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CN-122012430-A - Amine oxidase mutant and application thereof in synthesis of chiral compounds

CN122012430ACN 122012430 ACN122012430 ACN 122012430ACN-122012430-A

Abstract

The invention discloses an amine oxidase mutant and application thereof in synthesizing chiral compounds, and relates to the field of biology. The amine oxidase mutant provided by the invention has mutation at any one or more of the 47 th site, 182 th site, 183 th site, 275 th site, 282 rd site and 335 th site of a wild amino acid sequence, has higher catalytic activity on (S) -3-aminobutyric acid, has better stereoselectivity, can selectively oxidize (S) -3-aminobutyric acid under the condition of not influencing (R) -3-aminobutyric acid to generate 4-hydroxy-2-butanone, is applied to catalyzing (R) -3-aminobutyric acid feed liquid containing (S) -3-aminobutyric acid, can effectively improve the R-type ee value in the feed liquid, does not need additional refining and purifying steps, and has obviously reduced cost.

Inventors

  • CHENG LEIYU
  • WANG FUXIANG
  • HUANG GUODONG
  • CHEN JIANBO
  • WANG GUOFU
  • PENG JIANGEN
  • PENG CHEN
  • ZHANG YU

Assignees

  • 上虞新和成生物化工有限公司
  • 浙江新和成药业有限公司
  • 浙江新和成股份有限公司

Dates

Publication Date
20260512
Application Date
20260212

Claims (10)

  1. 1. An amine oxidase mutant, characterized in that the amine oxidase mutant has a mutation at any one or more of positions 47, 182, 183, 275, 282 and 335 of the amino acid sequence of a wild-type amine oxidase.
  2. 2. The amine oxidase mutant of claim 1, wherein the amine oxidase mutant has any one or a combination of mutations as set forth in N47A, T182S, L183V, M275Q, Q282S and F335Y relative to the wild-type amine oxidase.
  3. 3. The amine oxidase mutant according to claim 2, wherein the amine oxidase mutant has any combination of mutations as set forth in any of T182S/N47A, T S/L183V, T S/L183V/M275Q, T S/L183V/F335Y, T S/L183V/Q282S and T182S/L183V/Q282S/F335Y relative to the wild-type amine oxidase; Alternatively, the amino acid sequence of the wild-type amine oxidase has at least 80% identity with the sequence shown in SEQ ID NO. 1.
  4. 4. An isolated nucleic acid molecule encoding the amine oxidase mutant of any one of claims 1-3.
  5. 5. A recombinant vector comprising the isolated nucleic acid molecule of claim 4.
  6. 6. A recombinant cell comprising the isolated nucleic acid molecule of claim 4 or the recombinant vector of claim 5; alternatively, the recombinant cell comprises a recombinant bacterium.
  7. 7. A catalyst comprising the amine oxidase mutant according to any one of claims 1 to 3; Optionally, the catalyst comprises recombinant cells or a culture thereof according to claim 6.
  8. 8. The method for producing an amine oxidase mutant according to any one of claims 1 to 3, which comprises artificially synthesizing the amine oxidase mutant or culturing the recombinant cell according to claim 6.
  9. 9. Use of an amine oxidase mutant according to any of claims 1 to 3 or an isolated nucleic acid molecule according to claim 4 or a recombinant vector according to claim 5 or a recombinant cell according to claim 6 or a catalyst according to claim 7 for the preparation or purification of a selective oxidation (S) -3-aminobutanol or a chiral compound; optionally, the chiral compound comprises (R) -3-aminobutanol.
  10. 10. A method for purifying (R) -3-aminobutanol, which is characterized by comprising the steps of adding the amine oxidase mutant according to any one of claims 1-3 or the recombinant cell according to claim 6 or the catalyst according to claim 7 into a reaction system containing (S) -3-aminobutanol and (R) -3-aminobutanol for catalytic reaction; optionally, the reaction system containing (S) -3-aminobutanol and (R) -3-aminobutanol is a reaction system containing (R) -3-aminobutanol feed liquid; Optionally, the (R) -3-aminobutanol feed liquid comprises (R) -3-aminobutanol feed liquid produced by transaminase with 4-hydroxy-2-butanone as substrate; optionally, the conditions of the catalytic reaction comprise 20-40 ℃, 1-30 h and 0.1-2 VVM; optionally, adding 0.1-5 g of the recombinant cell or the culture thereof into the reaction system per 0.05-0.5 g of the (S) -3-aminobutanol; Optionally, the reaction system further comprises NAD oxidase or catalase, and/or a buffer solution; Optionally, the enzyme activity of the catalase in the reaction system is 50-1000U/L; Optionally, the pH of the buffer solution is 7.4-7.8; optionally, the buffer is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer.

Description

Amine oxidase mutant and application thereof in synthesis of chiral compounds Technical Field The invention relates to the field of biology, in particular to an amine oxidase mutant and application thereof in synthesizing chiral compounds. Background The (R) -3-amino butanol is used as a key chiral intermediate of dolutegravir which is an anti-AIDS drug, and the product quality of the (R) -3-amino butanol plays an important role in the quality of dolutegravir. With the development of the pharmaceutical industry, the purity of (R) -3-aminobutanol is increasingly demanding, in particular in terms of ee value and impurity content. At present, both chemical and biocatalytic methods are used for synthesizing high-purity (R) -3-aminobutanol, wherein biocatalysis has the remarkable advantages of mild reaction conditions, small environmental pollution, high economy and the like, and becomes a research and application hot spot. Various enzymes such as aminotransferase and amine dehydrogenase have been developed for biosynthesis of (R) -3-aminobutanol, wherein the aminotransferase route is to transfer amino groups on inexpensive amino donors such as isopropylamine, alanine and the like to carbonyl carbon of the substrate by an transamination reaction using 4-hydroxy-2-butanone as a substrate, and the yield of the product can reach 95%, but the ee value is only 99.8%, and the quality standard requirement (ee value 99.94%) that the isomer ratio is lower than 0.03% is not reached. At present, a step of tartaric acid chemical method resolution step is added after the catalytic process to improve the ee value, but the method not only consumes a large amount of resolution reagent, but also increases the subsequent extraction and purification process, and the production cost is increased by about 30%. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide an amine oxidase mutant and application thereof in synthesizing chiral compounds. The invention is realized in the following way: In a first aspect, embodiments of the present invention provide an amine oxidase mutant having a mutation at any one or more of positions 47, 182, 183, 275, 282 and 335 of the amino acid sequence of a wild-type amine oxidase. In a second aspect, embodiments of the invention provide an isolated nucleic acid molecule encoding an amine oxidase mutant as described in the previous embodiments. In a third aspect, embodiments of the invention provide a recombinant vector comprising an isolated nucleic acid molecule as described in the previous embodiments. In a fourth aspect, embodiments of the invention provide a recombinant cell comprising an isolated nucleic acid molecule as described in the previous embodiments or a recombinant vector as described in the previous embodiments. In a fifth aspect, embodiments of the present invention provide a catalyst comprising the amine oxidase mutant of the previous embodiments. In a sixth aspect, embodiments of the present invention provide a method for preparing an amine oxidase mutant as described in the previous embodiments, comprising artificially synthesizing the amine oxidase mutant or culturing the recombinant cell as described in the previous embodiments. In a seventh aspect, embodiments of the invention provide the use of an amine oxidase mutant as described in the preceding embodiments or an isolated nucleic acid molecule as described in the preceding embodiments or a recombinant vector as described in the preceding embodiments or a recombinant cell as described in the preceding embodiments or a catalyst as described in the preceding embodiments for the preparation or purification of a selective oxidation (S) -3-aminobutanol or a chiral compound. In an eighth aspect, an embodiment of the present invention provides a method for purifying (R) -3-aminobutanol, comprising the step of adding the amine oxidase mutant described in the previous embodiment or the recombinant cell described in the previous embodiment or the catalyst described in the previous embodiment to a reaction system containing (S) -3-aminobutanol and (R) -3-aminobutanol to perform a catalytic reaction. The invention has the following beneficial effects: The optimized amine oxidase mutant has higher catalytic activity on (S) -3-aminobutanol and better stereoselectivity, can selectively oxidize (S) -3-aminobutanol under the condition of not influencing (R) -3-aminobutanol to generate 4-hydroxy-2-butanone, can be applied to catalyzing (R) -3-aminobutanol feed liquid containing (S) -3-aminobutanol, can effectively improve the R-type ee value in the feed liquid in a short time, does not need additional refining and purifying steps, reduces the use of a large amount of resolution reagents in the existing tartaric acid chemical resolution method, and obviously reduces the cost. Drawings In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the