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Aminopherase mutant and application thereof in production of L-glufosinate-ammonium

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

Active Publication Date: 2018-11-27
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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  • Aminopherase mutant and application thereof in production of L-glufosinate-ammonium
  • Aminopherase mutant and application thereof in production of L-glufosinate-ammonium
  • Aminopherase mutant and application thereof in production of L-glufosinate-ammonium

Examples

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Embodiment 1

[0038] Example 1: Amplification of transaminase gene ABAT2

[0039] 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).

[0040] 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, cloning primer 1 and primer 2 each 1 μL at a concentration of 50 μM, genomic DNA 1 μL, Pfu DNA Polymerase 1 ...

Embodiment 2

[0043] Embodiment 2: Construction of recombinant Escherichia coli BL21(DE3) / pET28b-ABAT2

[0044] 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 priming of primer 3 and primer 4, utilize high-fidelity PfuDNA polymerase to amplify, use recombinant plasmid pMD18-T-ABAT2 as template (obtained in embodiment 1), obtain ABAT2 gene sequence, utilize Nde I and Xho The amplified fragment was treated with 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) to construct an expression Vector pET28b-ABAT2 ( figure 2 ). The constructed expression vector pET28b-ABAT2 was transformed into Escherichia coli BL21(DE3) (Invitrogen) (...

Embodiment 3

[0045] Embodiment 3: the preparation of recombinant transaminase (ABAT2) wet thalline

[0046] The recombinant Escherichia coli E.coliBL21(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 Cultivate at 200rpm for 12h, then inoculate with 1% (v / v) inoculum into fresh LB liquid medium containing kanamycin resistance at a final concentration of 50μg / ml, and cultivate at 37°C at 150rpm until the bacteria Body 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

(1) Technical field [0001] 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. (2) Background technology [0002] 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. [0003] [0004] Glufosinate-ammonium has two enantiomers, but only the L-configuration has...

Claims

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Application Information

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Patent Type & Authority Applications(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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