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Preparation method of L-glufosinate-ammonium

A technology based on glufosinate-ammonium and hydroxymethyl phosphine oxide, which can be applied in the biological field and can solve the problem of high cost of raw materials

Active Publication Date: 2019-07-26
SHANGHAI QIZHOU ZIYUE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0016] The technical problem to be solved by the present invention is that the existing method for preparing L-glufosinate-ammonium has defects such as high cost of raw materials, so the present invention provides a method for preparing L-glufosinate-ammonium

Method used

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  • Preparation method of L-glufosinate-ammonium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Acquisition of D amino acid oxidase (DAAO) gene

[0081] The DAAO enzyme is fully synthesized according to the gene sequence of the AC302 DAAO enzyme described in the patent US9834802B2.

Embodiment 2

[0082] Example 2 Expression of D amino acid oxidase (DAAO) gene

[0083] The DAAO enzyme gene was enzyme-linked to pET28a, the restriction site NdeI&HindIII, and the enzyme-linked vector was transformed into host Escherichia coli BL21 competent cells. The TB culture for strain inoculation is based on shaking at 200 rpm at 37°C, adding IPTG at a concentration of 0.1 mM to induce overnight, and harvesting the bacteria. After adding sterile glycerol with a final concentration of 25%, the strain was stored in a -80°C low-temperature refrigerator for future use.

Embodiment 3

[0084] Example 3 Cultivation of D amino acid oxidase (DAAO) bacterial strain and preparation of crude enzyme solution and enzyme activity assay

[0085] Composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, sterilized at 121°C for 20min, ready for use.

[0086]After the engineering bacteria containing the DAAO enzyme gene were activated by streaking on the plate, a single colony was picked and inoculated into 5ml LB liquid medium containing 50μg / ml kanamycin, and cultured with shaking at 37°C for 12h. Transfer to 50ml of fresh LB liquid medium containing 50μg / ml kanamycin according to 2% inoculum amount, shake at 37°C until OD600 reaches about 0.8, add IPTG to its final concentration of 0.5mM, induce culture at 18°C 16h. After the cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected and stored in a -80°C u...

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Abstract

The invention discloses a preparation method of L-glufosinate ammonium. The preparation method comprises the steps: in the presence of L-amino acid dehydrogenase, an inorganic amino donor and a reductive coenzyme nicotinamide adenine dinucleotide, 2-oxo-4-(hydroxymethoxyphosphonyl)butyrate is subjected to ammoniation reaction, and then the prepared L-glufosinate ammonium is subjected to acidification reaction; the L-amino acid dehydrogenase is L-amino acid dehydrogenase derived from bacillus stearophilus and / or L-amino acid dehydrogenase derived from intermediate high temperature actinomycetes. The preparation method of L-glufosinate-ammonium is simple to operate, does not use expensive reductive coenzyme NADPH, adopts a coenzyme NADH, reduces the cost of reaction and is beneficial to industrialized production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of L-glufosinate-ammonium. Background technique [0002] Glufosinate-ammonium is a broad-spectrum contact herbicide developed by Hearst in the 1980s. At present, the three major herbicides in the world are glyphosate, glufosinate-ammonium, and paraquat. Compared with glyphosate and paraquat, glufosinate-ammonium has excellent herbicidal performance and less side effects. Glufosinate-ammonium has two optical isomers, namely D-glufosinate-ammonium and L-glufosinate-ammonium, but only L-glufosinate-ammonium has herbicidal activity, so the method of developing L-glufosinate-ammonium is important for improving atom economy , It is of great significance to reduce the cost of use and reduce the pressure on the environment. [0003] At present, the methods for preparing L-glufosinate-ammonium mainly include chiral resolution, chemical synthesis and biocatal...

Claims

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Application Information

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IPC IPC(8): C12P13/04
CPCC12P13/04
Inventor 田振华程占冰丁少南徐文选黄瑶
Owner SHANGHAI QIZHOU ZIYUE BIOTECHNOLOGY CO LTD
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