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L-glutamate dehydrogenase mutant and application thereof

A technology for 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: 2019-10-18
SHANGHAI QIZHOU ZIYUE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] The technical problem to be solved by the present invention is that existing L-glutamic acid dehydrogenase has defects such as low catalytic efficiency when preparing L-glufosinate-ammonium, so the present invention provides a kind of L-glutamic acid dehydrogenase mutant and Its application in the preparation of L-glufosinate-ammonium

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
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Effect test

Embodiment 1

[0099] Example 1 Construction of L-glutamate dehydrogenase mutant library

[0100] Table 3 shows the primer sequences designed for the construction of the mutant library for mutating the 166th, 376th, and 196th positions of SEQ ID NO.1 in the sequence listing.

[0101] table 3

[0102]

[0103] Wherein, N represents any nucleotide in A, G, C, T, M represents A or C, and K represents G or T; it is selected according to the coding nucleotide of the amino acid to be mutated into at the site , For example, NNK in the A166-forward primer can represent AAG (lysine), AAT (aspartic acid), AGG (arginine) or AGT (serine), etc. The nucleotides corresponding to specific amino acids can be found in Table 2.

[0104] The gene cgGLUDH was synthesized according to the sequence of SEQ ID NO.1 in the sequence listing. The gene synthesis company was Suzhou Jinweizhi Biotechnology Co., Ltd. (Building C3, Bio-Nano Science and Technology Park, No. 218, Xinghu Street, Suzhou Industrial Park). T...

Embodiment 2

[0110] Embodiment 2 High-throughput screening mutant library

[0111] Screen according to the following experimental steps:

[0112] The transformants were inoculated in 96-well plates and induced overnight at 30°C with IPTG. Afterwards, the bacteria were harvested, cracked with bugbusterprotein extraction reagent, and centrifuged to obtain the enzyme solution.

[0113] Prepare final concentrations of 20mM PPO and 200mM NH 4 For the reaction solution of Cl and 0.37mM NADPH, take 180 μL of the above reaction solution on a microplate plate and then add 20 μL enzyme solution, the total system is 200 μL, and read the OD in a microplate reader 340 time value. With the wild type as the reference system, positive clones were selected, sequenced and detected for their enzyme activity. The sequencing company is Sangon Bioengineering (Shanghai) Co., Ltd., No. 698, Xiangmin Road, Songjiang District, Shanghai.

[0114] The selected positive clones were cultured as follows:

[0115] ...

Embodiment 3

[0125] Example 3 Acquisition of D amino acid oxidase (DAAO) gene

[0126] According to the gene sequence of AC302 DAAO enzyme recorded in the patent US9834802B2, the DAAO enzyme gene is fully synthesized. 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 and application thereof. The sequence of the L-glutamate dehydrogenase mutant comprises a sequence obtained by mutating amino acid residue Aat position 166 and / or amino acid residue V at position 376 in a sequence shown in SEQ ID NO. 1 into a basic, hydrophilic or small hindered amino acid residue. The application comprises the followingsteps: subjecting 2-oxo-4-(hydroxymethylphosphinyl)butanoate to amination reaction in the presence of an L-amino acid dehydrogenase mutant, an inorganic amino donor and a reduced coenzyme NADPH to obtain L-glufosinate salt; and further acidifying the obtained L-glufosinate salt to obtain L-glufosinate. Compared with wild-type L-glutamate dehydrogenase, the L-glutamate dehydrogenase mutant of theinvention has a higher catalyzed substrate concentration when used for preparing the L-glufosinate, thus the enzyme action efficiency is improved, the reaction 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 mutant of L-glutamic acid dehydrogenase and application thereof. 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 methods for preparing L-glufosinate-ammonium mainly include chiral resolution, chemical syn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12P13/04
CPCC12N9/0016C12P13/04C12Y104/01004C12N9/0028
Inventor 田振华程占冰丁少南徐文选王茹茹焦琦黄瑶
Owner SHANGHAI QIZHOU ZIYUE BIOTECHNOLOGY CO LTD
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