Xaa386; Xaa388; Xaa390; Xaa405; Xaa430; Xaa465; Xaa468; Xaa488; Xaa489; Xaa490; Xaa497; and Xaa501 of the amino acid sequence shown in SEQ ID NO: 2;
wherein
at Xaa143, L is replaced with A or P;
at Xaa326, K is replaced with C or F;
at Xaa426, L is replaced with F or P;
at Xaa432, F is replaced with L or I;
at Xaa433, T is replaced with C or A;
at Xaa435, L is replaced with S;
at Xaa438, S is replaced with I; and
at Xaa505, F is replaced with L.

2. The monooxygenase according to claim 1 , wherein the mutations further comprise any one of the following:
a) replacement of amino acid residue(s) at any 1, 2, 3, 4, or 5 positions other than the specified positions;
b) deletion of amino acid residue(s) at any 1, 2, 3, 4, or 5 positions other than the specified positions; and
c) insertion of amino acid residue(s) at any 1, 2, 3, 4, or 5 positions other than the specified positions.

3. The monooxygenase according to claim 1 , wherein the replacements of the amino acid residues at the specified positions further comprise any one or more of the following replacements:
at the Xaa21, S is replaced with G;
at the Xaa40, T is replaced with A;
at the Xaa55, L is replaced with Y, W, F or N;
at the Xaa70, E is replaced with G;
at the Xaa145, A is replaced with S;
at the Xaa156, E is replaced with G;
at the Xaa185, G is replaced with A or S;
at the Xaa220, M is replaced with R;
at the Xaa244, L is replaced with V or I;
at the Xaa246, F is replaced with Y;
at the Xaa248, L is replaced with E, N, A or W;
at the Xaa249, N is replaced with S;
at the Xaa277, F is replaced with L, V, Y, I or D;
at the Xaa281, F is replaced with V or A;
at the Xaa386, N is replaced with S;
at the Xaa388, I is replaced with F, C, K, G;
at the Xaa390, M is replaced with S, V, I;
at the Xaa405, K is replaced with M;
at the Xaa430, G is replaced with T or S;
at the Xaa465, K is replaced with R;
at the Xaa468, V is replaced with A;
at the Xaa488, E is replaced with K;
at the Xaa489, S is replaced with C;
at the Xaa490, W is replaced with R;
at the Xaa497, P is replaced with S; and
at the Xaa501, N is replaced with Y.

4. The monooxygenase according to claim 3 , wherein the replacements of the amino acid residues comprise any of the following replacements:
1) 8 replacements, wherein at the Xaa143, the L is replaced with the P; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the S; at the Xaa438, the S is replaced with the I; at the Xaa505, the F is replaced with the L;
2) 8 replacements, wherein at the Xaa143, the L is replaced with the A; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the S; at the Xaa438, the S is replaced with the I; at the Xaa505, the F is replaced with the L;
3) 9 replacements, wherein at the Xaa143, the L is replaced with the P; at the Xaa244, the L is replaced with the V; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the S; at the Xaa438, the S is replaced with the I; at the Xaa505, the F is replaced with the L;
4) 9 replacements, wherein at the Xaa143, the L is replaced with the P; at the Xaa244, the L is replaced with the I; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the S; at the Xaa438, the S is replaced with the I; at the Xaa505, the F is replaced with the L;
5) 9 replacements, wherein at the Xaa143, the L is replaced with the P; at the Xaa248, the L is replaced with the E; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the S; at the Xaa438, the S is replaced with the I; at the Xaa505, the F is replaced with the L;
6) 9 replacements, wherein at the Xaa143, the L is replaced with the P; at the Xaa248, the L is replaced with the N; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the S; at the Xaa438, the S is replaced with the I; at the Xaa505, the F is replaced with the L;
7) 9 replacements, wherein at the Xaa143, the L is replaced with the P; at the Xaa248, the L is replaced with the A; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the S; at the Xaa438, the S is replaced with the I; at the Xaa505, the F is replaced with the L;
8) 9 replacements, wherein at the Xaa143, the L is replaced with the P; at the Xaa248, the L is replaced with the W; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the S; at the Xaa438, the S is replaced with the I; at the Xaa505, the F is replaced with the L;
9) 9 replacements, wherein at the Xaa143, the L is replaced with the P; at the Xaa277, the F is replaced with the L; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the S; at the Xaa438, the S is replaced with the I; at the Xaa505, the F is replaced with the L;
10) 9 replacements, wherein at the Xaa143, the L is replaced with the P; at the Xaa277, the F is replaced with the V; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the S; at the Xaa438, the S is replaced with the I; at the Xaa505, the F is replaced with the L;
11) 9 replacements, wherein at the Xaa143, the L is replaced with the P; at the Xaa277, the F is replaced with the Y; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the S; at the Xaa438, the S is replaced with the I; at the Xaa505, the F is replaced with the L;
12) 9 replacements, wherein at the Xaa143, the L is replaced with the P; at the Xaa277, the F is replaced with the I; at the Xaa326, the K is replaced with the C; at the Xaa426, the L is replaced with the F; at the Xaa432, the F is replaced with the L; at the Xaa433, the T is replaced with the A; at the Xaa435, the L is replaced with the

Assignees

• Jiangsu Aosaikang Pharm Co Ltd
• Univ East China Science & Tech

Inventors

• Zhang Yan
• Yu Huilei
• Ren Shimiao
• Zhao Peng
• Xu Jianhe
• Wu Yinqi
• Zhao Qian

Classifications

• C12P41/002

  by oxidation/reduction reactions · CPC title

• C12Y114/14001 Primary

  Unspecific monooxygenase (1.14.14.1) · CPC title

• C12N9/0071 Primary

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12P17/165

  Heterorings having nitrogen atoms as the only ring heteroatoms · CPC title

• C12R2001/01

  Bacteria or Actinomycetales {; using bacteria or Actinomycetales} · CPC title