A kind of transaminase mutant and its application in producing l-glufosinate-ammonium

A mutant, transaminase technology, applied in the field of bioengineering, can solve the problems of only 52% conversion rate and low process efficiency

Active Publication Date: 2020-07-28
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But this process efficiency is low, when the concentration of substrate PPO is 552mmol / L, under the situation of almost completely consuming about 700mmol / L raw material L-aspartic acid, only generate the product L-PPT of 251.9mmol / L, and At the same time, about 234.5mmol / L of impurity pyruvic acid was generated, and the conversion rate of raw material PPO was only 52%.

Method used

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  • A kind of transaminase mutant and its application in producing l-glufosinate-ammonium
  • A kind of transaminase mutant and its application in producing l-glufosinate-ammonium
  • A kind of transaminase mutant and its application in producing l-glufosinate-ammonium

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1: Amplification of transaminase gene ABAT2

[0044] Pseudomonas fluorescens ZJB09-108 is isolated from the soil and stored in the China Center for Type Culture Collection (preservation number CCTCC NO: M2012539, which has been disclosed in the patent application, application number CN 201210593105.3, published (announcement ) No. CN103131649A).

[0045] Based on the transaminase gene sequencing information from Pseudomonas (WP_076423369.1) included in Genbank, a rapid nucleic acid extraction instrument was used to extract the total genomic DNA of Pseudomonas (thalline, and the genomic DNA was used as a template, and primer 1 (5'-ATGAACACCAACAACGCTC-3') and primer 2 (5'-TTAAGCCTGTTTAGCTTC-3') for PCR amplification. PCR reaction system (total volume 50 μL): 10×Pfu DNA Polymerase Buffer 5 μL, 10 mM dNTP mixture (dATP , dCTP, dGTP, and dTTP each 2.5 mM) 1 μL, 1 μL each of cloning primer 1 and primer 2 at a concentration of 50 μM, 1 μL of genomic DNA, 1 μL of Pfu D...

Embodiment 2

[0048] Embodiment 2: Construction of recombinant Escherichia coli BL21(DE3) / pET28b-ABAT2

[0049] According to the ABAT2 gene sequence design primer 3 (5'-CCG in embodiment 1 CATATG AACACCAAACAAC-3), Primer 4 (5'-TTG CTCGAG TTAAGCCTGTTTAGC-3'), and Nde I and Xho I restriction enzyme sites (underlined) were introduced into primer 3 and primer 4, respectively. Under the initiation of primer 3 and primer 4, high-fidelity Pfu DNA polymerase was used to amplify, and the recombinant plasmid pMD18-T-ABAT2 was used as a template (obtained in Example 1) to obtain the ABAT2 gene sequence, which was sequenced using Nde I and The amplified fragment was treated with Xho I restriction endonuclease (TaKaRa), and the fragment was ligated with the commercial vector pET28b (Invitrogen) treated with the same restriction endonuclease using T4 DNA ligase (TaKaRa), The expression vector pET28b-ABAT2 was constructed ( figure 2 ). The constructed expression vector pET28b-ABAT2 was transformed ...

Embodiment 3

[0050] Embodiment 3: the preparation of recombinant transaminase (ABAT2) wet thalline

[0051] The recombinant Escherichia coli E. coli BL21(DE3) / pET28a-ABAT2 bacterial cell containing the expression recombinant plasmid pET28a-ABAT2 obtained in Example 2 was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, 37°C, cultured at 200rpm for 12h, then inoculated with 1% (v / v) inoculum into fresh LB liquid medium containing kanamycin resistance at a final concentration of 50 μg / ml, and cultivated at 37°C at 150rpm until Cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.1mM, induce culture at 28°C for 12h, centrifuge at 4°C and 8000rpm for 10min, discard the supernatant, collect the precipitate, and obtain recombinant Escherichia coli BL21( DE3) / pET28a-ABAT2 wet cells. The bacterium can be used directly as a biocatalyst or for protein purification.

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Abstract

The invention discloses an aminopherase mutant and an application thereof in the production of L-glufosinate-ammonium. The application of the aminopherase mutant in the production of L-glufosinate-ammonium is as follows: a reaction system is formed by using a wet thallus obtained through the fermentation culture of recombinant escherichia coli containing a aminopherase mutant coding gene as a biocatalyst, 2-carbonyl-4 (methyl hydroxyl phosphoryl)-butyrate as a substrate, pyridoxal phosphate as a coenzyme, an amino donor as a cosubstrate, and a buffer solution with the pH value of 6-9 as a reaction medium, and is subjected to biocatalytic reaction at the temperature of 40 to 50 DEG C and the stirring speed of 150 to 250r / min to obtain the L-glufosinate-ammonium. According to the method, thetotal yield is 98%, and the e. e. value of the product reaches 99%.

Description

[0001] (1) Technical field [0002] The invention belongs to the technical field of bioengineering, and relates to a transaminase gene, an encoding enzyme, a recombinant vector containing the gene, a recombinant genetically engineered bacterium and a recombinant enzyme obtained by transforming the recombinant vector, and the ω-aminotransferase used in the preparation of optically pure hand The application of the sexual pesticide L-glufosinate-ammonium. [0003] (2) Background technology [0004] Glufosinate-ammonium, English name: Phosphinothricin (abbreviated as PPT), generally refers to the compound 2-amino-4-[hydroxyl (methyl) phosphono]-butyric acid or its salt formed with a basic compound. It is a high-efficiency, broad-spectrum, low-toxic non-selective herbicide developed by Bayer in the 1980s. It is an ideal herbicide for transgenic resistant crops and has a very broad application prospect. [0005] [0006] Glufosinate-ammonium has two enantiomers, but only the L-co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/1096C12N15/70C12P13/04C12Y206/01
Inventor 郑裕国程峰金利群彭凤薛亚平
Owner ZHEJIANG UNIV OF TECH
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