CN-121991910-A - Amine dehydrogenase mutant and application thereof in preparation of chiral compounds

Abstract

The invention discloses an amine dehydrogenase mutant and application thereof in preparation of chiral compounds, and relates to the field of biology. The amine dehydrogenase mutant provided by the embodiment of the invention has mutation at any one or more of 179 th, 202 nd, 223 rd, 224 th, 226 th, 227 th and 242 th sites of a wild amino acid sequence, has high activity and high enantioselectivity, has the enzyme catalytic efficiency obviously superior to that of the parent enzyme and the existing amine dehydrogenase, can catalyze high-concentration substrates, reaches more than 60g/L, has the advantages of high yield and high enantiomeric excess value (ee value), and provides a high-performance biocatalyst and industrialized application foundation for the efficient asymmetric synthesis of R-3-aminobutanol.

Inventors

• CHENG LEIYU
• WANG FUXIANG
• HUANG GUODONG
• CHEN JIANBO
• WANG GUOFU
• PENG JIANGEN
• PENG CHEN
• Bai Junjian

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

20260508

Application Date

20260214

Claims (10)

1. 1. An amine dehydrogenase mutant, characterized in that the amine dehydrogenase mutant has a mutation at any one or more of positions 179, 202, 223, 224, 226, 227 and 242 of the amino acid sequence of a wild-type amine dehydrogenase.
2. 2. The amine dehydrogenase mutant of claim 1, wherein the amine dehydrogenase mutant has any one or a combination of Q179S, T202S, P223A, A224E, I226L, Y227L and V242I relative to the wild-type amine dehydrogenase.
3. 3. The amine dehydrogenase mutant of claim 2, wherein the amine dehydrogenase mutant has a combination of mutations relative to the wild-type amine dehydrogenase of any of P223A/Y227L/T202S, A E/I226L/Q179S and P223A/A224E/V242I.
4. 4. The amine dehydrogenase mutant according to any one of claims 1 to 3, wherein the amino acid sequence of the wild-type amine dehydrogenase has at least 80% identity to the sequence shown in SEQ ID NO. 1.
5. 5. A biomaterial, characterized in that it is selected from any one of the following: (I) An isolated nucleic acid molecule encoding the amine dehydrogenase mutant of any one of claims 1-4; (II) a recombinant vector comprising said isolated nucleic acid molecule; (III) recombinant cells containing said recombinant vector.
6. 6. The biomaterial of claim 5, wherein the recombinant cell is a recombinant bacterium.
7. 7. A catalyst comprising the amine dehydrogenase mutant according to any one of claims 1 to 4; optionally, the catalyst comprises recombinant cells or a culture thereof according to claim 5.
8. 8. The method for producing an amine dehydrogenase mutant according to any one of claims 1 to 4, which comprises artificially synthesizing an amine dehydrogenase mutant or culturing the recombinant cell according to claim 5.
9. 9. Use of the amine dehydrogenase mutant according to any one of claims 1 to 4 or the biomaterial according to claim 5 or 6 or the catalyst according to claim 7 for the preparation of chiral compounds; Optionally, the chiral compound comprises (R) -3-aminobutanol.
10. 10. A process for preparing (R) -3-aminobutanol, which comprises adding the amine dehydrogenase mutant according to any one of claims 1 to 4 or the recombinant cell according to claim 6 or the catalyst according to claim 7 to a reaction system containing 4-hydroxy-2-butanone for catalytic reaction; Optionally, the temperature of the catalytic reaction is 30-50 ℃ and the pH is 8-9; optionally, adding 10-30 g of the recombinant cells or the culture thereof per 60g of the 4-hydroxy-2-butanone; Optionally, the reaction system also comprises formate dehydrogenase, ammonium formate and NAD + ; Optionally, 10-30 g of wet bacterial cells expressing formate dehydrogenase and 0.01-0.5 g of NAD + powder are added to 60g of 4-hydroxy-2-butanone.

Description

Amine dehydrogenase mutant and application thereof in preparation of chiral compounds Technical Field The invention relates to the field of biology, in particular to an amine dehydrogenase mutant and application thereof in preparation of chiral compounds. Background The (R) -3-aminobutanol has a vital role as a key chiral intermediate for preparing an anti-AIDS drug dolutegravir. The market demand exceeds 200 tons, and the product quality plays a decisive role in the quality of dolutegravir. In recent years, extensive research has been conducted in the fields of chemical catalysis and biocatalysis for synthetic strategies for high purity (R) -3-aminobutanol. Chemical synthesis methods, such as kinetic resolution and reduction reactions based on chiral starting materials, can achieve the preparation of (R) -3-aminobutanol, but have significant drawbacks. Such methods often rely on expensive transition metal catalysts, involve complex protection and deprotection steps in the reaction process, and often require high pressure equipment to maintain the reaction conditions, which not only greatly increases production costs, but also presents serious challenges for operational safety and equipment requirements. In comparison, biocatalytic technology has been a more potential green synthetic route by virtue of its unique advantages. The method has the advantages of mild biocatalysis reaction conditions, usually being carried out in an environment close to normal temperature and normal pressure, reducing energy consumption and reducing dependence on special equipment, and simultaneously, the method has high enantioselectivity, can accurately synthesize a target chiral product, effectively reduces the generation of byproducts, reduces the subsequent separation and purification cost, and shows good economy and environmental friendliness. In the biological preparation of (R) -3-aminobutanol, there are mainly two routes, namely, the first route is to use crotonic acid as an initial substrate, and to produce (R) -3-aminobutanol by means of the catalysis of aspartase, and then further reduce the reaction product by using a chemical catalyst such as ruthenium carbon, etc., so as to finally obtain the target product (R) -3-aminobutanol. However, the chemical reduction step in this process requires the use of hazardous reagents, which not only increases the operational risk, but may also lead to environmental pollution problems. And Ding Tongchun (4-hydroxy-2-butanone) is used as a substrate, and omega-aminotransferase is used for one-step catalytic reaction to directly prepare (R) -3-aminobutanol. The method has simple steps and is superior to the crotonic acid route in the aspect of atom economy. However, this method relies on pyridoxal phosphate, a coenzyme, to participate in the transamination process, and is costly, and at the same time, in order to drive the reaction forward, an excessive amount of organic ammonia donor is required, and a certain vacuum is maintained to remove the acetone produced by the reaction, which limits its wide application in large-scale industrial production. The strategy of amine dehydrogenase catalyzing asymmetric reductive amination reactions of ketones with ammonia to synthesize chiral amines presents significant advantages over the above biocatalytic methods. The method avoids the harsh reaction conditions such as high temperature, high pressure, metal catalyst and the like required by a chemical method, simultaneously uses cheap inorganic ammonia as an amino donor for the reaction, synthesizes (R) -3-aminobutanol by a coupled Formate Dehydrogenase (FDH) cofactor regeneration system through one-pot reaction, only generates H 2 O and CO 2 as byproducts, and has easy separation of the products and almost irreversible reaction. In the prior art, the amine dehydrogenase has obvious limitations in the process of preparing (R) -3-aminobutanol by catalyzing asymmetric reduction, on one hand, the catalytic activity of the enzyme is lower, the catalytic efficiency is low, the substrate concentration of a reaction system can only reach 10-20g/L, the low substrate concentration not only leads to low production efficiency, the catalyst and the energy consumption cost of unit products are higher, but also increases the process difficulty of separating and purifying the subsequent products due to the low substrate concentration and limited conversion rate, and severely restricts the application of the enzyme in industrial production, on the other hand, the enantioselectivity of the enzyme is insufficient, and the requirement that the ee value of the product is more than 99.9% is not met. In view of this, the present invention has been made. Disclosure of Invention The invention aims at providing an amine dehydrogenase mutant and application thereof in preparing chiral compounds. The invention is realized in the following way: In a first aspect, embodiments of the present invention provide an amine dehy