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Production method of L-phosphinothricin

A technology for the regeneration of glufosinate-ammonium and coenzyme, applied in the biological field, can solve the problems of only 52% reaction conversion rate, little advantage compared with chemical synthesis, low optical purity of products, etc. Overall yield and strong stereoselectivity

Active Publication Date: 2017-07-04
ZHEJIANG UNIV
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  • Abstract
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Problems solved by technology

[0004] However, the first two herbicides have encountered great problems: the long-term and large-scale use of glyphosate, firstly, it causes a large number of weeds to develop resistance, making glyphosate tend to be ineffective; secondly, it causes serious soil erosion and soil compaction ; Due to its high toxicity, paraquat has been included in the Rotterdam Convention, and more and more countries around the world have banned or restricted its use. The Announcement No. 1745 jointly issued by the Ministry of Agriculture and other ministries and commissions of China has stated that paraquat water agent was released in 2014. Production ceased on July 1, 2016 and prohibited on July 1, 2016
This type of biocatalytic process still has obvious defects: glufosinate-ammonium derivatives need to be synthesized, the product is difficult to separate, the optical purity of the product is not high, the process is complicated, and the advantages are not large compared with chemical synthesis
But this process efficiency is low, when the concentration of substrate PPO is 552mmol / L, under the situation of almost completely consuming about 700mmol / L raw material L-aspartic acid, only generate the product L-PPT of 251.9mmol / L, and At the same time, about 234.5mmol / L of impurity alanine was generated, and the reaction conversion rate of raw material PPO was only 52%.

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0057] Embodiment 1 Genetic Engineering Bacteria Strain Construction

[0058] 1.1 Construction of genetically engineered bacteria expressing γ-aminobutyric acid / α-ketoglutarate aminotransferase

[0059] The transaminase genes were cloned from the genomes of Escherichia coli E.coli K12W3110, Bacillus subtilis 168 and Bacillus magaterium YYBM1 respectively. ) to design corresponding PCR upstream primers and downstream primers.

[0060] Primers for transaminases derived from E.coli:

[0061] EC-F sequence: 5'-CCG GAATTC ATGAGCAACAATGAATTCCATC-3' (EcoRI)

[0062] EC-R sequence: 5'-CCG CTCGAG TTAATCGCTCAGCGCATCC-3'(XholI)

[0063] Primers for transaminases from Bacillus subtilis:

[0064] BS-F sequence: 5'-CCC GAGCTC ATGAGTCAAAACAACAGCAAGCATCA-3'(SacI)

[0065] BS-R sequence: 5'-CCC AAGCTT TTAAGCTCGCAGGCCCGCCT-3' (HindIII)

[0066] Primers for transaminases from Bacillus magaterium:

[0067] BM-F sequence: 5'-CGC GGATCC ATGAGTCAAACTTTTAGCAA-3' (BamHI)

[0068] BM-...

Embodiment 2

[0105] 2.1 Culture of microorganisms

[0106] 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.

[0107] The genetically engineered bacteria E.coli BL21(DE3) containing the transaminase gene was 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 0.3mM, and induce culture at 28°C for 20h. 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.

[0108] 2.2 Preparation of crude enzyme solution

[0109] The bacterial cells collected after the cultivation were...

Embodiment 3

[0119] γ-aminobutyric acid / α-ketoglutarate aminotransferase derived from Escherichia coli K12W3110, glutamate dehydrogenase derived from Escherichia coli K12W3110, glucose dehydrogenase derived from Bacillus megaterium DSM319, Refer to Example 1 for strain construction. The cells were collected after microbial culture, and the enzyme activity of the wet cells was measured. The enzyme activity assay method refers to Example 2. The enzyme activities of the three strains of cells are 932.5U / g wet cells, 338.3U / g wet cells, and 993.3U / g respectively. wet cell

[0120] A solution containing 100 mM of PPO, 5 mM of glutamic acid, 1 mM of pyridoxal phosphate, 120 mM of glucose and 0.1 mM of NADH was placed in a warm bath at 37°C, and the pH of the solution was adjusted to 7.5 with 30% ammonia water. Add 5 g / L of engineering bacterial cells expressing γ-aminobutyric acid / α-ketoglutarate aminotransferase derived from Escherichia coli E.coli K12W3110, and engineering bacteria expressing...

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Abstract

The invention discloses a production method of L-phosphinothricin. According to the method, 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid is used as a substrate and is catalyzed by an enzyme catalytic system to obtain L-phosphinothricin, and the enzyme catalytic system is composed of gamma-aminobutyric acid / alpha-ketoglutarate transaminase, glutamate dehydrogenase and a coenzyme regeneration system. The method utilizes the advantages of high catalytic activity, strong stereoselectivity and the like of transaminase, and also solves the problem of incomplete transaminase catalytic reaction, so that the catalytic reaction can completely convert the substrate 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid into L-phosphinothricin, and the conversion rate can reach 100%; no by-product alpha-ketoglutarate is accumulated in the final product of the method, and the residual amount of other substances such as the raw material glutamic acid in the product after the end of the reaction is extremely low, so the subsequent refining process of L-phosphinothricin is greatly simplified, and the total yield of the product is increased.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a new method for producing optically pure L-glufosinate-ammonium by biological enzyme method. Background technique [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 broad-spectrum herbicide developed by Hoechst in the 1980s (later owned by Bayer). [0003] As we all know, the total herbicide market is huge, accounting for 60 to 70% of the entire herbicide market, especially in tropical and subtropical regions, where the usage is huge. At present, the three major herbicides in the world are glyphosate, paraquat and glufosinate-ammonium. In terms of market usage, glyphosate takes the lead, with an annual global usage of about 850,000 tons in recent years; paraquat follows closely, with a market share of about 100,000 tons in 2015...

Claims

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Application Information

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IPC IPC(8): C12P13/04
Inventor 杨立荣周海胜蒙丽钧尹新坚徐刚吴坚平
Owner ZHEJIANG UNIV
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