Method for preparing L-phosphinothricin through de-racemization by biological enzyme method, phosphinothricin dehydrogenase mutant and application

A biological enzyme method, glufosinate-ammonium technology, applied in the biological field, can solve the problems of complex process, waste of raw materials, expensive chiral resolution reagents, etc., and achieve the effect of good catalytic efficiency and yield improvement.

Active Publication Date: 2020-07-03
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process mainly has the following disadvantages: it needs to use expensive chiral resolution reagents, the theoretical yield can only reach 50%, the single resolution rate is low, and the process is relat

Method used

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  • Method for preparing L-phosphinothricin through de-racemization by biological enzyme method, phosphinothricin dehydrogenase mutant and application
  • Method for preparing L-phosphinothricin through de-racemization by biological enzyme method, phosphinothricin dehydrogenase mutant and application
  • Method for preparing L-phosphinothricin through de-racemization by biological enzyme method, phosphinothricin dehydrogenase mutant and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. The cultivation of engineered bacteria

[0042] After the engineering bacteria were activated by streaking on a plate, 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 to 50mL LB liquid medium also containing 50μg / mL kanamycin according to 2% inoculum amount, and culture with shaking at 37°C until OD 600 When it reaches about 0.8, add IPTG with a final concentration of 0.5mM, and culture with shaking at 28°C 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.

[0043] 2. Preparation of crude enzyme solution

[0044] Wash the collected cells with pH 8 phosphate buffer (50 mM pH=8 phosphate buffer) twice, then resuspend the cells in pH 8 phosphate buffer (50 mM) Cells were ultrasonically broken 30 ...

Embodiment 2

[0049] Determination of specific enzyme activity of glufosinate-ammonium dehydrogenase and its mutants.

[0050] Enzyme activity unit (U) is defined as: under the conditions of 35°C and pH 7.4, the amount of enzyme required to generate 1 μmol of L-glufosinate-ammonium per minute is defined as an enzyme activity unit, U. Specific enzyme activity is defined as the number of activity units per mg of enzyme protein, U / mg.

[0051] Standard conditions for enzyme activity detection: 100mM 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid, 10mM NADPH, appropriate amount of enzyme solution, 30°C, pH 7.4, 600 rpm for 10 minutes, sample treatment And carry out HPLC detection analysis.

[0052] Protein concentration was determined with BCA protein assay kit (Nanjing KGI Biotechnology Development Co., Ltd., Nanjing).

Embodiment 3

[0054] Construction and screening of glufosinate-ammonium dehydrogenase mutant library.

[0055] 1. Construction of genetically engineered bacteria

[0056] The gene sequence of glufosinate-ammonium dehydrogenase (GenBank No.: WP_101496154) derived from Thiopseudomonas denitrificans was codon-optimized and sent to Shenggong Bioengineering (Shanghai) Co., Ltd. for whole gene synthesis. and cloned into the recombinant expression plasmid pETduet-1 to construct the plasmid pETduet-1-GluDH. After the recombinant plasmid was verified to be correct by sequencing, it was transferred into the expression host E. coli BL21 (DE3) for subsequent expression of the recombinant glufosinate-ammonium dehydrogenase. The codon-optimized glufosinate-ammonium dehydrogenase gene sequence is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0057] 2. Construction of glufosinate-ammonium dehydrogenase mutant library

[0058] The first step is to construct a glufosinate-am...

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Abstract

The invention discloses a method for preparing L-phosphinothricin through de-racemization by a biological enzyme method, a phosphinothricin dehydrogenase mutant and an application. According to the method for preparing L-phosphinothricin through de-racemization by a biological enzyme method, D,L-phosphinothricin is used as a raw material, through catalysis with a multienzyme catalysis system, theL-phosphinothricin is obtained, wherein the enzyme catalysis system comprises D-amino acid oxidase which is used for catalyzing D-phosphinothricin in the D,L-phosphinothricin into 4-(hydroxymethylphosphinyl)-2-oxobutanoic, and the phosphinothricin dehydrogenase mutant which is used for performing catalytic reduction on the 4-(hydroxymethylphosphinyl)-2-oxobutanoic to obtain the L-phosphinothricin,the phosphinothricin dehydrogenase mutant is obtained through mutation of phosphinothricin dehydrogenase in wild mushrooms Thiopseudomonas denitrificans, and a mutation site is V377S. The phosphinothricin dehydrogenase mutant disclosed by the invention has better catalysis efficiency, when a catalytic reaction is performed by using racemation D,L-phosphinothricin as a substrate, the conversion rate is far higher than that of a catalytic reaction performed by using a wild type enzyme as a substrate, and the PPO yield is also substantially increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing L-glufosinate-ammonium by biological enzymatic deracemization, a mutant of glufosinate-ammonium dehydrogenase and its application. Background technique [0002] Glufosinate-ammonium, also known as glufosinate, the English name is Phosphinothricin (abbreviated as PPT), the chemical name is 2-amino-4-[hydroxy (methyl) phosphono] butyric acid, it is the second largest transgenic crop resistant to weeding in the world Agent, developed and produced by Hearst (now owned by Bayer after several mergers). Glufosinate-ammonium is a phosphonic acid herbicide, a glutamine synthetase inhibitor, and a non-selective (killing) contact herbicide. [0003] As we all know, the total herbicide market is huge. 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-...

Claims

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Application Information

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IPC IPC(8): C12P41/00C12P13/04C12N9/08C12N9/06C12N9/04C12N15/53
CPCC12P41/002C12P13/04C12N9/0024C12N9/0065C12N9/0002C12N9/0006C12Y104/03003C12Y111/01006C12Y102/01002C12Y101/9901C12Y101/01001C12N9/0008C12N9/0016C12Y104/01C12R2001/01
Inventor 薛亚平程峰曹成浩郑裕国
Owner ZHEJIANG UNIV OF TECH
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