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A d-amino acid oxidase mutant and its application

An amino acid and oxidase technology, applied in the direction of oxidoreductase, application, enzyme, etc., can solve the problems of low enzymatic activity, poor ammonium resistance, and poor enzyme activity stability of D-amino acid oxidase

Active Publication Date: 2020-12-08
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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  • A d-amino acid oxidase mutant and its application
  • A d-amino acid oxidase mutant and its application
  • A d-amino acid oxidase mutant and its application

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. (Floor C3, 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 co...

Embodiment 2

[0047] Example 2 Construction of D-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). pET28a-N 2 DAAO was used as a template, and the target band was amplified by PCR.

[0049] Wherein, the primer sequence of designing PCR is constructed and designed for the mutant library that is mutated at the 211th and 213rd positions of the muta...

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. Record the...

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Abstract

The invention discloses a mutant of D-amino acid oxidase, the sequence of which comprises amino acid residue N at position 54 of the sequence shown in SEQ ID NO.1 or a sequence having at least 76% identity with SEQ ID NO.1, The mutated sequence of the 58th amino acid residue F, the 211th amino acid residue C and the 213th amino acid residue M; the D-amino acid oxidase mutant has a higher enzyme than the wild-type D-amino acid oxidase activity, enzymatic stability and / or ammonium resistance. The invention also discloses the application of the D-amino acid oxidase mutant in the preparation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid. The D-amino acid oxidase mutant of the present invention has high enzyme activity, improved enzyme activity stability and / or enhanced ammonium resistance, thereby reducing costs and being beneficial to industrial production.

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 preparing 2-oxo-4-(hydroxymethylphosphinyl)butyric 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-glufosinate-ammoniu...

Claims

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Application Information

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Patent Type & Authority Patents(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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