Glufosinate-ammonium dehydrogenase mutant, engineering bacterium, immobilized cell and application

A technology of immobilized cells and glufosinate-ammonium, applied in applications, genetic engineering, immobilized on or in inorganic carriers, etc., can solve the problems of high cost, low reduction activity and low catalytic efficiency of L-glufosinate-ammonium , to achieve the effect of improving thermal stability and operational stability, realizing repeated use and high stability

Pending Publication Date: 2022-04-15
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0013] The purpose of the present invention is to provide a kind of glufosinate-ammonium dehydrogenase for the problem of low activity and poor stability of 2-carbonyl-4-(hydroxymethylphosphono)-butyric acid asymmetric amination reduction of existing glufosinate-ammonium dehydrogenase NADH-dependent glufosinate-ammonium dehydrogenase mutants, engineering bacteria, immobilized cells and applications for L-glufosinate-ammonium chiral biosynthesis solve the problem of high cost of L-glufosinate-ammonium preparation by asymmetric amination reduction. The problem of low catalytic efficiency

Method used

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  • Glufosinate-ammonium dehydrogenase mutant, engineering bacterium, immobilized cell and application
  • Glufosinate-ammonium dehydrogenase mutant, engineering bacterium, immobilized cell and application
  • Glufosinate-ammonium dehydrogenase mutant, engineering bacterium, immobilized cell and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Construction and screening of glufosinate-ammonium dehydrogenase mutant library

[0038] 1. Parent engineering bacteria of glufosinate-ammonium dehydrogenase

[0039] The nucleotide sequence of wild-type glufosinate-ammonium dehydrogenase KmGDH (NCBI accession number WP_010290083.1) from Kurthia massiliensis was codon-optimized and synthesized by Hangzhou Qingke Biotechnology Co., Ltd. , the obtained KmGDH gene (the nucleotide sequence is shown in SEQ ID No.1, the amino acid sequence of the encoded protein is shown in SEQ ID No.2), cloned on the NcoI of MCS1 (multiple cloning site 1) of the plasmid pETDuet , construct the recombinant expression vector pETDuet-KmGDH, retain the His-Tag gene of the plasmid itself, transform it into Escherichia coli E.coli BL21(DE3), and send it to Hangzhou Qingke Biotechnology Co., Ltd. to synthesize the glufosinate-ammonium dehydrogenase female engineering bacterium E .coli BL21(DE3) / pETDuet-KmGDH.

[0040] SEQ ID NO.1

[0...

Embodiment 2

[0059] Embodiment 2: Induced expression of glufosinate-ammonium dehydrogenase mutant engineering bacteria

[0060] The parent engineering bacteria of glufosinate-ammonium dehydrogenase in Example 1, the parent of glufosinate-ammonium dehydrogenase and formate dehydrogenase set out to co-express strains and the co-expression of glufosinate-ammonium dehydrogenase mutants with formate dehydrogenase The strains were inoculated into LB liquid medium containing a final concentration of 50 μg / mL ampicillin, cultured at 37°C for 8 hours, and inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL ampicillin at an inoculum volume concentration of 2%. In the culture medium, culture at 37°C and 180 rpm for 2 hours, then add 0.1mM IPTG to the culture medium at a final concentration of 0.1mM, cultivate at 18°C ​​for 14 hours, centrifuge at 4°C and 8000 rpm for 10 minutes to obtain the corresponding wet bacteria. The composition of LB liquid medium: 10g / L pepton...

Embodiment 3

[0063] Example 3: Mutant library screening

[0064]The glufosinate-ammonium dehydrogenase parent engineering bacteria prepared by the method in Example 2, the glufosinate-ammonium dehydrogenase parent and formate dehydrogenase co-express wet bacteria or the glufosinate-ammonium dehydrogenase mutant and formate dehydrogenation Enzyme co-expression wet bacteria as a catalyst, 2-carbonyl-4-(hydroxymethylphosphono)-butyric acid as a substrate, ammonium formate as a coenzyme regeneration substrate, and a small amount of NAD added exogenously + , with pH 7.4, 100mM phosphate buffer as the reaction medium to form a 1mL reaction system, the catalyst dosage is based on the weight of wet bacteria, the final concentration is 10g / L, the final substrate concentration is 100mM, the final concentration of ammonium formate is 125mM, NAD + The final concentration was 1 mM, and the reaction was carried out at 35°C and 600 rpm for 5 min. Take 50 μL of the reaction solution, add 5 μL hydrochlori...

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Abstract

The invention discloses a glufosinate-ammonium dehydrogenase mutant, an engineering bacterium, an immobilized cell and application. The mutant is obtained by carrying out single mutation or multiple mutations on the 145th site, the 384th site, the 348th site, the 292th site, the 202 site, the 70th site and the 78th site of a glufosinate-ammonium dehydrogenase amino acid sequence shown in SEQ ID No.2. According to the immobilized cell of the co-expression engineering bacterium of the glufosinate-ammonium dehydrogenase mutant and the formate dehydrogenase, the L-glufosinate-ammonium is obtained through catalytic reduction by using a 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid substrate, the asymmetric synthesis of the L-glufosinate-ammonium is realized, an expensive chemical resolution reagent is not needed, the thermal stability and the operation stability of the L-glufosinate-ammonium are improved, and the repeated use is realized. The enzyme activity of the immobilized cells is 162.5 U / g, the enzyme activity recovery rate is 78.1%, and the immobilized cells can be recovered through filtration and can be repeatedly used for more than 20 batches to keep 100% conversion rate, so that the production cost is greatly reduced.

Description

(1) Technical field [0001] The invention relates to an NADH-dependent glufosinate-ammonium dehydrogenase mutant, engineering bacteria, immobilized cells and applications thereof. (2) Background technology [0002] Glufosinate-ammonium (phosphinothricin, also known as glufosinate, referred to as PPT), the chemical name is 2-amino-4-[hydroxy (methyl) phosphono]-butyric acid, which is the second most resistant herbicide in genetically modified crops in the world. Developed and produced by Sturt (now owned by Bayer after several mergers), it is also known as glufosinate ammonium salt, Basta, Buster, etc. It is a phosphonic acid herbicide, and the non-selective (killing) contact herbicide is glutamine Synthetic enzyme inhibitors. [0003] Glufosinate-ammonium has two optical isomers, L-glufosinate-ammonium and D-glufosinate-ammonium. But only the L-type has physiological activity, and is easy to decompose in the soil. It is less toxic to humans and animals, has a wide weeding s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N11/14C12N1/21C12P13/04C12R1/19
Inventor 薛亚平张嘉敏程峰郑裕国
Owner ZHEJIANG UNIV OF TECH
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