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L-glutamate dehydrogenase mutant and application thereof

A technology of glutamate dehydrogenase and mutants, which is applied in the field of L-glutamate dehydrogenase mutants and can solve problems such as low catalytic efficiency

Active Publication Date: 2020-11-24
SHANGHAI QIZHOU ZIYUE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] The technical problem to be solved by the present invention is that the existing L-glutamate dehydrogenase has defects such as low catalytic efficiency when preparing L-glufosinate-ammonium or its salt, so the present invention provides a kind of L-glutamate dehydrogenase Mutant and its application in the preparation of L-glufosinate-ammonium or its salt

Method used

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  • L-glutamate dehydrogenase mutant and application thereof
  • L-glutamate dehydrogenase mutant and application thereof
  • L-glutamate dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1 Obtaining of L-glutamate dehydrogenase mutant enzyme

[0103] From NCBI retrieved from Lysinibacillus sphaericus glutamate dehydrogenase (hereinafter referred to as LsGluDH) sequence SEQ ID NO.1, Genbank accession number WP_012293812.1, according to the nucleotide sequence SEQ ID NO of the mutant gene in Table 3 .4, SEQ ID NO.6, SEQ ID NO.8, and SEQ ID NO.10 synthesized genes, the gene synthesis company is Suzhou Jinweizhi Biotechnology Co., Ltd. (Building C3, Bio-Nano Technology Park, No. 218, Xinghu Street, Suzhou Industrial Park).

[0104] Then, the mutant genes were enzyme-linked to pET28a, and the restriction sites NdeI&HindIII were respectively enzyme-linked, and the enzyme-linked vectors were transformed into host Escherichia coli BL21 competent cells. Inoculate the constructed strain into TB culture based on 37°C, 200rpm shaker, induce overnight with IPTG concentration of 0.1mM, harvest the strain, and obtain the engineered strain containing the glutam...

Embodiment 2

[0109] The specific enzyme activity detection of embodiment 2 mutant enzyme

[0110] Substrate solution configuration: add 355 μL of 2.25M PPO (final concentration 20mM) (made by the inventor, preparation method reference US8017797B, Figure 6 its corresponding mass spectrum) and 0.4g NH 4 Cl (final concentration 200mM), adjust the pH to 8.5 with ammonia water, and adjust the volume to 40ml with 50mM Tris-HCl buffer solution at pH 8.5.

[0111] Enzyme activity detection method:

[0112] The total reaction system is 1ml, and the absorbance value is measured at OD340nm. Add 940μL substrate solution to 1ml cuvette successively, adjust to zero, then add 10μL 25mM NADPH, and finally add 50μl crude enzyme solution, record the value change from 0-10min, every Take a value every 30s, take the reaction time as the abscissa, and the absorption value at 340nm wavelength as the ordinate to draw a curve, take the slope, calculate the reduction rate of NADPH, and calculate the enzyme acti...

Embodiment 3

[0120] Example 3 Acquisition of D amino acid oxidase (DAAO) gene

[0121] The DAAO enzyme gene is fully synthesized according to the gene sequence of the AC302 DAAO enzyme described in the patent US9834802B2. The synthesis company is Suzhou Jinweizhi Biotechnology Co., Ltd., No. 211 Pubin Road, Yanchuang Park, Jiangbei New District, Nanjing, Jiangsu Province.

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Abstract

The invention discloses an L-glutamate dehydrogenase mutant. The sequence of the L-glutamate dehydrogenase mutant is obtained by mutating a 175 amino acid residue A of the sequence shown in SEQ IDNO. 1 into G and mutating a 386 amino acid residue V into an amino acid residue with smaller steric hindrance. The invention also discloses application of the L-glutamate dehydrogenase mutant inpreparation of L glufosinate-ammonium or salts thereof. When the L-glutamate dehydrogenase mutant is used for preparing the L-glufosinate-ammonium or salts thereof, compared with the L-glutamate dehydrogenase mutant which is only mutated at the 175 site or 386 site, the L-glutamate dehydrogenase mutant has higher enzyme activity, thereby improving the action efficiency of the enzyme, decreasing the reaction cost and being beneficial to industrial production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a mutant of L-glutamic acid dehydrogenase and application thereof. Background technique [0002] Glufosinate-ammonium (2-amino-4-[hydroxy(methyl)phosphono]butyric acid) 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 methods for preparing L-glufosinate-ammo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12P13/04
CPCC12N9/0016C12P13/04C12Y104/01004C12R2001/01C12N15/70C12Y104/01002C12N9/0028C12P9/00
Inventor 田振华程占冰丁少南焦琦徐文选黄瑶江枫
Owner SHANGHAI QIZHOU ZIYUE BIOTECHNOLOGY CO LTD
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