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Fatty acid photodecarboxylase mutant and application thereof to synthesis of L-phosphinothricin

A photodecarboxylase, fatty acid technology, applied in the direction of application, enzyme, lyase, etc., can solve the problems of low substrate concentration, low L-PPT activity, etc., and achieve the effect of good application value

Active Publication Date: 2020-12-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a stereoselective fatty acid photodecarboxylase mutant and A method for preparing L-glufosinate-ammonium using the recombinant bacteria of the fatty acid photodecarboxylase mutant gene and its crude enzyme solution as a biocatalyst; the mutant has the characteristics of high enzyme activity and can efficiently catalyze D,L-PPT to prepare L- Glufosinate-ammonium

Method used

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  • Fatty acid photodecarboxylase mutant and application thereof to synthesis of L-phosphinothricin
  • Fatty acid photodecarboxylase mutant and application thereof to synthesis of L-phosphinothricin
  • Fatty acid photodecarboxylase mutant and application thereof to synthesis of L-phosphinothricin

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Experimental program
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Effect test

Embodiment 1

[0040] 1. Construction and screening of fatty acid photodecarboxylase mutant library

[0041] The fatty acid photodecarboxylase gene (the amino acid sequence is shown in SEQ ID No.2, the nucleotide sequence is shown in SEQ ID No.1) was constructed as an expression vector pET28b-CvFAP, transformed into Escherichia coli, and the starting strain E.coli BL21 (DE3 ) / pET28b-CvFAP.

[0042] The fatty acid photodecarboxylase mutant library was prepared by three rounds of site-directed saturation mutagenesis, and the primers were designed as shown in Table 1.

[0043] In the first round, the vector pET28b-CvFAP was used as a template, and the site-directed saturation mutation primers G402NYT-F and G402NYT-R in Table 1 were used as primers, and the fatty acid photodecarboxylase amino acid sequence shown in SEQ ID No.2 was subjected to saturation mutation PCR. The 402th threonine is mutated into one of alanine, isoleucine, phenylalanine, proline, serine, threonine and valine, and transf...

Embodiment 2

[0062] Example 2 Fatty acid photodecarboxylase mutant CvFAP-G402F-T370R-S513G strain catalyzes the decarboxylation of D-glufosinate-ammonium in racemic glufosinate-ammonium to prepare L-glufosinate-ammonium

[0063] Use the CvFAP-G402F / T370R / S513G mutant to produce optically pure L-PPT in grams at 25 °C in a total volume of 50 mL pH 6, 100 mM phosphate buffer, 10 μL 100 mM alkane molecule solution, 25 mL CvFAP crude enzyme solution (0.25 g wet cells in 10 mL pH 8.5, 100 mM phosphate buffer) and their solution for photobiocatalytic decarboxylation by D / L-PPT reaction. Add 100 mg D / L-PPT to a clear glass beaker (total volume 100 mL). Under gentle magnetic stirring, the beaker was exposed to blue LED light. After 12 h, an aliquot was taken for determination of conversion and e.e.

[0064] 0.1 g of D / L-PPT was dissolved in pH 6 phosphate buffer (100 mM) and decarboxylated under blue light. After 12 hours, the conversion rate was 50 and the e.e value was 96%. The product has bee...

Embodiment 3

[0065] Example 3 Fatty Acid Photodecarboxylase Mutant CvFAP-G402F-T370R-S513G Strain Whole Cell Catalyzed Decarboxylation of D-Glufosinate in Racemic Glufosinate-ammonium to Prepare L-glufosinate-ammonium

[0066] Use the CvFAP-G402F / T370R / S513G mutant to produce optically pure L-PPT in grams at 25 °C in a total volume of 50 mL pH 7, 100 mM phosphate buffer, 10 μL 100 mM alkane molecule solution, 25 mL CvFAP crude enzyme solution (0.75 g wet cells in 10 mL pH 7, 100 mM phosphate buffer) and their solution for photobiocatalytic decarboxylation by D / L-PPT reaction. Add 500 mg D / L-PPT to a clear glass beaker (total volume 100 mL). Under gentle magnetic stirring, the beaker was exposed to blue LED light. After 16 h, an aliquot was taken for determination of conversion and e.e.

[0067] 0.5g D / L-PPT was dissolved in pH 7 phosphate buffer (100mM) and decarboxylated under blue light. After 16h, the conversion rate was 50 and the e.e value was 96%.

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Abstract

The invention discloses a fatty acid photodecarboxylase mutant and an application thereof to synthesis of L-phosphinothricin. The fatty acid photodecarboxylase mutant is obtained by mutating glycine at the 402nd site of amino acid shown in SEQ ID No.2 into phenylalanine; or, mutating threonine at the 370 site into arginine when mutating on the 402nd site; or mutating serine at the 513rd sitewhen mutating on the 402nd site and the 370 site into glycine and the like. A fatty acid photodecarboxylase gene is mutated by utilizing a site-specific saturation mutation technology, the 370th site, the 371st site, the 402nd site, the 513rd site and the 514 site are found to be key sites influencing enzyme activity and stereoselectivity, a mutant of which the enzyme activity and ee valueare far higher than those of female parent fatty acid photodecarboxylase is obtained, the highest conversion rate is 50%, and the product ee value is 96%.

Description

technical field [0001] The invention relates to the technical field of L-glufosinate-ammonium production, and mainly relates to a fatty acid photodecarboxylase mutant and its application in the synthesis of L-glufosinate-ammonium. 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 h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P41/00C12P13/04C12R1/19
CPCC12N9/88C12Y401/01C12N15/70C12P41/00C12P13/04
Inventor 薛亚平程峰吴冬阳李举谋郑裕国
Owner ZHEJIANG UNIV OF TECH
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