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CN-121975756-A - Hydrogen peroxide-tolerant D-amino acid oxidase mutant and preparation and application thereof

CN121975756ACN 121975756 ACN121975756 ACN 121975756ACN-121975756-A

Abstract

The application relates to the technical field of bioengineering, in particular to a hydrogen peroxide-tolerant D-amino acid oxidase mutant and preparation and application thereof. The D-amino acid oxidase mutant has mutation of one or more amino acids of 52 th, 54 th, 91 st, 195 th, 241 th and 366 th relative to the amino acid sequence of wild D-amino acid oxidase, wherein the mutated amino acid is selected from valine, asparagine, isoleucine or alanine, and the amino acid sequence of the wild D-amino acid oxidase is shown as SEQ ID NO. 1. Compared with wild D-amino acid oxidase, the D-amino acid oxidase mutant has the characteristics of high enzyme activity and high hydrogen peroxide tolerance, and when racemic D, L-glufosinate is used as a substrate for catalytic reaction, the conversion rate is far higher than that of the wild D-amino acid oxidase mutant, the D-amino acid oxidase mutant has higher catalytic activity, and the PPO yield is also greatly improved. The application also provides a process for preparing the L-glufosinate-ammonium by one-pot oxidation-reduction, which solves the inhibition effect among multiple enzymes and ensures that the catalytic efficiency reaches the optimal level of industry.

Inventors

  • YU LIJUN
  • HE XUEFENG
  • LI ZHIHENG
  • JIANG HONGYU
  • WANG YINGBIN

Assignees

  • 上海奥萝拉医药科技有限公司

Dates

Publication Date
20260505
Application Date
20251222

Claims (10)

  1. 1. A D-amino acid oxidase mutant has an amino acid sequence which is mutated at one or more amino acids of 52 th, 54 th, 91 st, 195 th, 241 th and 366 th relative to the amino acid sequence of wild D-amino acid oxidase, wherein the mutated amino acid is selected from valine, asparagine, isoleucine or alanine, and the amino acid sequence of the wild D-amino acid oxidase is shown as SEQ ID NO. 1.
  2. 2. The D-amino acid oxidase mutant according to claim 1, wherein the D-amino acid oxidase mutant comprises any one or more of the following mutations: 1) Asparagine at position 52 is mutated to valine; 2) Methionine at position 54 is mutated to asparagine; 3) Threonine at position 91 is mutated to isoleucine; 4) The cysteine at position 195 is mutated to alanine; 5) An alanine mutation at position 241 to isoleucine; 6) The cysteine at position 366 is mutated to alanine.
  3. 3. The D-amino acid oxidase mutant according to claim 1, wherein the amino acid sequence of the D-amino acid oxidase mutant comprises any of the following: 1) Asparagine at position 52 to valine and methionine at position 54 to asparagine; 2) Asparagine at position 52 to valine, methionine at position 54 to asparagine, threonine at position 91 to isoleucine; 3) Asparagine at position 52 to valine, methionine at position 54 to asparagine, and cysteine at position 195 to alanine; 4) Asparagine at position 52 is mutated to valine, methionine at position 54 is mutated to asparagine, cysteine at position 195 is mutated to alanine, and alanine at position 241 is mutated to isoleucine; 5) Asparagine at position 52 is mutated to valine, methionine at position 54 is mutated to asparagine, cysteine at position 195 is mutated to alanine, alanine at position 241 is mutated to isoleucine, and cysteine at position 366 is mutated to alanine.
  4. 4. The mutant D-amino acid oxidase of claim 1, wherein the amino acid sequence of the mutant D-amino acid oxidase is selected from the group consisting of any of SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14.
  5. 5. An isolated polynucleotide encoding the D-amino acid oxidase mutant of any one of claims 1-4.
  6. 6. A construct, characterized in that, A polynucleotide according to claim 5.
  7. 7. A host cell comprising the construct or genome of claim 6 having the polynucleotide of claim 5 integrated therein.
  8. 8. A method for producing a D-amino acid oxidase mutant comprising culturing the host cell of claim 7 under conditions suitable for expression of the D-amino acid oxidase mutant of any of claims 1-4 to obtain the D-amino acid oxidase mutant.
  9. 9. Use of a D-amino acid oxidase mutant according to any of claims 1-4, a polynucleotide according to claim 5, a construct according to claim 6 or a host cell according to claim 7 for the preparation of L-glufosinate.
  10. 10. A method for preparing L-glufosinate by catalysis, comprising preparing L-glufosinate by using the D-amino acid oxidase mutant of any of claims 1-4, the polynucleotide of claim 5, the construct of claim 6 or the host cell of claim 7.

Description

Hydrogen peroxide-tolerant D-amino acid oxidase mutant and preparation and application thereof Technical Field The application relates to the technical field of bioengineering, in particular to a hydrogen peroxide-tolerant D-amino acid oxidase mutant and preparation and application thereof. Background D-amino acid oxidase (D-amino acid oxidase, DAAO) is a key enzyme for oxidation of D-type amino acids. In the reaction, the amino group of D-amino acid is oxidized and deaminated to generate alpha-keto acid (such as D-pyruvic acid), meanwhile, the dehydrogenation reaction reoxidizes reduced FAD into oxidized FAD (hydrogen peroxide is generated), the reaction process releases ammonia and hydrogen peroxide, the generated alpha-keto acid can enter tricarboxylic acid for circulatory metabolism, the ammonia is discharged out of the body through urea circulation, and physiological processes such as metabolism of exogenous amino acid, regulation of neurotransmitter D-serine level, maintenance of cell wall structural integrity and the like are involved in the organism. DAAO has stereoisomerism specificity to hydrophobic D-amino acid (such as D-alanine and D-phenylalanine), and is widely applied to the preparation process of keto acid and derivatives thereof at present. For example, in the antibiotic preparation process, DAAO and 7-aminocephalosporanic acid acylase (7-ACA acylase) are used for producing 7-aminocephalosporanic acid (7-ACA) which is an important raw material of cephalosporin in a two-step method. The D-amino acid oxidase expressed by methanol yeast is used for constructing high-expression pichia pastoris recombinant bacteria, and the fermentation activity of the pichia pastoris recombinant bacteria in a 14L tank reaches 8000-1 2000U/L. The application is wider at present, and the field of preparing the L-glufosinate by reducing the D-glufosinate by reductase after the DAAO oxidizes the D-glufosinate. Glufosinate is a low-toxicity, high-efficiency organophosphorus herbicide developed by the german bayer company. Glufosinate is also known as bialaphos and glufosinate, and the English name is phosphinothricin (PPT for short) and the chemical name is 2-amino-4- [ hydroxy (methyl) phosphono ] butanoic acid. The main mechanism of glufosinate weeding is to inhibit glutamine synthetase in plants, so that the reversible reaction of glutamic acid is interrupted, and plants are poisoned by ammonia and cannot synthesize chlorophyll, so that the plants die. The glufosinate is mainly used for preventing and controlling annual and perennial grassy weeds and dicotyledonous weeds, and has huge application market as herbicide. At present, three herbicides in the world are paraquat, glyphosate and glufosinate respectively. Among them, glyphosate and paraquat have been gradually introduced into herbicide markets due to the resistance and high toxicity of weeds, while glufosinate has excellent herbicidal properties and less phytotoxicity side effects, and has a huge application market in the future. The glufosinate has two optical isomers, namely L-glufosinate and D-glufosinate, and the weeding activity of the L-glufosinate is obviously higher than that of the D-form, so that the glufosinate has small biological toxicity, wide weeding spectrum and small damage to the environment. At present, the main stream product of glufosinate sold in the market is usually DL-glufosinate mixed solution, so that the use amount of glufosinate is obviously increased. Therefore, scientists develop L-glufosinate, and the using amount of the glufosinate can be obviously reduced, which has important significance for improving the atom economy, reducing the using cost and relieving the environmental pressure. At present, three preparation methods of L-glufosinate-ammonium are mainly adopted, namely a chiral resolution method, a chemical synthesis method and a biocatalysis method. The biocatalysis method has absolute cost and process advantages, is a main stream production process at present, and the L-glufosinate produced by enzyme catalysis has the advantages of strict stereoselectivity, mild reaction conditions, high yield and the like, and the path mainly comprises the following four types: (1) The L-glufosinate derivative is used as a substrate and is obtained by direct hydrolysis through an enzymatic method, and the method has the main advantages of high conversion rate and higher chiral value of the product, but expensive and difficult-to-obtain chiral raw materials are needed as precursors; (2) Taking homoserine as a chiral source donor, taking methyl phosphinic acid as a phosphorus donor, and adopting one-step or multi-step enzymatic catalysis to synthesize the L-glufosinate-ammonium, wherein the process is currently in a pre-research and development stage and has no obvious technical breakthrough; (3) The method takes the precursor of the racemic glufosinate-ammonium as a substrate, and is obtained through selective resolution of enzyme,