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Application of omega-transaminase and method for preparing L-glufosinate-ammonium through deracemization by biological enzyme process

A transaminase, glufosinate-ammonium technology, applied in the biological field, can solve the problems of increased process cost, inactivation of enzyme protein, lack of industrialization potential, etc., and achieves the effect of improving utilization rate and low overall process cost

Active Publication Date: 2021-03-26
ZHEJIANG UNIV HANGZHOU GLOBAL SCI & TECH INNOVATION CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, D-amino acid oxidase will produce hydrogen peroxide during the reaction process, which will easily lead to the inactivation of the enzyme protein; although hydrogen peroxide can be removed by adding additional catalase, the disadvantages of hydrogen peroxide cannot be completely eliminated. impact, and the use of catalase increases process cost
[0007] U.S. patent US20180030487 adopts a two-step method to deracemize D, L-glufosinate-ammonium to generate L-glufosinate-ammonium, first use D-amino acid oxidase or D-amino acid dehydrogenase or chemical method to oxidize D-glufosinate-ammonium 2-carbonyl-4-(hydroxymethylphosphono)butanoic acid, and then reduce 2-carbonyl-4-(hydroxymethylphosphono)butanoic acid to L- Glufosinate-ammonium, but the types of D-amino acid dehydrogenase are rare, the enzyme activity is low, and an equivalent amount of expensive coenzyme NADPH needs to be consumed, so it does not have industrialization potential

Method used

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  • Application of omega-transaminase and method for preparing L-glufosinate-ammonium through deracemization by biological enzyme process
  • Application of omega-transaminase and method for preparing L-glufosinate-ammonium through deracemization by biological enzyme process
  • Application of omega-transaminase and method for preparing L-glufosinate-ammonium through deracemization by biological enzyme process

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Experimental program
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Effect test

Embodiment 1

[0042] The construction of embodiment 1 genetically engineered bacteria

[0043] 1. Construction and screening of ω-transaminase library

[0044] Through literature research, a known R-ω-transaminase was selected, and its gene sequence was obtained from NCBI; the above gene sequence was codon-optimized and sent to Qingke Bioengineering Co., Ltd. for whole gene synthesis, and recombined into pET- 28a(+) plasmid; after correct sequencing, it was introduced into the expression host Escherichia coli E.coil BL21(DE3), and recombinant bacteria were constructed for the subsequent expression of ω-transaminase.

[0045] With D, L-glufosinate-ammonium as the substrate, enzymes with catalytic activity to D-glufosinate-ammonium were screened by HPLC high performance liquid chromatography, and the results are shown in Table 1.

[0046] Table 1 Screening of ω-transaminase

[0047] Numbering source NCBI accession number Enzyme activity U / L TA1 Aspergillus terreus NIH...

Embodiment 2

[0095] 1. The cultivation of microorganisms

[0096] 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.

[0097] 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.

[0098] 2. Preparation of crude enzyme solution

[0099] The bacterial cells collected after the cultivation were washed wit...

Embodiment 3

[0106] Select the ω-transaminase derived from Bacillus.Sp YM-01 obtained in Example 1, the glutamic acid dehydrogenase derived from Pseudomonasputida KT2440, and the alcohol dehydrogenase derived from Clostridium beijerinckii, and obtain the crude enzyme liquid after culturing and breaking cells. The enzyme activities were 4.2U / L, 116.9U / L, and 1126.3U / L, respectively.

[0107] Use 0.1M disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 8.0) to prepare 100mM D, L-glufosinate-ammonium, 250mM isopropanol, 500mM ammonium sulfate, 20mM NADP + , 10mM PLP respectively take 2mL, 2mL, 1mL, 1mL, 2mL of the above solution, then add ω-transaminase 1mL, glutamate dehydrogenase and alcohol dehydrogenase 500μL each. The reaction flask was placed in a constant temperature water bath at 40° C. for shaking reaction, and the concentrations of L-glufosinate-ammonium and D-glufosinate-ammonium were detected by HPLC.

[0108] After 72 hours of reaction, the data are as follows: th...

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Abstract

The invention discloses application of omega-transaminase and a method for preparing L-glufosinate-ammonium through deracemization by a biological enzyme process. An amino acid sequence of the omega-transaminase is as shown in SEQ ID NO.1. The omega-transaminase has the activity of catalyzing D-glufosinate-ammonium to be converted into 4-(methylhydroxyphosphinyl)-2-oxobutyric acid, the D-glufosinate-ammonium and the L-glufosinate-ammonium serve as raw materials, and the L-glufosinate-ammonium is obtained through catalysis of an enzyme catalysis system. The enzyme catalysis system comprises theomega-transaminase used for converting the D-glufosinate-ammonium into the 4-(methylhydroxyphosphinyl)-2-oxobutyric acid, and glutamate dehydrogenase used for converting the 4-(methylhydroxyphosphinyl)-2-oxobutyric acid into the L-glufosinate-ammonium. According to the method, the D-glufosinate-ammonium and the L-glufosinate-ammonium can be directly used as the raw materials for deracemization, so that expensive resolution reagents are not needed; and an auxiliary raw material isopropanol can be converted into a byproduct isopropylamine, and the isopropylamine can be recycled and is an important pesticide chemical raw material, so that the process disclosed by the invention is lower in total cost and has industrial application potential.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an application of ω-transaminase and a method for preparing L-glufosinate-ammonium by biological enzymatic deracemization. Background technique [0002] Glufosinate (Glufosinate, also known as glufosinate phosphinothricin), chemical name: 2-amino-4 (hydroxymethylphosphono) butyric acid, is the first naturally occurring non-protein amino acid found in several Streptomyces, It was first chemically synthesized and named by the German Hearst Company. Glufosinate-ammonium is very similar to glutamic acid in molecular structure, so it can reversibly combine with the active site of glutamine synthetase to inhibit the biological activity of glutamine synthetase. Glutamine synthetase is an enzyme necessary for plant production of glutamine and ammonia detoxification. The use of glufosinate-ammonium in plants can reduce the level of glutamine and increase the content of ammonia in tissues, w...

Claims

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Application Information

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IPC IPC(8): C12P41/00C12P13/00C12P9/00
CPCC12P41/001C12P41/006C12P13/001C12P9/00
Inventor 杨立荣王子渊周海胜张红玉许金玲吴坚平
Owner ZHEJIANG UNIV HANGZHOU GLOBAL SCI & TECH INNOVATION CENT
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