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Glufosinate-ammonium dehydrogenase mutant and application thereof to production of L-glufosinate-ammonium through oxidation-reduction of multienzymes

A mutant, glufosinate-ammonium technology, applied in the production field of chiral pure L-glufosinate-ammonium, can solve the problems of insufficient activity and limited industrial application, and achieve improved catalytic activity, easy separation and purification, and high yield Effect

Inactive Publication Date: 2019-12-20
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activity of the glufosinate-ammonium dehydrogenase wild enzyme to 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid is not high enough, thereby limiting its industrial application

Method used

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  • Glufosinate-ammonium dehydrogenase mutant and application thereof to production of L-glufosinate-ammonium through oxidation-reduction of multienzymes
  • Glufosinate-ammonium dehydrogenase mutant and application thereof to production of L-glufosinate-ammonium through oxidation-reduction of multienzymes
  • Glufosinate-ammonium dehydrogenase mutant and application thereof to production of L-glufosinate-ammonium through oxidation-reduction of multienzymes

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Experimental program
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Effect test

Embodiment 1

[0043] The construction of embodiment 1 expression vector and engineering bacterium

[0044] (1) Recombinant Escherichia coli E.coli BL21(DE3) / pETDuet-1-PPTGDH

[0045] Through literature reports and gene sequence homology, a glufosinate-ammonium dehydrogenase strain derived from Pseudomonasmoorei was selected in the NCBI database, and the NCBI accession number was WP_090325311.1, and the whole gene was synthesized. The nucleotide sequence is shown in Figure SEQ Shown in ID NO.1, the amino acid sequence is shown in SEQ ID NO.2.

[0046] Primers were designed according to the nucleotide sequence shown in SEQ ID NO.1, and Sac I and NotI restriction enzyme sites were introduced in the primers respectively:

[0047] Upstream primer: 5'-GAGCTCATGATTGAGAGCGTCGAGTCT-3';

[0048] Downstream primer: 5'-GCGGCCGCTTAGACGACCCCCTGTGCC-3';

[0049] Using the pETDuet-1 plasmid as the expression vector, construct E. coli E.coli BL21(DE3) / pETDuet-1-PPTGDH:

[0050]Construction of the expres...

Embodiment 2

[0060] Example 2 Glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase were recombined with glufosinate-ammonium dehydrogenase to obtain recombinant Escherichia coli E. coli BL21(DE3) / pETDuet-1-PPTGDH-GDH, E. coli BL21(DE3) / pETDuet-1-PPTGDH-ADH, E. coli BL21(DE3) / pETDuet-1-PPTGDH-FDH.

[0061] 1. Glucose dehydrogenase (NCBI accession number: KM817194.1), formate dehydrogenase (NCBI accession number: WP_013726924.1), and alcohol dehydrogenase (NCBI accession number: CAD66648.1) were respectively cloned by one-step cloning method To the second multiple cloning site of plasmid pETDuet-1:

[0062] Acquisition of glucose dehydrogenase gene, formate dehydrogenase gene and alcohol dehydrogenase gene with homologous sequences: each 15-20bp sequence at the beginning and end of the linearized vector pETDuet-1 is used as a homologous sequence to E.coli BL21(DE3) / pET28b-GDH, E.coli BL21(DE3) / pET28b-FDH and E.coli BL21(DE3) / pET28b-ADH (according to NCBI accession number, usi...

Embodiment 3

[0082] Embodiment 3: Induced expression of glufosinate-ammonium dehydrogenase-glucose dehydrogenase or alcohol dehydrogenase or formate dehydrogenase recombinant bacteria, D-amino acid oxidase

[0083] 1. Wet thallus containing glufosinate-ammonium dehydrogenase gene-glucose dehydrogenase gene or alcohol dehydrogenase gene or formate dehydrogenase gene: the recombinant Escherichia coli E.coli BL21 (DE3) obtained in Example 1 was respectively / pETDuet-1-PPTGDH-GDH, E.coli BL21(DE3) / pETDuet-1-PPTGDH-ADH, E.coli BL21(DE3) / pETDuet-1-PPTGDH-FDH, inoculated to contain 50μg / mL ampicillin The resistant LB liquid medium was cultured at 37°C for 12 hours at 200rpm, and then inoculated into fresh LB liquid medium containing 50 μg / mL ampicillin resistance with a 1% (v / v) inoculation amount, and incubated at 37°C , Cultivated to OD of the bacteria at 150rpm 600After reaching 0.6-0.8, add IPTG with a final concentration of 0.1mM, induce culture at 18°C ​​for 16h, centrifuge at 8000rpm at 4...

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Abstract

The invention discloses a glufosinate-ammonium dehydrogenase mutant and an application thereof to production of L-glufosinate-ammonium through oxidation-reduction of multienzymes. According to the method, a method for preparing an L-glufosinate-ammonium precursor through D-amino acid oxidase is utilized, D, L-glufosinate-ammonium or D-glufosinate-ammonium is used as substrate, and in aerobic environment or under the situation that catalase exists, through excised D-amino acid oxidase or in vitro expression of cells of D-amino acid oxidase, the D-glufosinate-ammonium is catalyzed to obtain 4-(hydroxymethylphosphinyl)-2-oxobutanoic; and under the action of glufosinate-ammonium dehydrogenase, the 4-(hydroxymethylphosphinyl)-2-oxobutanoic is catalyzed to generate the L-glufosinate-ammonium, the activity of a catalyst is improved by about 10 times, and the concentration of the substrate is increased by 5 times. The method is high in raw material conversion rate and high in yield, and products are easy to separate and purify.

Description

[0001] (1) Technical field [0002] The invention relates to the field of biochemical industry, and relates to a production method of chiral pure L-glufosinate-ammonium; it is a method for producing optically pure L-glufosinate-ammonium by coupling amino acid oxidase derived from microorganisms and glufosinate-ammonium dehydrogenase Methods. [0003] (2) Technical background [0004] Glufosinate-ammonium (glufosinate-ammonium) is the world's second most resistant herbicide to genetically modified crops. It is developed and produced by Hearst (now owned by Bayer after several mergers). Its chemical name is 4-[hydroxy (methyl) phosphono ]-DL-homoalanine, also known as glufosinate-ammonium salt, Basta, Buster, etc., is a phosphonic acid herbicide, a glutamine synthetase inhibitor, and a non-selective (killing) contact herbicide. [0005] At present, the three major herbicide species in the world are glyphosate, glufosinate-ammonium and paraquat. With the rapid development of glu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N1/21C12P13/04C12R1/19
CPCC12N9/0016C12N9/0024C12P13/04C12Y104/03003
Inventor 薛亚平程峰李清华郑裕国
Owner ZHEJIANG UNIV OF TECH
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