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A method for preparing l-glufosinate-ammonium by deracemization by biological enzymatic method, glufosinate-ammonium dehydrogenase mutant and application

A biological enzyme method, glufosinate-ammonium technology, applied in the biological field, can solve the problems of complex process, expensive chiral resolution reagents, low single resolution rate, etc., and achieve the effect of good catalytic efficiency

Active Publication Date: 2022-08-05
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 relatively complicated
The main advantage is that the raw materials are relatively easy to obtain and the catalyst activity is high, but the theoretical yield can only reach 50%, resulting in waste of raw materials

Method used

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  • A method for preparing l-glufosinate-ammonium by deracemization by biological enzymatic method, glufosinate-ammonium dehydrogenase mutant and application
  • A method for preparing l-glufosinate-ammonium by deracemization by biological enzymatic method, glufosinate-ammonium dehydrogenase mutant and application
  • A method for preparing l-glufosinate-ammonium by deracemization by biological enzymatic method, glufosinate-ammonium dehydrogenase mutant and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Cultivation of engineered bacteria

[0042] After the engineered bacteria were activated by streaking on a plate, a single colony was inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin, and incubated at 37°C with shaking for 10 h. Transfer to 50 mL of LB liquid medium containing 50 μg / mL kanamycin at 2% of the inoculum, and shake to OD at 37 °C. 600 When it reached about 0.8, IPTG with a final concentration of 0.5 mM was added, and the culture was shaken at 28 °C for 12 h. After the cultivation, the culture solution was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, the cells were collected, and stored in a -80°C ultra-low temperature refrigerator until use.

[0043] 2. Preparation of crude enzyme solution

[0044] The cells collected after the culture were washed twice with pH 8 phosphate buffer (50 mM pH=8 phosphate buffer), and then the cells were resuspended by adding pH=8 phosphate buffer (50 mM). Cells were disrupted ...

Embodiment 2

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

[0050] The unit of enzyme activity (U) is defined as: the amount of enzyme required to generate 1 μmol of L-glufosinate per minute at 35° C. and pH 7.4 is defined as one unit of enzyme activity, U. Specific enzyme activity is defined as the unit of activity per milligram of enzyme protein, U / mg.

[0051] Standard conditions for enzyme activity detection: 100 mM 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid, 10 mM NADPH, appropriate amount of enzyme solution, 30 °C, pH 7.4, 600 rpm for 10 minutes, sample treatment And carry out HPLC detection and analysis. The protein concentration was determined with a BCA protein assay kit (Nanjing Keygen Biotechnology Development Co., Ltd., Nanjing).

Embodiment 3

[0053] Construction and screening of glufosinate-ammonium dehydrogenase mutant library.

[0054] 1. Construction of genetically engineered bacteria

[0055] The gene sequence of glufosinate-ammonium dehydrogenase (GenBank No.: WP_101496154) derived from polyculture denitrifying sulfur bacteria (Thiopseudomonas denitrificans) was codon-optimized and sent to Sangon Bioengineering (Shanghai) Co., Ltd. for full gene synthesis, And cloned into the recombinant expression plasmid pETduet-1 to construct the plasmid pETduet-1-GluDH. After the recombinant plasmid was verified by sequencing, it was transferred into the expression host E. coli BL21 (DE3) for subsequent expression of recombinant glufosinate 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.

[0056] 2. Construction of a library of glufosinate dehydrogenase mutants

[0057] The first step is to construct a glufo...

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Abstract

The invention discloses a method for preparing L-glufosinate-ammonium by de-racemization by biological enzymatic method, a glufosinate-ammonium dehydrogenase mutant and application. A method for preparing L-glufosinate-ammonium by biological enzymatic de-racemization, using D, L-glufosinate-ammonium as raw materials, and obtaining L-glufosinate-ammonium catalyzed by a multi-enzyme catalytic system, wherein the enzyme catalytic system comprises a method for D-amino acid oxidase for catalyzing D-glufosinate in D,L-glufosinate to 2-carbonyl-4-[hydroxy(methyl)phosphono]butanoic acid, and for converting 2-carbonyl-4 -[Hydroxy(methyl)phosphono]butyric acid catalytic reduction to L-glufosinate-ammonium dehydrogenase mutant of glufosinate Dehydrogenase mutation, the mutation site is: V377S. The glufosinate-ammonium dehydrogenase mutant of the present invention has better catalytic efficiency, and when the racemic D,L-glufosinate-ammonium is used as the substrate for the catalytic reaction, the conversion rate is much higher than that of the wild-type enzyme, and the PPO yield is also greatly improved .

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing L-glufosinate-ammonium by de-racemization by biological enzymatic method, a glufosinate-ammonium dehydrogenase mutant and application. Background technique [0002] Glufosinate-ammonium, also known as glufosinate, English name Phosphinothricin (PPT), chemical name 2-amino-4-[hydroxy(methyl)phosphono]butyric acid, is the world's second largest genetically modified crop tolerant to herbicide It was developed and produced by Hearst Corporation (now owned by Bayer Corporation after several mergers). Glufosinate-ammonium is a phosphonic acid herbicide, a glutamine synthase inhibitor, a non-selective (killing) contact herbicide. [0003] As we all know, the market for biocide herbicides is huge. At present, the three major herbicides in the world are paraquat, glyphosate, and glufosinate. In terms of market use, glyphosate comes out on top, but due to its long-ter...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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