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Method of utilizing chemistry-enzyme method to produce L-glufosinate ammonium

A technology of glufosinate-ammonium and enzymatic method, which is applied in the field of chemical-enzymatic production of L-glufosinate-ammonium, which can solve the problems of reduced yield and achieve the effects of easy separation, simple reaction steps and high optical selectivity

Active Publication Date: 2018-10-23
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reversible reaction often leads to a decrease in the yield, and it is necessary to add an excess of amino donors, which brings a great burden to the later product separation.

Method used

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  • Method of utilizing chemistry-enzyme method to produce L-glufosinate ammonium
  • Method of utilizing chemistry-enzyme method to produce L-glufosinate ammonium
  • Method of utilizing chemistry-enzyme method to produce L-glufosinate ammonium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: the synthesis of racemic N-acetylglufosinate-ammonium

[0026] Take 5g of racemic glufosinate-ammonium, put it into a 250mL three-neck flask, add 100g of acetic acid, keep stirring at 40°C, add 5g of acetic anhydride, and react until the solution is clear and transparent. Rotary evaporator 80 ℃, vacuum 0.075 ~ 0.085MPa, evaporate acetic acid. The concentrate was dissolved in water, and the aqueous sodium hydroxide solution was adjusted to neutrality (pH 8.5), and ethanol was added to stand still to precipitate crystals, and after drying, racemic N-acetylglufosinate-ammonium was obtained. Utilize liquid chromatography (Shimadzu LC-16) to detect, the liquid phase spectrogram is figure 2 , mass spectrometry confirmed that it was N-acetylglufosinate-ammonium ( image 3 ). The column is C-18column (250mm×4.6mm, 5μm), the mobile phase is 10mM phosphoric acid solution (pH 2.1), the flow rate is 0.75mL / min, the ultraviolet detection wavelength is 210nm, and ...

Embodiment 2

[0027] Embodiment 2: the construction of engineering bacteria

[0028] A strain derived from Stenotrophomonas sp., annotated as the sequence of carboxypeptidase Ss1, was codon-optimized for whole gene synthesis, and the expression plasmid was pET-28b. The stop codon was site-directedly mutated so that the tail (C-terminus) of the expressed recombinase contained the histidine tag in pET28b. The inserted sequence (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2) was verified by sequencing and then transferred into the expression host E. coli BL21 (DE3) for subsequent recombinase expression.

Embodiment 3

[0029] Embodiment 3: Induced expression of recombinase

[0030] The engineered bacterium constructed in Example 2 was inoculated into LB liquid medium containing 50 μg / mL kanamycin, cultivated overnight at 37° C., and then inoculated into 50 μg / mL kanamycin containing 50 μg / mL kanamycin with 1% inoculum size (v / v). In the LB liquid medium, 37 ℃, 150rpm cultivated to the cell concentration OD 600 To about 0.6, add IPTG with a final concentration of 0.1mM, induce culture at 28°C for 12h, centrifuge at 8000rpm for 10min at 4°C, and collect wet bacterial cells.

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Abstract

The invention discloses a method of utilizing a chemistry-enzyme method to produce L-glufosinate ammonium. According to the method, racemic N-acetyl glufosinate ammonium is taken as the substrate, engineering bacteria containing carboxyl-peptidase genes are subjected to fermentation culture to obtain wet cells, wet cells or pure enzymes, which are obtained by steps of grinding wet cells through ultrasonic waves and extracting grinded wet cells, are taken as the catalyst, a buffer solution with a pH value of 5-10 is taken as the reaction medium, reactions are performed in a water bath with a temperature of 45 DEG C at a rotation speed of 200 rpm, after complete reactions, a reaction solution containing D-N-acetyl glufosinate ammonium and L-glufosinate ammonium is obtained, the reaction solution is separated and purified, and collected D-N-acetyl glufosinate ammonium is subjected to racemization and then split in cycles to obtain L-glufosinate ammonium at the same time. The provided method does not need a coenzyme circulation system or an amino donor with a structure that is similar with the product; the reaction steps are simple, the reaction product is easy to separate, the opticalselectivity is high (ee value is 99%), the separation effect of the ion exchange column is obvious, and L-glufosinate ammonium with higher purity can be obtained more easily (purity is 98%).

Description

(1) Technical field [0001] The invention relates to the field of biochemical industry, in particular to a chemical-enzymatic method for producing L-glufosinate-ammonium. (2) Background technology [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 that of racemic g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/04C12P41/00
CPCC12P13/04C12P41/001
Inventor 薛亚平郑裕国曹成浩徐建妙吴哲明
Owner ZHEJIANG UNIV OF TECH
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