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Enzyme combination for producing L-phosphinothricin, and production method of L-phosphinothricin

A production method, glufosinate-ammonium technology, is applied in the field of enzyme engineering to achieve the effects of reducing production costs, increasing product concentration and production intensity, and being extremely easy to remove

Inactive Publication Date: 2020-05-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the problem of low concentration of synthesized L-glufosinate-ammonium and difficult separation of impurities in the prior art, the present invention provides a complete L-glufosinate-ammonium production process, providing multiple coenzyme regeneration substrates and coenzyme regeneration enzymes in L-glufosinate The application in the synthesis of ammonium phosphine solves the problem of separating the by-products of the coenzyme regeneration system from the product L-glufosinate-ammonium and improves the production efficiency of L-glufosinate-ammonium

Method used

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  • Enzyme combination for producing L-phosphinothricin, and production method of L-phosphinothricin
  • Enzyme combination for producing L-phosphinothricin, and production method of L-phosphinothricin
  • Enzyme combination for producing L-phosphinothricin, and production method of L-phosphinothricin

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: strain construction and enzyme activity assay

[0034] 1. Strain construction

[0035] (1) Construction of genetically engineered bacteria expressing glutamate dehydrogenase

[0036] Glutamate dehydrogenase with higher catalytic activity is selected as the catalyst for preparing L-glufosinate-ammonium by the method of the present invention, and its amino acid sequence is shown in SEQ ID NO.1. The base sequence of this glutamate dehydrogenase (the glutamate dehydrogenase mutant strain (PpGluDH-T121N / L123Y) in the published patent CN 110184246A) is cloned on the relevant expression plasmid, and the expression plasmid includes: pRSFDuet, pET One of -24a(+), pET-28a(+) and pET-30a(+), the insertion sites are BamHI and HindIII; the recombinant plasmid is transformed into the expression host Escherichia coli E.coli after sequencing and verification In BL21(DE3), construct corresponding genetically engineered bacteria expressing glutamate dehydrogenase, such as E....

Embodiment 2

[0048] Embodiment 2: Adopt genetically engineered bacteria E.coli BL21(DE3) / pRSFDuet-GluDH-ADH to produce L-glufosinate-ammonium

[0049] (1) Strain activation: use an inoculation loop to take the genetically engineered bacteria E.coli BL21(DE3) / pRSFDuet-GluDH co-expressing glutamic acid dehydrogenase and alcohol dehydrogenase preserved in the -80°C glycerol tube in the strain tube - ADH strains are marked on the surface of LB-Kan-agar solid medium (plate or eggplant bottle), and the plate is inverted and placed in a constant temperature incubator at 37°C for 12 hours;

[0050] (2) Seed cultivation: re-inoculate the activated strains into 40 mL of primary seed medium, culture with shaking at 37°C for 12 hours to obtain primary seed liquid; then, transfer the primary seed liquid into 400 mL of secondary seed culture Medium, cultured with shaking at 35°C for 4 hours to obtain secondary seed liquid;

[0051] Among them, the primary seed medium is LB medium, 10g / L peptone, 5g / L y...

Embodiment 3

[0057] Embodiment 3: Adopt genetically engineered bacteria E.coli BL21(DE3) / pET-24a(+)-GluDH-ADH to produce L-glufosinate-ammonium

[0058] (1) The fermentation of the engineered bacteria and the acquisition of the sludge were the same as in Example 2. After fermentation, get OD 600 =296, the enzyme activity of glutamate dehydrogenase was measured as 1269U / mL in the fermentation broth, and the activity of alcohol dehydrogenase was 947U / mL.

[0059] (2) Inject 2.5L of pure water into a 10.0L reactor, add 1.5kg of bacteria slime, add water to 4.0L, start stirring, adjust the pH to 7.5 with ammonia water, and control the reaction temperature to 40°C with circulating cooling water. 2.0kg of solid PPO powder with a purity of 95% and 500mL of isopropanol were configured into 5.0L of substrate feed solution, which was continuously added to the reaction. Join NADP + Use 0.5 g to start the reaction, use ammonia water to control the pH of the reaction solution to 7.5±0.1, take sample...

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Abstract

The invention discloses an enzyme combination for producing L-phosphinothricin. The enzyme combination comprises glutamate dehydrogenase and a coenzyme regenerating enzyme, wherein the coenzyme regenerating enzyme is alcohol dehydrogenase, formate dehydrogenase and phosphite dehydrogenase. The invention also discloses a production method of L-phosphinothricin, 4-(methylhydroxyphosphinyl)-2-oxobutyric acid is used as a raw material, NH<4><+>, a coenzyme NADP<+> / NADP, and a coenzyme regeneration substrate are added, and then the enzyme combination is used for catalysis, wherein the glutamate dehydrogenase is used to catalyze a reaction of 4-(methylhydroxyphosphinyl)-2-oxobutyric acid to obtain L-phosphinothricin, and the coenzyme regenerating enzyme is used to reduce NADP<+> to NADP. According to the enzyme combination and the production method of L-phosphinothricin provided by the invention, by-products produced are very easy to remove, a post-treatment process of the product is simplified, the total yield of the product is greater than 95%, and the production cost of L-phosphinothricin is reduced, so that the method is a green, environment-friendly, and low-carbon process route, and is suitable for large-scale industrial production applications.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to an enzyme combination for producing L-glufosinate-ammonium and a production method of 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. The long-term and large-scale use of glyphosate, first, causes a large number of weeds to develop resistance, making glyphosate tend to be ineffective; second, it causes serious soil erosion and soil compaction; paraquat has been listed in the "Rotterdam" due to its high toxicity. More and more countries around the world have banned or restricted their use. Glufosinate-ammonium is the best substitute for...

Claims

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Application Information

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IPC IPC(8): C12P13/04C12N9/06C12N9/04C12N9/02
CPCC12N9/0004C12N9/0006C12N9/0008C12N9/0016C12P13/04C12Y101/01C12Y102/01002C12Y104/01002C12Y120/01001
Inventor 杨立荣周海胜尹新坚吴坚平张红玉
Owner ZHEJIANG UNIV
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