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D-amino acid oxidase mutant and applications thereof

An amino acid and oxidase technology, applied in oxidoreductase, application, enzyme and other directions, can solve the problems of low D-amino acid oxidase enzyme activity, poor ammonium resistance, poor enzyme activity stability, etc. The effect of enhancing ammonium and improving the stability of enzyme activity

Active Publication Date: 2020-04-17
SHANGHAI QIZHOU ZIYUE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The technical problem to be solved by the present invention is to overcome defects such as low enzyme activity, poor enzyme activity stability or poor ammonium tolerance of D-amino acid oxidase in the prior art. The present invention provides a D-amino acid oxidase mutant and Its application in the preparation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid

Method used

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  • D-amino acid oxidase mutant and applications thereof
  • D-amino acid oxidase mutant and applications thereof
  • D-amino acid oxidase mutant and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The preparation of embodiment 1 wild-type D-amino acid oxidase (DAAO)

[0042] N from Rhodotorula sp.JG-1b retrieved from NCBI with GenBank accession number KWU45700 2 DAAO enzyme, fully synthetic wild-type (wt) N 2 DAAO enzyme gene, the gene synthesis company is Suzhou Jinweizhi Biotechnology Co., Ltd. (C3 Building, Bio-Nano Science and Technology Park, No. 218, Xinghu Street, Suzhou Industrial Park).

[0043] The synthesized wtN 2 DAAO enzyme gene connection plasmid pET28a (see J.Am.Chem.Soc., 2017, 139(32), 11241-11247 for specific methods), restriction sites NdeI and HindIII, transform the enzyme-linked vector into the host large intestine Bacillus BL21 competent cells; inoculate them into LB liquid culture based on 37°C, 200rpm shaker culture, until OD 600 When the temperature reaches about 0.8, take the bacterial solution and add sterile glycerol with a final concentration of 25%, and store it in a -80°C low-temperature refrigerator for later use.

[0044] The...

Embodiment 2

[0047] Construction of embodiment 2D-amino acid oxidase (DAAO) mutant library (position 211, position 213)

[0048] wtN as described in Example 1 2 On the basis of the DAAO sequence, the 54th and 58th positions (specifically N54V, F58Q) were mutated to obtain a mutated D-amino acid oxidase sequence, and the gene N was synthesized according to the sequence 2 DAAO (N54V, F58Q), the gene synthesis company is Suzhou Jinweizhi Biotechnology Co., Ltd. (Building C3, Bio-Nano Science and Technology Park, No. 218, Xinghu Street, Suzhou Industrial Park). Then introduce plasmid pET28a with NdeI and HindIII restriction sites to construct plasmid pET28a-N 2 DAAO (see J.Am.Chem.Soc., 2017, 139(32), 11241-11247 for plasmid construction methods). With plasmid pET28a-N 2 DAAO was used as a template, and the target band was amplified by PCR.

[0049] Wherein, the 211st, 213rd positions of the mutated D-amino acid oxidase sequence (N54V, F58Q) are mutated for the mutant library construction ...

Embodiment 3

[0058] Example 3 High-throughput screening mutant library

[0059] Screen according to the following experimental steps:

[0060] The transformant obtained in Example 2 was inoculated into a 96-well plate for culture, and induced overnight at 30° C. with IPTG. Afterwards, the bacteria were harvested, cracked with bugbuster protein extraction reagent, and centrifuged to obtain the DAAO mutant enzyme solution.

[0061] Microplate reader detection method: take 100 μL of 100 mM substrate (racemic glufosinate-ammonium, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) with a pH of 8.0, and add 50 μL of chromogenic solution (containing 60 mg / mL of TBHBA (3 -hydroxy-2,4,6-tribromobenzoic acid) and 100 mg / mL of 4-AAP (4-aminoantipyrine)) and 25 μL of HRP (horseradish peroxidase, 0.1 mg / mL), and finally Add 25 μL of the above-mentioned DAAO mutant enzyme solution to obtain a 200 μL reaction system on a microtiter plate, and analyze it at 30° C. and pH 8.0. The absorb...

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Abstract

The invention discloses a D-amino acid oxidase mutant, wherein the sequence of the D-amino acid oxidase mutant comprises a sequence obtained by mutating the 54th amino acid residue N, the 58th amino acid residue F, the 211st amino acid residue C and the 213th amino acid residue M of a sequence represented by SEQ ID NO.1 or a sequence having at least 76% identity with the SEQ ID NO.1, and the D-amino acid oxidase mutant has high enzyme activity, high enzyme activity stability and / or high ammonium tolerance compared with wild type D-amino acid oxidase. The invention also discloses applications of the D-amino acid oxidase mutant in preparation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid. The D-amino acid oxidase mutant is high in enzyme activity, and has the enhanced enzyme activity stability and / or ammonium tolerance, so that the cost is reduced, and industrial production is facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a D-amino acid oxidase mutant and its application in the preparation of 2-oxo-4-(hydroxymethylphosphinyl)butanoic acid (PPO). Background technique [0002] Glufosinate-ammonium is a broad-spectrum contact herbicide developed by Hearst in the 1980s. At present, the three major herbicides in the world are glyphosate, glufosinate-ammonium, and paraquat. Compared with glyphosate and paraquat, glufosinate-ammonium has excellent herbicidal performance and less side effects. Glufosinate-ammonium has two optical isomers, namely D-glufosinate-ammonium and L-glufosinate-ammonium, but only L-glufosinate-ammonium has herbicidal activity, so the method of developing L-glufosinate-ammonium is important for improving atom economy , It is of great significance to reduce the cost of use and reduce the pressure on the environment. [0003] At present, the method for preparing L-glufosina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12P9/00
CPCC12N9/0024C12P9/00C12Y104/03003C12N15/00
Inventor 田振华程占冰徐艳冰
Owner SHANGHAI QIZHOU ZIYUE BIOTECHNOLOGY CO LTD
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