Method for preparing L-phosphinothricin through deracemization and by biological enzyme method

A biological enzyme method and glufosinate-ammonium technology, applied in the biological field, can solve problems such as complex process, soil compaction, and no advantages, and achieve the effect of a low-carbon process route

Active Publication Date: 2017-12-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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
Chinese patent (CN103275896) uses racemic 2-amino-4 (hydroxymethylphosphoryl)-butyroc

Method used

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  • Method for preparing L-phosphinothricin through deracemization and by biological enzyme method
  • Method for preparing L-phosphinothricin through deracemization and by biological enzyme method
  • Method for preparing L-phosphinothricin through deracemization and by biological enzyme method

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Experimental program
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Embodiment 1

[0050] The construction of embodiment 1 genetically engineered bacteria

[0051] 1. Construction and screening of D-amino acid oxidase library and construction of genetically engineered bacteria expressing D-amino acid oxidase

[0052] D-Amino-acid oxidase gene from Rhodotorula gracilis (Rhodotorula gracilis) ATCC26217 by known NCBI accession number AAB51107.1 (Jorge Alonso, et al. D-Amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC26217 .Microbiology (1998), 144, 1905-1101), screen the gene sequence with higher similarity with RgDAAO from the NCBI database.

[0053] The gene sequence of the above D-amino acid oxidase, after codon optimization, was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for whole gene synthesis, and cloned into the recombinant expression plasmid pET-28a(+). After the recombinant plasmid was verified to be correct by sequencing, it was transferred into the expression host E. coli BL21 (DE3) for the subsequent expres...

Embodiment 2

[0108] 1. Culture of microorganisms

[0109] 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.

[0110] Genetically engineered bacteria E.coli BL21 (DE3) 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 until its concentration is 0.1-0.3mM, and induce culture at 18-28°C for about 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.

[0111] 2. Preparation of Crude Enzyme Solution

[0112] The bacterial cells collected after the cultivation were washed with 50 mM ...

Embodiment 3

[0121]D-amino acid oxidase derived from Neurospora crassa OR74A, glutamate dehydrogenase derived from Pseudomonas putida KT2440, glucose dehydrogenase derived from Bacillus megaterium DSM319 were selected, and reference example 1 was made for strain construction.

[0122] Collect the cells after microbial culture, obtain the crude enzyme liquid and measure the enzyme activity. For the specific method, refer to Example 2. The crude enzyme liquid activities of the three enzymes are 3.741U / L, 22.17U / L, and 1012.65U / L respectively.

[0123] With 0.1M NH 3 ·NH 4 Cl buffer solution (pH=8.0) was prepared with 100mM D,L-glufosinate-ammonium solution, and 0.1M NH 3 ·NH 4 Prepare 100mM glucose solution in Cl buffer solution (pH=8.0), take 150μL of 100mM D,L-glufosinate-ammonium solution and 180μL of 100mM glucose solution, add them to 2mL EP tube, then add D-amino acid oxidase crude enzyme solution 700 μL, glutamate dehydrogenase crude enzyme solution 165 μL, glucose dehydrogenase cr...

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Abstract

The invention discloses a method for preparing L-phosphinothricin through deracemization and by a biological enzyme method. According to the method, the L-phosphinothricin is obtained by taking D,L-phosphinothricin as a raw material and through catalysis by an enzyme catalysis system, and the enzyme catalysis system consists of D-amino acid oxidase, amino acid dehydrogenase and coenzyme regeneration systems. By the method, racemized D,L-phosphinothricin serves as the raw material, the D-phosphinothricin is oxidized into 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid by the D-amino acid oxidase, and the L-phosphinothricin does not take part in the reaction and is completely retained; but the 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid is catalytically reduced into the L-phosphinothricin by the amino acid dehydrogenase, so that in-situ deracemization of the D,L-phosphinothricin is realized, the obtained L-phosphinothricin does not contain other side products, the total yield of the products is more than or equal to 99 percent, and the optical purify can exceed 99 percent.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing L-glufosinate-ammonium by biological enzymatic deracemization; specifically, it relates to a biocatalytic method for splitting glufosinate-ammonium racemic mixture to prepare optically pure L-glufosinate. - the method of glufosinate-ammonium. 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 world's three major herbicides are glyphosate, paraquat and glufosinate-ammonium. In terms of mar...

Claims

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Application Information

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IPC IPC(8): C12P41/00C12P13/04
CPCC12P13/04C12P41/00Y02P20/582
Inventor 杨立荣周海胜居述云尹新坚徐刚吴坚平
Owner ZHEJIANG UNIV
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