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Method for preparing L-glufosinate-ammonium by use of amino acid dehydrogenase

A technology of glutamate dehydrogenase and dehydrogenase, which is applied in the production of chiral pure L-glufosinate-ammonium and the field of optically pure L-glufosinate-ammonium, can solve complex process, expensive chiral resolution reagents, L-glufosinate-ammonium separation troubles and other problems, to achieve the effect of easy separation and purification, high conversion rate of raw materials, and simple process

Active Publication Date: 2017-07-25
ZHEJIANG UNIV
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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 yield is low, and the process is relatively complicated
But utilize transaminase to prepare L-glufosinate-ammonium and there are two big defects, and one is that raw material PPO can not be completely converted into L-PPT, and the conversion rate is the highest only 90%; More than 4 times the equivalent of L-glutamic acid is needed as the amino donor, and the excess glutamic acid brings great trouble to the separation of L-glufosinate-ammonium

Method used

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  • Method for preparing L-glufosinate-ammonium by use of amino acid dehydrogenase

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Experimental program
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Effect test

Embodiment 1

[0064] The construction of embodiment 1 amino acid dehydrogenase

[0065] 1. Activation of wild bacteria and genome extraction

[0066] All wild bacteria were activated and cultured with LB medium, the formula was: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then sterilized at 121°C for 20min, ready for use. The solid medium is LB medium with 2% agar added.

[0067] Streak the glycerol tube containing the wild bacteria onto a plate containing LB solid medium, and culture it statically at 30°C for 2-3 days. Pick colonies from the plates and transfer them into Erlenmeyer flasks containing 50 mL of LB medium, and culture them at 30°C and 200 rpm for 2 to 3 days. After obtaining the culture medium, the whole genome was extracted according to the operating instructions of the genome extraction kit. The obtained genome can be directly used for the amplification of the target gene or stored at -20°C for a long time.

[0068] 2. Amplification of ...

Embodiment 2

[0097] The cultivation of embodiment 2 thalline and the preparation of crude enzyme liquid

[0098] 1. Bacteria culture

[0099] Composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, sterilized at 121°C for 20min, ready for use.

[0100] After the engineering bacteria containing the amino acid dehydrogenase gene were activated by streaking on a plate, a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37°C for 12 hours. Transfer to 50mL fresh LB liquid medium also containing 50μg / ml Kan at 2% inoculum size, shake culture to OD at 37°C 600 When it reaches about 0.8, add IPTG to its final concentration of 0.5mM, and induce culture at 18°C ​​for 16h. After the cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, the bacteria were collected, and stored in a -80°C ultra-l...

Embodiment 3

[0103] The mensuration of embodiment 3 enzyme storehouse enzyme activity

[0104] Standard enzyme activity detection system: 25g / L wet bacteria (ultrasonic disruption), 10mM substrate, 20mM coenzyme (NADH or NADPH), 750mM NH 4 Cl, the total system volume is 400 μL, and the reaction medium is pH 8.0 phosphate buffer. Definition of unit enzyme activity: under standard reaction conditions, the amount of enzyme required to generate 1 μmol L-glufosinate-ammonium per minute.

[0105] The reaction solution was prepared according to the above-mentioned standard enzyme activity detection system, reacted in a metal bath shaking reactor at 30°C for 6 hours, added 40 μL of 5M NaOH to terminate the reaction, and stored on ice. After the sample was diluted by a certain number of times, the concentration of L-glufosinate-ammonium was detected by pre-column derivatization high-performance liquid chromatography, and the enzyme activity was calculated.

[0106] Table 4 enzyme library enzyme a...

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Abstract

The invention discloses a method for preparing L-glufosinate-ammonium by use of amino acid dehydrogenase. The method comprises the following steps: taking 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid or a salt thereof as a substrate, taking a cell of the isolated glutamate dehydrogenase or in vitro expression glutamate dehydrogenase as a catalyst to perform reductive amination reaction under the condition of the existence of inorganic amino donor and reduced coenzyme, thereby acquiring the L-glufosinate-ammonium. The method disclosed by the invention is high in raw material conversion rate and high in yield, the product is easy to separate and purify, and the chirality purity is high; compared with the transaminase and like catalysis technology, the process is relatively simple, and the raw material conversion rate reaches up to 100%.

Description

technical field [0001] The invention relates to the field of biochemical technology, in particular to a production method of chiral pure L-glufosinate-ammonium; specifically, a method for producing optically pure L-glufosinate-ammonium by using amino acid dehydrogenase derived from microorganisms. [0002] technical background [0003] Glufosinate-ammonium (glufosinate-ammonium) is the world's second most resistant herbicide to genetically modified crops. It is developed and produced by Hearst (now owned by Bayer after several mergers). Its chemical name is 4-[hydroxy (methyl) phosphono ]-DL-homoalanine, also known as glufosinate-ammonium salt, Basta, Buster, etc., is a phosphonic acid herbicide, a glutamine synthetase inhibitor, and a non-selective (killing) contact herbicide. [0004] At present, the three major herbicide species in the world are glyphosate, glufosinate-ammonium and paraquat. With the rapid development of glufosinate-ammonium transgenic crops, the market d...

Claims

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Application Information

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IPC IPC(8): C12P13/04
CPCC12P13/04
Inventor 杨立荣尹新坚蒙丽钧周海胜吴坚平徐刚
Owner ZHEJIANG UNIV
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