Glufosinate-ammonium dehydrogenase mutant and application thereof to synthetizing L- glufosinate-ammonium

A mutant, glufosinate-ammonium technology, applied in the production field of chiral pure L-glufosinate-ammonium, can solve the problems of low reduction activity and low substrate concentration, and achieve easy separation and purification, high yield and simple process Effect

Active Publication Date: 2019-04-12
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The present invention aims at the problem that the existing glufosinate-ammonium dehydrogenase has low asymmetric amination reduction activity of 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid and low substrate concentration, and provides a glufosinate-ammonium The phosphine dehydrogenase mutant and the genetic recombinant bacteria using the glufosinate-ammonium dehydrogenase mutant and its crude enzyme solution are used as biocatalysts for the chiral biosynthesis of L-glufosinate-ammonium, and the activity of the catalyst is increased by nearly 10 times. 5-fold increase in substrate concentration

Method used

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  • Glufosinate-ammonium dehydrogenase mutant and application thereof to synthetizing L- glufosinate-ammonium
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  • Glufosinate-ammonium dehydrogenase mutant and application thereof to synthetizing L- glufosinate-ammonium

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Experimental program
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Effect test

Embodiment 1

[0036] The construction of embodiment 1 expression vector and engineering bacterium

[0037] Based on literature reports and gene sequence homology, four strains of dehydrogenases were selected from the NCBI database, which were derived from Pseudomonas, Pseudomonas extremaustralis, Pseudomonas moorei and Pseudomonassaudiphocaensis respectively. Or WP_037025837.1, and carry out whole gene synthesis, respectively obtain the glufosinate-ammonium dehydrogenase E1 (nucleotide sequence is shown in SEQ ID NO.1, aminoacid sequence is shown in SEQ ID NO.2) derived from Pseudomonas, Glufosinate-ammonium dehydrogenase E2 derived from Pseudomonasextremaustralis (the nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4), the glufosinate-ammonium dehydrogenase E3 derived from Pseudomonas moorei (nuclear The nucleotide sequence is shown in SEQ ID NO.5, the amino acid sequence is shown in SEQ ID NO.6) and the glufosinate-ammonium dehydrogenase E4 d...

Embodiment 2

[0051] Embodiment 2: Induced expression of glufosinate-ammonium dehydrogenase, glucose dehydrogenase, SDS-PAGE analysis

[0052] 1. Wet cells containing glufosinate-ammonium dehydrogenase: the recombinant Escherichia coli E.coli BL21(DE3) / pETDuet-1-PPTGDHE1, E.coli BL21(DE3) / pETDuet-1- PPTGDHE2, E.coli BL21(DE3) / pETDuet-1-PPTGDHE3, E.coli BL21(DE3) / pETDuet-1-PPTGDHE4, inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance, 37°C, Cultivate at 200rpm for 12h, then inoculate 1% (v / v) inoculum into fresh LB liquid medium containing 50μg / mL ampicillin resistance, and cultivate at 37°C and 150rpm until the OD600 of the bacteria reaches 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°C for 20min, discard the supernatant, collect the precipitate, and wash twice with pH 7.5, 20mM phosphate buffer saline (PBS) , that is, to obtain Escherichia coli E.coli BL21(DE3) / pETDuet-1-PPTGDHE1, E.coli BL21(DE...

Embodiment 3

[0066] Example 3 Glufosinate-ammonium dehydrogenase catalyzes PPO to prepare L-glufosinate-ammonium

[0067] Crude enzyme solution: add the wet cells containing glufosinate-ammonium dehydrogenase and the wet cells containing glucose dehydrogenase prepared by the method in Example 2 to resuspend the cells in PBS with pH 7.5 and 100 mM, and put them on the ice-water mixture Ultrasonic crushing for 10 min, ultrasonic crushing conditions: power 400W, crushing for 1 s, pausing for 5 s, to obtain crude enzyme solution.

[0068] In 30mL PBS buffer (100mM, pH 7.5), add the crude enzyme solution of glufosinate-ammonium dehydrogenase (the amount of bacteria is 25g / L buffer), 2-carbonyl-4-(hydroxymethylphosphinyl)-butyl Acid (final concentration 100mmol / L), inorganic amino donor (NH 4 ) 2 SO 4(final concentration 500mM), glucose (final concentration 120mmol / L), NADPH (final concentration 1mmol / L), GDH crude enzyme solution (the amount of bacteria is 50g / L buffer) constitute the reacti...

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Abstract

The invention discloses a glufosinate-ammonium dehydrogenase mutant and an application thereof to synthetizing L- glufosinate-ammonium. According to a method, 4-(hydroxymethylphosphinyl)-2-oxobutanoicor salts are used as a substrate, under the condition that an inorganic amino donor and reduced coenzyme exist, glufosinate-ammonium dehydrogenase or cells containing the glufosinate-ammonium dehydrogenase are used as a biocatalyst, and a reductamination reaction is performed so as to obtain the L-glufosinate-ammonium. The method is higher in raw material conversion rate and yield, products are easy to separate and purify, and the chirality purity is high. Compared with other catalyzing technologies, the technology is simpler relatively, and the raw material conversion rate is as high as 100%.

Description

(1) Technical field [0001] 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 utilizing mutants of glufosinate-ammonium dehydrogenase derived from microorganisms. (2) Technical background [0002] 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. [0003] At present, the three major herbicide species in the world are glyphosate, glufosinate-ammonium and paraquat. With the rapid development of glufosinate-ammonium transgenic crops,...

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
CPCC12N9/0016C12P13/04C12Y104/01
Inventor 薛亚平程峰李清华郑裕国徐建妙
Owner ZHEJIANG UNIV OF TECH
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