Method for producing L-glufosinate-ammonium by using transaminase and ethylene-forming enzyme

An ethylene synthase and transaminase technology is applied in a new process for producing L-glufosinate-ammonium, in the field of improving the production efficiency of optically pure L-glufosinate-ammonium, and can solve the problems of low process efficiency, long time consumption, large alanine consumption and the like , to achieve the effect of simplifying the refining process, simple reaction process and reducing the cost of raw materials

Active Publication Date: 2017-09-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] In the patent (publication number CN105603015A) that Yang Lirong etc. applied for in China, proposed a new technology (as Figure 4 ), they use 2-carbonyl-4-(hydroxymethylphosphono)butanoic acid (PPO for short) as the raw material, and use L-alanine or the salt formed by

Method used

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  • Method for producing L-glufosinate-ammonium by using transaminase and ethylene-forming enzyme
  • Method for producing L-glufosinate-ammonium by using transaminase and ethylene-forming enzyme
  • Method for producing L-glufosinate-ammonium by using transaminase and ethylene-forming enzyme

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1.1 Construction of genetically engineered bacteria expressing transaminase

[0051] Transaminase genes were cloned from the genomes of Bacillus subtilis 168, Bacillus magaterium YYBM1, Escherichia coli K12W3110, Bacillus stearothermophilus and Streptomyces sp. DSM 40736 respectively, according to the corresponding genomic DNA sequences ( GenBank accession numbers are CP010052.1, CP001982.1, CP012868.1, AE016877.1 and EFL35314.1) to design corresponding PCR upstream primers and downstream primers.

[0052] Primers for transaminases from Bacillus subtilis:

[0053] BS-F sequence: 5'-CCC GAGCTC ATGAGTCAAAACAACAGCAAGCATCA-3'(SacI)

[0054] BS-R sequence: 5'-CCC AAGCTT TTAAGCTCGCAGGCCCGCCT-3' (HindIII)

[0055] Primers for transaminases from Bacillus magaterium:

[0056] BM-F sequence: 5'-CGC GGATCC ATGAGTCAAACTTTTAGCAA-3' (BamHI)

[0057] BM-R sequence: 5'-CCC AAGCTT TTACACTTCAACCGTTTGCT-3' (HindIII)

[0058] Primers for transaminases derived from E.coli:

[...

Embodiment 2

[0099] 2.1 Culture of microorganisms

[0100] 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.

[0101] The genetically engineered bacteria E.coli BL21(DE3) containing transaminase gene and ethylene synthase were inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37°C for 12 hours. Transfer to 500mL fresh LB liquid medium also containing 50μg / mL Kan, shake culture at 37°C until OD 600 When it reaches about 0.8, add IPTG to its concentration of 0.1mM, and induce culture at 18°C ​​for 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 -70°C ultra-low temperature refrigerator until use.

[0102] 2.2 Preparation of pure enzyme

[0103] The bacterial cells collected after the cul...

Embodiment 3

[0111] Embodiment 3 contrasts the reaction effect of transaminase and ethylene synthetase coupling

[0112] 3.1 Qualitative reaction of whole cells

[0113] Configure substrate solution: quantitatively weigh PPO, L-glutamic acid, L-arginine, FeSO 4 And pyridoxal phosphate into the beaker, adjust pH=8.5 with 30% ammonia water, add in the volumetric flask, and use deionized water to make the final concentration of PPO 20mM, the final concentration of L-glutamic acid is 20mM, L-Arg concentration is 5mM, Fe 2+ Concentrations were 2 mM and pyridoxal phosphate 1 mM.

[0114] After the cultivation, the bacteria were collected by centrifugation, the supernatant was discarded, and the activity of the bacteria was measured after washing the bacteria. Mix transaminase (derived from Escherichia coli, GenBank accession number CP012868.1) and ethylene synthase (derived from Pseudomonas syringae, GenBank accession number D13182.1) according to the ratio of activity 1:1, For transaminase,...

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Abstract

The invention discloses a method for producing L-glufosinate-ammonium by using transaminase and an ethylene-forming enzyme. According to the method, 2-carbonyl-4-(hydroxyl methyl phosphoryl) butyric acid or salt thereof is taken as a raw material, and the L-glufosinate-ammonium is obtained through a catalytic reaction of the transaminase and the ethylene-forming enzyme under the condition of taking glutamic acid or salt thereof as an amidogen donor. According to the method, the 2-carbonyl-4-(hydroxyl methyl phosphoryl) butyric acid or the salt thereof is taken as a substrate, and the L-glufosinate-ammonium is obtained through the co-catalysis of the transaminase and the ethylene-forming enzyme under the condition of taking the glutamic acid or the salt thereof as the amidogen donor, so that by-product alpha-dibasic ketonic acid after a transamination reaction is completely converted into carbon dioxide and ethylene through catalysis of the ethylene-forming enzyme, under the condition of guaranteeing few using amount of the raw materials, the conversion rate of the raw materials is obviously improved, the cost of the raw materials is reduced, the subsequent purification technology is simplified, and the product total recovery is improved.

Description

technical field [0001] The invention belongs to the field of biochemical technology, and in particular relates to a new process for producing L-glufosinate-ammonium; specifically, it is a new method for improving the production efficiency of optically pure L-glufosinate-ammonium by utilizing two biological enzymes. Background technique [0002] Glufosinate-ammonium, English name: Phosphinothricin (referred to as PPT), generally refers to the compound 2-amino-4-[hydroxy (methyl) phosphono]-butyric acid (PPO) or its salt formed with a basic compound (such as Formula (1) shown). Glufosinate-ammonium is a broad-spectrum contact herbicide developed by Hearst in the 1980s (later owned by Bayer). It belongs to the phosphonic acid herbicide and is an inhibitor of glutamine synthesis. It is transferred in the leaves, but cannot Transferred elsewhere, the inhibition of glutamine synthesis leads to the accumulation of ammonium ions and the disintegration of chloroplasts, thereby inhib...

Claims

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Application Information

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IPC IPC(8): C12P13/04
CPCC12P13/04
Inventor 杨立荣蒙丽钧周海胜刘亚运尹新坚徐刚吴坚平
Owner ZHEJIANG UNIV
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