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Biosynthesis preparation method of L-glufosinate

A biosynthesis and bio-enzyme technology, applied in the direction of fermentation, can solve the problems of high risk, soil compaction, and demanding processing and manufacturing processes, and achieve the effects of high substrate utilization, simple process flow, and good catalytic effect

Inactive Publication Date: 2018-07-31
武汉茵茂特生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The synthesis process is at 500-600°C. If the control is unstable, it is easy to produce yellow phosphorus and phosphine, which are very easy to be natural, which is very dangerous.
In addition, the material is highly corrosive, and there are strict requirements on the material selection and manufacturing process of the reaction equipment. The current domestic processing and manufacturing level is difficult to meet the production requirements.
[0005] These methods are all methods for preparing glufosinate-ammonium, and glufosinate-ammonium is L / D mixed type, wherein the main role is L-type; L-glufosinate-ammonium can be degraded by microorganisms in soil, and D-type glufosinate-ammonium Difficult to degrade, can eventually lead to soil compaction

Method used

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  • Biosynthesis preparation method of L-glufosinate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Preparation of nitrile hydratase

[0037]Recombinant nitrile hydratase genetically engineered bacteria, the specific preparation method is: select the gene sequence of nitrile hydratase derived from Rhodococcus. Synthetic), cloned into the Nde I and XhoI restriction sites of the expression vector pET28a, and transformed the host strain E.coli BL21 (DE3) competent cells; after picking the positive transformant and identifying it by sequencing, the recombinant expression vector was obtained; the recombinant expression The vector is transferred into E. coli BL21 (DE3) strain, and the recombinant nitrile hydratase gene engineering bacteria capable of inducing the expression of the recombinant nitrile hydratase is obtained.

[0038] Inoculate the recombinant nitrile hydratase genetically engineered bacteria into LB medium containing kanamycin, and culture overnight at 37°C to obtain seed culture solution; inoculate the seed culture solution into TB medium containing kanam...

Embodiment 2

[0052] The biosynthetic preparation method of L-glufosinate-ammonium comprises the steps:

[0053] Step 1: The reaction was carried out in a 1L shake flask, with 30g of 2-amino-4-(ethoxymethylphosphono)-butyronitrile as the substrate, and 300mL of citric acid-sodium citrate buffer solution as the solvent for resuspension 3g whole cells of nitrile hydratase genetic engineering bacteria derived from Rhodococcus.rhodochrous, 3g whole cells of amide racemase genetic engineering bacteria derived from Achromobacter obae, 3g whole cells of L-amide hydrolase genetic engineering bacteria derived from Brevundimonas diminuta Cells, put pyridoxal phosphate into the shake flask, the input concentration of pyridoxal phosphate is 10mM, then add CoCl 2 , CoCl 2 The input concentration is 1mM, the pH value of the transformation system is controlled to be 6.5, and the temperature of the transformation system is controlled to be 35°C; the transformation reaction is carried out in a shaking tabl...

Embodiment 3

[0058] The biosynthetic preparation method of L-glufosinate-ammonium comprises the steps:

[0059] Step 1: The reaction was carried out in a 500mL shake flask, with 20g of 2-amino-4-(ethoxymethylphosphono)-butyronitrile as a substrate, and 200mL of phosphate buffer solution resuspended 2g of Rhodococcus. The whole cell of rhodochrous nitrile hydratase genetically engineered bacteria, the pH value of the transformation system is controlled to be 8, and the temperature of the transformation system is controlled to be 40°C; the transformation reaction is carried out in a shaking table, and the speed of the shaking table is controlled at 180r / min. The product was completely consumed, and purified to obtain 2-amino-4-(ethoxymethylphosphono)-butyramide. MS(ESI): m / z 209.11[M+H] + The reaction formula is as follows:

[0060]

[0061] Step 2: The reaction was carried out in a 500 mL shake flask, with 10 g of purified 2-amino-4-(ethoxymethylphosphono)-butyramide as the substrate, ...

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Abstract

The invention discloses a biosynthesis preparation method of L-glufosinate, and the method comprises the following steps: Step 1, constructing a reaction system by 2-amino-4-(Roxymethylphosphono)butanamide as a substrate and a solvent, adding a catalyst to the reaction system for bioconversion reaction to obtain a conversion liquid containing L-2-amino-4-(Rmethylphosphono)-butyric acid; and Step 2, removing a protecting group of the L-2-amino-4-(Rmethylphosphono)-butyric acid to obtain L-2-amino-4-(hydroxymethylphosphono)-butyric acid, namely the L-glufosinate. The biosynthesis preparation method is simple in process, has no special requirements for equipment, and is suitable for industrial production.

Description

technical field [0001] The invention relates to a preparation method of pesticides, in particular to a biosynthetic preparation method of L-glufosinate-ammonium. Background technique [0002] Glufosinate-ammonium, the chemical name is 4-[hydroxy(methyl)phosphono]-DL-homoalanine, which was developed and produced by Hearst (now Bayer, Germany) in the 1980s, and belongs to phosphonic acid Herbicides are glutamine synthesis inhibitors, non-selective contact herbicides, which were registered for use as herbicides in 1984. In 2004, my country only had the registration of glufosinate-ammonium technical, and in 2005, my country had product registration. [0003] Since glyphosate was widely used, glyphosate-resistant weeds have been increasing, and the harm has gradually increased. Paraquat is a strong herbicide that kills weeds and is highly toxic to humans and animals. On July 1, 2014, my country revoked the registration and production license of paraquat water solution, and sto...

Claims

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Application Information

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IPC IPC(8): C12P13/04
CPCC12P13/04
Inventor 曾超胡磊陈建华
Owner 武汉茵茂特生物技术有限公司
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