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Glutamate dehydrogenase mutants and application thereof

一种谷氨酸脱氢酶、突变体的技术，应用在催化2-羰基-4-丁酸制备L-草铵膦中的应用,谷氨酸脱氢酶突变体领域，能够解决2-羰基-4-(羟基甲基膦酰基)丁酸催化活力低等问题，达到良好催化效率、大工业应用前景、提高催化活力的效果

Active Publication Date: 2019-08-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the problem of low catalytic activity of glutamate dehydrogenase derived from Pseudomonas putida to non-natural substrate 2-carbonyl-4-(hydroxymethylphosphono)butanoic acid, and provides multiple glutamate enzymes with significantly improved enzyme activity. Amino acid dehydrogenase mutant and its application in the synthesis of L-glufosinate-ammonium

Method used

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  • Glutamate dehydrogenase mutants and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Example 1 Pseudomonas putida-derived glutamate dehydrogenase (PpGluDH) directed evolution based on error-prone PCR

[0053] Step 1: Activation of PpGluDH recombinant bacteria and plasmid extraction

[0054] The Escherichia coli engineering bacteria carrying the pET-28a(+)-PpGluDH recombinant plasmid were activated and cultured in LB medium.

[0055] The specific formula of LB medium is: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, sterilized at 121°C for 20min, ready for use. The solid medium is LB medium with 2% agar added.

[0056] Streak the glycerol tube containing the PpGluDH recombinant bacteria onto a plate containing LB solid medium (50 μg / mL kanamycin), and culture it statically at 37° C. for 12 hours. Pick a single colony from the plate and insert it into 5 mL LB medium containing 50 μg / mL kanamycin, and culture at 37 °C and 200 rpm for 12 h. After obtaining the culture medium, extract the plasmid acco...

Embodiment 2

[0086] Example 2 Construction and Screening of Focused Saturation Mutation Library

[0087] Analyzing the results of Example 1, saturation mutations were performed on amino acid residues 121, 123, 379, 383 and 402 of PpGluDH. Design primers (Table 6), use the pET-28a(+)-PpGluDH plasmid as a template, and use K402X-F, T121X / L123X-F or A379X / L383X-F and Aid-R as primer pairs for PCR to obtain linearized short fragments , and then use K402X-R, T121X / L123X-R or A379X / L383X-R and Aid-F as primer pairs to perform PCR to obtain linearized long fragments. Refer to the instruction manual of the recombination cloning kit (ClonExpress II One Step Cloning Kit) for subsequent DpnI digestion, gel recovery, recombination and transformation operations to construct a saturation mutation library at position 402 (K402X) and a combined mutation library at positions 121 and 123 (T121X / L123X), 379 and 383 combined mutation library (A379X / L383X) 3 mutation libraries.

[0088] Table 6 Primers used ...

Embodiment 3

[0095] Example 3 Glutamate dehydrogenase wild-type (PpGluDH), glucose dehydrogenase dual enzyme coupling preparation of L-glufosinate-ammonium

[0096] Bacterial culture and crude enzyme solution preparation: After the glycerol tubes of engineered bacteria containing wild-type PpGluDH and glucose dehydrogenase (BsGDH-2M, SEQ ID NO.2) were activated by streaking on a plate, single colonies were inoculated into In 50 mL LB liquid medium containing 50 μg / mL kanamycin, shake culture at 37°C for 12 hours. Transfer to 1L fresh LB liquid medium also containing 50μg / ml Kan according to 2% inoculum size, and culture with shaking at 37°C until OD 600 When it reaches about 0.6, add IPTG to its final concentration of 0.5mM, and induce culture at 18°C ​​for 16h. After the cultivation, the culture solution was centrifuged at 12000 g at 4° C. for 10 min, the bacteria were collected, and the cells were disrupted by ultrasonic to prepare the crude enzyme solution.

[0097] The reaction syste...

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Abstract

The present invention discloses glutamate dehydrogenase mutants and an application thereof. The glutamate dehydrogenase mutants are one of the following: mutating 402th lysine of an amino acid sequence shown in SEQ ID NO.1 to phenylalanine or aspartic acid; mutating 406th isoleucine to phenylalanine or threonine; conducting combined mutation of 121th threonine and 123th leucine; and conducting combined mutation of 379th alanine and 383th leucine. A molecular transformation method combining directed evolution with semi-rational design significantly improves catalytic activity of glutamate dehydrogenase derived from pseudomonas putida to 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) and solves a problem of low glutamate dehydrogenase activity in preparation of L-glufosinate by a reductive amination method.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to a glutamate dehydrogenase mutant and its application in catalyzing 2-carbonyl-4-(hydroxymethylphosphono)butyric acid to prepare L-glufosinate-ammonium. [0002] technical background [0003] Glufosinate-ammonium (Phosphinothricin, also known as Glufosinate) is a phosphorus-containing amino acid herbicide, the chemical name is 2-amino-4-(hydroxymethylphosphono)butyric acid, and it is the third largest herbicide in the world and the second largest genetically modified crop herbicide. Glufosinate-ammonium has the characteristics of broad herbicidal spectrum, low toxicity, high activity, partial conductivity and good environmental compatibility. It is widely used in the control of weeds in non-cultivated land, no-till land, farmland crops, aquatic fields, etc. Among the two configurations of glufosinate-ammonium, only the L-form has herbicidal activity (Herbicidal composit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12P13/00
CPCC12N9/0016C12P13/001C12Y104/01004C12P13/04C12P9/00C12N15/74C12Y104/01002
Inventor 杨立荣尹新坚吴坚平徐刚
Owner ZHEJIANG UNIV
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