Redox asymmetrical method for preparing L-phosphinothricin by biological multi-enzyme coupling

A technology of glufosinate-ammonium and coupling method, applied in biochemical equipment and methods, oxidoreductase, microorganism and other directions, can solve the problems of complex process, waste of raw materials, expensive chiral resolution reagents, etc., and achieve good catalytic efficiency, The effect of increasing the yield of PPO

Active Publication Date: 2020-06-23
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process mainly has the following disadvantages: it needs to use expensive chiral resolution reagents, the theoretical yield can only reach 50%, the single resolution rate is low, and the process is relatively complicated
The main advantage is that the raw materials are relatively easy to obtain and the catalyst activity is high, but the theoretical yield can only reach 50%, resulting in waste of raw materials

Method used

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  • Redox asymmetrical method for preparing L-phosphinothricin by biological multi-enzyme coupling
  • Redox asymmetrical method for preparing L-phosphinothricin by biological multi-enzyme coupling
  • Redox asymmetrical method for preparing L-phosphinothricin by biological multi-enzyme coupling

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Construction and screening of D-amino acid oxidase mutant library.

[0054] 1. Construction of genetically engineered bacteria

[0055] The gene sequence of D-amino acid oxidase (Gen Bank No.: POY70719.1) from Rhodotorula taiwanensis was codon-optimized and sent to Sangon Bioengineering (Shanghai) Co., Ltd. for whole gene synthesis and cloned to the recombinant expression plasmid pET-24a(+). After the recombinant plasmid was verified to be correct by sequencing, it was transferred into the expression host E. coli BL21 (DE3) for subsequent expression of the recombinant D-amino acid oxidase. The codon-optimized D-amino acid oxidase gene sequence is shown in SEQ ID No.2.

[0056] 2. Construction of D-amino acid oxidase mutant library

[0057] In the first round, the codon-optimized D-amino acid oxidase gene obtained by the above-mentioned whole gene synthesis was used as a template, and the primers used to mutate M213R and M213S in Table 1 were used for site-directed mu...

Embodiment 2

[0076] Construction of Genetically Engineered Bacteria Expressing Transaminase

[0077] 1. Amplification of target gene transaminase

[0078] The transaminase gene was cloned from the Pseudomonas genome, and the corresponding PCR upstream primers and downstream primers were designed according to the corresponding genomic DNA sequence (GenBank accession number WP_076423369.1).

[0079] Upstream primer: ATGAACACCAACAACGCTC

[0080] Downstream primer: TTAAGCCTGTTTAGCTTC

[0081] PCR amplification system:

[0082] 2×Phanta Max buffer: 25 μL;

[0083] dNTPs: 1 μL;

[0084] Upstream primer: 1 μL;

[0085] Downstream primer: 1 μL;

[0086] Template: 1 μL;

[0087] Phanta Super-Fidelity DNA Polymerase: 0.5 μL;

[0088] wxya 2 O: 20.5 μL.

[0089] PCR reaction conditions: pre-denaturation at 95°C for 5min; denaturation at 95°C for 30s, annealing at 60°C for 30s, extension at 72°C for 6min, a total of 30 cycles; post-extension at 72°C for 10min; storage at 4°C.

[0090] The P...

Embodiment 3

[0094] Culture of microorganisms

[0095] 1. Bacteria culture

[0096] After the engineering bacteria containing D-amino acid oxidase gene and transaminase gene were respectively activated by streaking on a plate, a single colony was picked and inoculated into 10 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37 °C for 10 h. Transfer to 50mL LB liquid medium also containing 50μg / mL kanamycin according to 2% inoculum amount, and culture with shaking at 37°C until OD 600 When it reaches about 0.8, add IPTG with a final concentration of 0.5mM, and culture with shaking at 28°C for 12h. After the cultivation, the culture solution was centrifuged at 8000rpm for 10min, the supernatant was discarded, the bacteria were collected, and stored in a -80°C ultra-low temperature refrigerator until use.

[0097] 2. Preparation of crude enzyme solution

[0098] After the culture, the collected cells were washed twice with pH 8 phosphate buffer (50 mM). After t...

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Abstract

The present invention discloses a redox asymmetrical method for preparing L-phosphinothricin by biological multi-enzyme coupling. D, L-phosphinothricin is used as a raw material, the L-phosphinothricin is obtained through catalysis of an enzyme catalysis system, the enzyme catalysis system comprises a D-amino acid oxidase mutant for catalyzing D-phosphinothricin in the D, L-phosphinothricin into 2-carbonyl-4-[hydroxy (methyl) phosphonyl] butyric acid and a transaminase for catalyzing and reducing the 2-carbonyl-4-[hydroxy (methyl) phosphonyl ] butyric acid into the L-phosphinothricin, the D-amino acid oxidase mutant is obtained by mutation of D-amino acid oxidase in wild rhodotorula taiwanensis, and mutation sites are selected from one of the following four types: (1) M213S; (2) M213S-N54V-F58E; (3) M213S-N54V-F58E-D207A; and (4) M213S-N54V-F58E-D207A-S60T. The D-amino acid oxidase mutant has better catalytic efficiency, a conversion rate is far higher than that of wild enzyme when theracemic D, L-phosphinothricin is used as a substrate, and yield of PPO is also greatly improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing L-glufosinate-ammonium by asymmetric oxidation-reduction method of biological multi-enzyme coupling method. Background technique [0002] Glufosinate-ammonium, also known as glufosinate, the English name is Phosphinothricin (abbreviated as PPT), the chemical name is 2-amino-4-[hydroxy (methyl) phosphono] butyric acid, it is the second largest transgenic crop resistant to weeding in the world Agent, developed and produced by Hearst (now owned by Bayer after several mergers). Glufosinate-ammonium is a phosphonic acid herbicide, a glutamine synthetase inhibitor, and a non-selective (killing) contact herbicide. [0003] As we all know, the total herbicide market is huge. At present, the world's three major herbicides are paraquat, glyphosate, and glufosinate-ammonium. In terms of market use, glyphosate is the champion, but due to its long-term use, a large numbe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P41/00C12P13/04
CPCC12P41/00C12P13/04C12Y104/03003C12N9/1096C12N15/52C12N9/0024C12Y206/01019C12N15/70C12N1/20C12N2511/00C12N2800/101C12P7/52
Inventor 薛亚平程峰王柳玉郑裕国
Owner ZHEJIANG UNIV OF TECH
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