Glufosinate dehydrogenase mutant and application thereof

A glufosinate-ammonium and dehydrogenase technology, applied in the field of glufosinate-ammonium dehydrogenase mutants, can solve the problems of low asymmetric reduction activity and low substrate concentration, and achieve the effect of good industrial application prospects.

Active Publication Date: 2019-05-14
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to provide a stereoselective method for the existing glufosinate-ammonium dehydrogenase to the problems of low asymmetric reduction activity of 2-carbonyl-4-(hydroxymethylphosphono) butyric acid and low substrate concentration. A glufosinate-ammonium dehydrogenase mutant and a method for preparing L-glufosinate-ammonium using the recombinant bacteria of the glufosinate-ammonium dehydrogenase mutant gene and its crude enzyme solution as a biocatalyst; the mutant has the characteristics of high enzyme activity, It can efficiently catalyze the asymmetric reduction of 2-carbonyl-4-(hydroxymethylphosphono)butanoic acid to L-glufosinate-ammonium

Method used

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  • Glufosinate dehydrogenase mutant and application thereof
  • Glufosinate dehydrogenase mutant and application thereof
  • Glufosinate dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 Construction and screening of glufosinate-ammonium dehydrogenase mutant library

[0052] The glufosinate-ammonium dehydrogenase gene (amino acid sequence shown in SEQ ID No.2 and nucleotide sequence shown in SEQ ID No.1) derived from baiji was constructed as expression vector pETDuet-1-lvPDH, transformed into Escherichia coli, The starting strain E.coli BL21(DE3) / pETDuet-1-lvPDH was obtained.

[0053] The glufosinate-ammonium dehydrogenase mutant library was prepared by three rounds of site-directed saturation mutagenesis, and the primers were designed as shown in Table 1.

[0054] In the first round, using the vector pETDuet-1-lvPDH as a template, using the site-directed saturation mutation primers K90-F and K90-R in Table 1 as primers, through saturation mutation PCR, the glufosinate-ammonium shown in SEQ ID No.2 was removed. The 90th lysine in the hydrogenase amino acid sequence was mutated into the remaining 19 amino acids, and transformed, plated, and...

Embodiment 2

[0065] Embodiment 2 Induced expression of glufosinate-ammonium dehydrogenase parent, mutant and glucose dehydrogenase

[0066] Glucose dehydrogenase gene gdh (nucleotide sequence shown in SEQ ID No.3, amino acid sequence shown in SEQ ID No.4) is synthesized from the whole gene and obtained from recombinant glucose dehydrogenase of the genus Exiguobacterium sibiricum Strain E. coli BL21(DE3) / pET28b-GDH.

[0067] Example 1 departure bacterial strain E.coli BL21(DE3) / pETDuet-1-lvPDH and glufosinate-ammonium dehydrogenase mutant strain and recombinant glucose dehydrogenase bacterial strain E.coli BL21(DE3) / pET28b-GDH were respectively inoculated into containing In LB liquid medium with a final concentration of 50 μg / mL ampicillin and kanamycin, culture at 37°C for 9 hours, and inoculate into fresh ampicillin containing a final concentration of 50 μg / mL at a volume fraction of 2% (v / v). In the LB liquid medium of kanamycin and kanamycin, culture at 37°C and 180 rpm for 1.5 hours, ...

Embodiment 3

[0068] Embodiment 3 mutation library screening

[0069] Mix the wet cells of the mutant strain induced and expressed in Example 2 and the wet cells of glucose dehydrogenase at a mass ratio of 3:1, and add the amount of 50 g / L of the total amount of cells into pH 7.5, 100 mM phosphate buffer for resuspension , ultrasonic crushing on ice-water mixture for 10 minutes, ultrasonic crushing conditions: power of 400W, crushing for 1 second, pausing for 1 second, to obtain the crude enzyme solution of the mutant strain. Under the same conditions, replace the wet cells of the mutant strain with the starting strain to prepare the crude enzyme solution of the starting strain.

[0070] The crude enzyme solution of the mutant strain or the crude enzyme solution of the original strain is used as a catalyst, 2-carbonyl-4-(hydroxymethylphosphono)butyric acid is used as a substrate, glucose is used as an auxiliary substrate, and no exogenous NADPH or NADP is added. + , using the endogenous NA...

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Abstract

The invention discloses a glufosinate dehydrogenase mutant and application thereof. The glufosinate dehydrogenase mutant is obtained by single mutation or multiple mutation of the 90th, 91st and 376thamino acid shown in SEQ ID No.2; the 90th lysine is mutated to serine; the 91th glycine is mutated to serine or valine; the 376th serine is mutated into arginine. The mutant utilizes a site-directedsaturation mutation technique for mutation of the glufosinate dehydrogenase gene shown in SEQ ID No.1, and finds that the 90th, 91st, and 376th positions are key sites affecting the enzyme activity, the obtained specific enzyme activity is much higher than the mutant of the parental glufosinate dehydrogenase, the specific enzyme activity of the mutant lvPDH-K90S-G91P-S376R is 8.4 times higher thanthat of the parental glufosinate dehydrogenase, and the mutant has industrial application prospects.

Description

technical field [0001] The invention relates to the technical field of glufosinate-ammonium production, in particular to a glufosinate-ammonium dehydrogenase mutant and application thereof. Background technique [0002] Glufosinate-ammonium, also known as glufosinate, English name: Phosphinothricin (abbreviated as PPT), chemical name is 2-amino-4-[hydroxy (methyl) phosphono]-butyric acid. Glufosinate-ammonium is a systemic herbicide with broad-spectrum herbicidal activity. Herbicides are widely used, and the domestic and foreign markets are huge. Glufosinate-ammonium is one of the three major herbicides. In recent years, due to its mechanism of action and transgenic technology, its market share is expected to further break through. [0003] Glufosinate-ammonium currently in the market is mainly racemic. Glufosinate-ammonium has two optical isomers: L-glufosinate-ammonium and D-glufosinate-ammonium. But only L-glufosinate-ammonium has herbicidal activity, which is twice th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N1/21C12P13/00C12R1/19
Inventor 薛亚平程峰曹成浩徐建妙郑裕国
Owner ZHEJIANG UNIV OF TECH
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