NADH kinase mutant, coding gene and application

A technology of mutants and kinases, applied in the field of NADH kinase mutants and their coding genes, can solve the problems of unavailable, expensive, and reduced utilization of raw materials, and achieve the effect of reducing the amount of use

Active Publication Date: 2022-01-11
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] (1) The enzyme directly hydrolyzes the derivative of L-glufosinate-ammonium, which has the advantages of high conversion rate and high ee value of the product, but the raw material used is an expensive and difficult-to-obtain chiral precursor
[0007] (2) The selective resolution of racemic glufosinate-ammonium by enzyme has the advantage that the raw material is relatively easy to get and the catalytic efficiency is high, but the theoretical yield can only reach 50%, which greatly reduces the raw material utilization rate

Method used

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  • NADH kinase mutant, coding gene and application
  • NADH kinase mutant, coding gene and application
  • NADH kinase mutant, coding gene and application

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Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1: Construction of NADH kinase genetically engineered bacteria

[0041] The gene sequence of NADH kinase (Gen Bank No.: NC_001148.4) from Saccharomyces cerevisiae was codon-optimized and sent to Sangon Bioengineering (Shanghai) Co., Ltd. for whole gene synthesis, and cloned into the recombinant expression plasmid pET-28a (+) on. The codon-optimized NADH kinase gene sequence is shown in SEQ ID No.2.

[0042] Since the expression vector used for glutamate dehydrogenase and formate dehydrogenase is the pETDuet-1 plasmid, which cannot coexist with the pET-28a(+) plasmid, the recombinant expression vector of NADH kinase was replaced with the pCDFDuet plasmid. The empty pCDFDuet plasmid was used as a template, and pCDFDuet-Pf and pCDFDuet-Pr in Table 1 were used as primers for PCR. This process was called plasmid linearization. Using the synthetic recombinant plasmid pET-28a(+) as a template, NADHK-Pf and NADHK-Pr in :1 are used as primers for amplification, and ...

Embodiment 2

[0046] Embodiment 2: Construction of NADH kinase mutant library

[0047] In the first round, the above-mentioned preserved bacterial solution was cultured in a test tube, and the plasmid was extracted as a template for site-directed mutation PCR of NADH kinase mutation, and T175H-Pf and T175H-Pr in Table 2 were used as mutation primers to perform site-directed mutation PCR. The PCR product was transformed, plated, and the T175H mutant was obtained by screening, which was named T175H.

[0048] In the second round, site-directed mutagenesis PCR was performed using T175H as a template and R252A-Pf and R252A-Pr in Table 2 as primers. The PCR product was transformed, plated, and the mutant of T175H mutation and R252A was obtained by screening, which was named as T175H-R252A.

[0049] In the third round, T175H-R252A was used as a template, and I332K-Pf and I332K-Pr in Table 2 were used as primers for site-directed mutagenesis PCR. The PCR product was transformed, plated, and the T...

Embodiment 3

[0055] Embodiment 3: the construction of double plasmid engineering bacterium

[0056] The glutamic acid dehydrogenase gene and the formate dehydrogenase gene are respectively located at the first multiple cloning site and the second multiple cloning site on the plasmid PetduetI, and the plasmid is named PetduetI-GluDH-FDH. The NADH kinase gene is located in a multiple cloning site of the PcdfduetI plasmid, named PcdfduetI-NADHK. The two plasmids were extracted using a plasmid extraction kit, introduced into the same BL21 competent cell, spread on a plate containing both streptomycin and ampicillin resistance, and cultured for 12 hours. Pick a single colony and inoculate it into 10ml LB liquid medium containing 50ul / ml streptomycin and 50ul / ml ampicillin resistance, and culture with shaking at 37°C for 10h. Add 30% glycerol to the bacterial solution at a ratio of 1:1, and store in a -80°C refrigerator.

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Abstract

The invention relates to an NADH kinase mutant, a coding gene thereof and application of the NADH kinase mutant in biosynthesis of L-glufosinate-ammonium, D-amino acid and L-aminobutyric acid through a multi-enzyme coupling method. The mutant is obtained by NADH kinase as shown in SEQ ID No.1 through one of the following mutations that (1) threonine T at a 175th site is mutated into histidine H; (2) threonine T at a 175th site is mutated into histidine H, and arginine R at a 252nd site is mutated into alanine A; and (3) threonine T at a 175th site is mutated into histidine H, arginine R at a 252nd site is mutated into alanine A, and isoleucine I at a 332nd site is mutated into lysine K. The NADH kinase mutant has desirable catalytic efficiency, and when intermediate product PPO is used as a substrate for a catalytic reaction, the conversion rate is far higher than that of an original enzyme, and the yield of the L-glufosinate-ammonium is also greatly increased; the defects of more process steps, harsh reaction conditions, expensive chiral raw materials and the like for synthesis of L-PPT through a chemical method are overcome; and a high-efficiency, low-cost and environment-friendly production mode is provided.

Description

(1) Technical field [0001] The invention relates to an NADH kinase mutant and its coding gene, and its application in the biosynthesis of L-glufosinate-ammonium, D-amino acid and L-aminobutyric acid by multi-enzyme coupling method. (2) Background technology [0002] Chiral amino acids are widely used chiral compounds and are of great significance in active pharmaceutical intermediates such as peptides and single enantiomers. The synthesis of chiral amino acids by bio-enzyme has the advantages of simple operation, mild reaction conditions, high product yield and high optical activity value, and is currently a research hotspot in the asymmetric synthesis of chiral amino acids. The asymmetric reduction of prochiral ketones by amino acid dehydrogenase is the most common method, but amino acid dehydrogenase needs to consume NADPH in the asymmetric reduction, and the high price of NADPH limits its application at the industrial level. Constructing a NADPH regeneration cycle system...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12P41/00C12P13/06C12P13/04
CPCC12N9/1205C12P41/001C12P41/002C12P13/06C12P13/04C12Y207/01086
Inventor 邹树平伍小艳薛亚平
Owner ZHEJIANG UNIV OF TECH
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