Glutamate dehydrogenase mutant for producing L-glufosinate-ammonium and L-glufosinate-ammonium production method

A technology of glutamate dehydrogenase and production method, applied in the preparation of L-glufosinate-ammonium, in the field of glutamate dehydrogenase mutants, to achieve the effect of mild reaction conditions

Active Publication Date: 2021-07-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009]Aiming at the problem that glutamate dehydrogenase is NADPH or NADP+ coenzyme-dependent in the prior art

Method used

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  • Glutamate dehydrogenase mutant for producing L-glufosinate-ammonium and L-glufosinate-ammonium production method
  • Glutamate dehydrogenase mutant for producing L-glufosinate-ammonium and L-glufosinate-ammonium production method
  • Glutamate dehydrogenase mutant for producing L-glufosinate-ammonium and L-glufosinate-ammonium production method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: microbial cultivation and enzyme activity assay

[0046] 1. Microbial culture

[0047] (1) Composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then adjusted to volume, sterilized at 121°C for 20min, ready for use. LB liquid solid medium composition (plate): add agar powder 20g / L on the basis of LB liquid medium, sterilize at 121°C for 20min, cool to 50-60°C, add 50μg / mL kanamycin (Kan), Import into a Petri dish, cool and solidify before use.

[0048] The genetically engineered bacteria E.coli BL21(DE3) was 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 isopropylthiogalactoside (IPTG) to a concentration of 0.3mM, and induce culture at 28°C for 20h. After the cultivation, the culture solution ...

Embodiment 2

[0058] Embodiment 2: the construction of genetically engineered bacteria

[0059] 1. Construction of genetically engineered bacteria expressing the original glutamate dehydrogenase

[0060] By comparing the literature data, the original gene sequence of the glutamate dehydrogenase that has high catalytic activity to the natural substrate α-ketoglutarate (the product is L-glutamic acid) and can use NADH as a coenzyme is obtained, such as Table 1 shows.

[0061] By querying the gene database NCBI, the gene sequence of the above-mentioned original glutamate dehydrogenase was obtained, sent to Shenggong Bioengineering (Shanghai) Co., Ltd. for whole gene synthesis, and cloned into the recombinant expression plasmid pET-28a(+), inserted into The enzyme cutting sites are BamH I and Xho I. After the recombinant plasmid was verified to be correct by sequencing, it was transferred into the expression host Escherichia coli E.coli BL21 (DE3) for the expression of the original glutamate ...

Embodiment 3

[0081] Example 3: Preparation of L-glufosinate-ammonium using strains co-expressing E10 and E21

[0082] The genetic engineering that co-expresses E10 and E21 is selected, and the cells are collected after being cultured by microorganisms according to the operation in Example 1. The collected bacterial cells were ultrasonically disrupted and centrifuged to remove the precipitate to obtain a crude enzyme solution. Put a solution containing about 200mM PPO and about 250mM glucose in a magnetically stirred reactor, adjust the pH of the solution to 7.5 with 30% ammonia water, and then add NAD + 0.001mM; then add a crude enzyme solution equivalent to a wet cell concentration of 20g / L, control the reaction temperature in a water bath to 20°C, start stirring, use ammonia water to control pH = 7.5, and react for 24 hours. The formation concentration of phosphine was 197.9mM, and the ee value of the detected product glufosinate-ammonium was 99.1%.

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Abstract

The invention discloses a glutamate dehydrogenase mutant for producing L-glufosinate-ammonium, and belongs to the technical field of gene engineering, and the amino acid sequence after mutation is shown as SEQ ID NO.1-SEQ ID NO.11. The invention also discloses a production method of L-glufosinate-ammonium, which comprises the following steps: by taking 2-carbonyl-4-[hydroxyl (methyl) phosphonyl]-butyric acid as a raw material, adding NH4<+> and coenzyme NADH / NAD<+>, then catalyzing by using the glutamate dehydrogenase mutant, and reducing and aminating the 2-carbonyl-4-[hydroxyl (methyl) phosphonyl]-butyric acid into L-glufosinate-ammonium by using glutamate dehydrogenase. A large number of glutamate dehydrogenase mutant genes are developed through the method, L-glufosinate-ammonium can be prepared from glutamate dehydrogenase mutants by using nicotinamide adenine dinucleotide (NADH or NAD<+>) which is low in price as coenzyme, the substrate conversion rate is larger than or equal to 99%, the optical purity of the product exceeds 99%, reaction conditions are mild, and the method is a green, environment-friendly and low-carbon process route, and is suitable for large-scale industrial production and application.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a glutamate dehydrogenase mutant and its application in preparing L-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. [0003] At present, the world's three major herbicides are glyphosate, paraquat and glufosinate-ammonium. At present, 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 banne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/0016C12Y104/01002C12N15/70C12P13/04
Inventor 杨立荣周海胜陆利兵吴坚平张红玉
Owner ZHEJIANG UNIV
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