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Method for preparing L-glufosinate ammonium by using biological multi-enzyme coupling method

A technology of glufosinate-ammonium and recombinant microorganisms, which is applied in the biological field, can solve the problems of difficult separation and purification of products, difficulty in increasing the dosage of substrates, etc., and achieve the effects of high product yield, low production cost, and simple separation and purification process

Active Publication Date: 2021-02-26
YONGNONG BIOSCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this process realizes the dynamic kinetic resolution of glufosinate-ammonium racemate, there are obvious defects in this process: first, it is difficult to increase the substrate dosage (only 300mM D, L-glufosinate); The reaction of PPO to L-glufosinate-ammonium guided by the above-mentioned method can only achieve a conversion rate of 90% due to the influence of reversible reactions; the third is that because the amine donor is L-glutamic acid, there is still a large amount of residue after the reaction, and the product is separated and purified difficulty

Method used

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  • Method for preparing L-glufosinate ammonium by using biological multi-enzyme coupling method
  • Method for preparing L-glufosinate ammonium by using biological multi-enzyme coupling method
  • Method for preparing L-glufosinate ammonium by using biological multi-enzyme coupling method

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1: the cultivation of engineering bacterium thalline

[0049] The engineering bacteria E.coli BL21(DE3) / pCDFduet-1-APH1, E.coli BL21(DE3) / pCDFduet-1-EN5, E.coli BL21(DE3) / pCDFduet-1-APH1-EN5 were streaked on the plate After activation, a single colony was picked and inoculated into 10 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37°C for 10 h. Transfer 2% of the inoculum into 50 mL of LB liquid medium also containing 50 μg / mL kanamycin, culture with shaking at 37 °C until the OD600 reaches about 0.8, add IPTG with a final concentration of 0.5 mM, and shake at 28 °C Cultivate for 12h. After the cultivation, the culture solution was centrifuged at 8000rpm for 10min, the supernatant was discarded, the bacteria were collected, and stored in a -80°C ultra-low temperature refrigerator until use.

Embodiment 2

[0050] Embodiment 2: Enzyme sequence synthesis and bacterial strain construction

[0051] After the sequence (R)-transaminase (APH1) annotated from Pseudarthrobacter chlorophenolicus (the amino acid sequence is shown in SEQ ID NO.1, the nucleotide sequence is shown in SEQ ID NO.2) after the whole gene synthesis, insert The expression plasmid pET-28a(+) was used to obtain pET28a-APH1. After sequencing verification, pET28a-APH1 was transferred into the expression host E. coli BL21 (DE3) for subsequent expression of the recombinase.

[0052] The sequence derived from Corynebacterium vitaeruminis DSM 20294 annotated as (S)-transaminase (EN5) (the amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4) After the whole gene synthesis, Insert the expression plasmid pET-28a(+) to obtain pET28a-EN5. After sequencing verification, pET28a-EN3 was transferred into the expression host E. coli BL21 (DE3) for the subsequent expression of the recomb...

Embodiment 3

[0053] Embodiment 3: Contain the construction of the co-expression strain of (R)-transaminase and (S)-transaminase system:

[0054] The APH1 gene used in Example 2 was connected to the multi-cloning site vector pCDFduet-1 by a one-step cloning kit, the restriction sites were HindIII and XhoI, and the one-step cloning primers were C1-F and C1-R (Table 1) , and the plasmid pCDFduet-1-APH1 was constructed. On the basis of the pCDFduet-1-APH1 plasmid, the EN5 used in Example 2 was connected to the second cloning site of the multi-cloning site vector pCDFduet-1 through a one-step cloning kit, and the restriction sites were NdeI and XhoI, the one-step cloning primers are C2-F and C2-R, the plasmid pCDFduet-1-APH1-EN5 is constructed, and the co-expression strain E.coli BL21(DE3) / pCDFduet-1-APH1-EN5 is constructed. APH1-EN5 constructs such as figure 2 shown.

[0055] Table 1: Cloning primer sequences

[0056] Primer sequence C1-F CCCAAGCTTAAGGAGATATACATATGACCTC...

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Abstract

The invention relates to a method for preparing L-glufosinate ammonium by using a biological multi-enzyme coupling method. The method comprises converting D, L-glufosinate ammonium into L-glufosinateammonium in a presence of (R)-transaminase, (S)-transaminase, an amino receptor and an amino donor. The method can realize a high-efficiency resolution of high-concentration D, L glufosinate ammoniumto prepare the L-glufosinate ammonium.

Description

technical field [0001] The application relates to the field of biotechnology, in particular to a method for preparing L-glufosinate-ammonium by using a biological multi-enzyme coupling method. Background technique [0002] Glufosinate-ammonium (also known as bialaphos, glufosinate, trade names include Baoshida, Baisuton, etc., English name is phosphinothricin (abbreviated as PPT), chemical name is 2-amino-4-[hydroxyl (methyl ) Phosphono] butyric acid), the world's second largest genetically modified crops resistant to herbicides, developed and produced by Hearst. Glufosinate-ammonium is a phosphonic acid herbicide, a glutamine synthetase inhibitor, and a non-selective (killing) contact herbicide. At present, the world's three major herbicides are paraquat, glyphosate, and glufosinate-ammonium. In terms of market use, glyphosate is the champion, but due to its long-term use, a large number of weeds have developed resistance, and glyphosate tends to be ineffective; paraquat ...

Claims

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Application Information

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IPC IPC(8): C12P13/04
CPCC12P13/04C12N9/1096C12N15/70C12Y206/01
Inventor 王华磊魏东芝吴承骏刘清海罗中华张长雷
Owner YONGNONG BIOSCI
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