Transaminase, mutant and application thereof in production of L-glufosinate ammonium

The technology of transaminase and mutant is applied in the application field of preparing asymmetric chiral amine compounds, and can solve the problems of low stereoselectivity, high catalyst price, difficult recovery of solvent and the like

Active Publication Date: 2018-10-16
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aiming at the reported asymmetric synthesis of L-glufosinate-ammonium with low stereoselectivity, high catalyst price and difficult solvent recovery, the present invention provides a transaminase with high catalytic activity and strong stereoselectivity for catalytic synthesis of L-glufosinate. - Glufosinate-ammonium

Method used

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  • Transaminase, mutant and application thereof in production of L-glufosinate ammonium
  • Transaminase, mutant and application thereof in production of L-glufosinate ammonium
  • Transaminase, mutant and application thereof in production of L-glufosinate ammonium

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Embodiment 1

[0032] Embodiment 1: the amplification of transaminase gene ata3

[0033] 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).

[0034] According to the transaminase gene sequencing information from Pseudomonas (WP_076423369.1) included in Genbank, the total genomic DNA of Pseudomonas CCTCC NO: M2012539 was extracted with a rapid nucleic acid extraction instrument, and the genomic DNA was used as a template. 1 (5'-ATGTCTAAAAACGAATCTCTGCTGCAGC-3') and primer 2 (5'-TTAAGCCAGTTCGTCGAAGCATTC-3') for PCR amplification. PCR reaction system (total volume 50 μL): 5 μL of 10×Pfu DNA Polymerase Buffer, 1 μL of 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP), 1 μL of cloning primer 1 and primer 2 each at a concentration of 50 μM, geno...

Embodiment 2

[0037] Embodiment 2: Construction of recombinant Escherichia coli E.coli BL21(DE3) / pET28b-ata3

[0038] Design primer 3 (5'-CCG according to embodiment 1ata3 gene sequence CATATG TCTAAAAACGAATCTC-3'), primer 4 (5'-TTG CTCGAG TTAAGCCAGTTCGTCG-3'), and Nde I and Xho I restriction enzyme sites (underlined) were introduced into primer 3 and primer 4, respectively. Under the priming of primers 3 and 4, high-fidelity Pfu DNA polymerase was used to amplify, and the recombinant plasmid pMD18-T-ata3 was used as a template (obtained in Example 1) to obtain the ATA3 gene sequence, which was sequenced using NdeI 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-ata3 ( figure 2 ). The constructed expression vector pET28b-ata3 was transformed into Escherichia c...

Embodiment 3

[0039] Example 3: Induced expression of transaminase (ata3)

[0040] The recombinant Escherichia coli E. coli BL21(DE3) / pET28b-ata3 obtained in Example 2 was inoculated into LB liquid medium containing 50 μg / ml kanamycin resistance, cultivated at 37° C. for 12 hours at 200 rpm, and then added with 1% (v / v) Inoculum amount was inoculated into fresh LB liquid medium containing 50 μg / ml kanamycin resistance, and cultivated at 37°C and 150 rpm until the cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.1mM, induce culture at 25°C for 18h, centrifuge at 8000rpm at 4°C for 20min, discard the supernatant, collect the precipitate, and obtain the recombinant Escherichia coli E. coli containing the expression recombinant plasmid. coli BL21 / pET28b-ata3 wet cells. The bacteria can be directly used as a biocatalyst.

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Abstract

The invention discloses transaminase, a mutant and application thereof in production of L-glufosinate ammonium. The mutant is characterized in that isoleucine at the 62nd site of amino acid in SEQ IDNO:2 is substituted by valine; serine at the 74th site is substituted by threonine; methionine at the 93rd site is substituted by isoleucine; tyrosine at the 167th site is substituted by phenylalanine; alanine at the 220th site is substituted by proline; arginine at the 282nd site is substituted by lysine; alanine at the 353rd site is substituted by serine; isoleucine at the 355th site is substituted by valine. The transaminase is characterized in that 4-(methyl hydroxyphosphoryl)-2-carbonyl-butyric acid is used as a primer to prepare the L-glufosinate ammonium, the transaminase product ee isgreater than 99.9%; the 4h primer conversion rate is 56.31%; the transaminase mutant product ee is greater than 99.9%, and the 4h primer conversion rate is 95.42%.

Description

(1) Technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a transaminase sequence Pseudomonas fluorescens ZJB09-108 gene, an expression vector, a genetic engineering bacterium and an application thereof in preparing an asymmetric chiral amine compound. (2) Background technology [0002] Transaminase (Amine Transaminase, ATA, EC2.6.1.X) belongs to the transferase class, which catalyzes the transfer of the amino group on the amino donor to the latent chiral ketone compound to obtain chiral amine and by-product ketone or α-keto acid A class of enzymes, the reaction process needs the participation of pyridoxal phosphate (Pyridoxal phosphate, PLP), and the reaction it catalyzes is reversible. Transaminases widely exist in nature and play an important role in the process of nitrogen metabolism in cells. In general, all transaminases can be classified according to the amino acid substrates used in their reversible reactions, and name...

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 金利群郑裕国彭凤程峰柳志强薛亚平贾东旭
Owner ZHEJIANG UNIV OF TECH
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