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High-density fermentation method of recombinant phosphinothricin dehydrogenase engineering bacteria under phosphorus emission limitation

A glufosinate-ammonium and dehydrogenase technology, applied in microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problems of difficult wastewater treatment, unfavorable industrial application, and high phosphorus content, and achieve easy wastewater treatment and realization. High-density fermentation, less environmental pollution

Active Publication Date: 2021-03-19
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] The purpose of the present invention is to provide a high-density fermentation method of recombinant glufosinate-ammonium dehydrogenase engineering bacteria under the limitation of phosphorus discharge, by adding glycerol (replacing phosphoric acid) in the fermentation process and providing a method for recombinant glufosinate-ammonium dehydrogenase on this basis The high-density fermentation method of dehydrogenase engineering bacteria solves the problems of adjusting the pH value with phosphoric acid alone in the fermentation process, resulting in excessive phosphorus content in the fermentation process, resulting in difficult wastewater treatment, which is not conducive to industrial applications, etc.

Method used

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  • High-density fermentation method of recombinant phosphinothricin dehydrogenase engineering bacteria under phosphorus emission limitation
  • High-density fermentation method of recombinant phosphinothricin dehydrogenase engineering bacteria under phosphorus emission limitation
  • High-density fermentation method of recombinant phosphinothricin dehydrogenase engineering bacteria under phosphorus emission limitation

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Effect test

Embodiment 1

[0041] Embodiment 1, the construction of recombinant glufosinate-ammonium dehydrogenase engineering bacteria

[0042] Refer to the patent ZL201811585674.7 "a glufosinate-ammonium dehydrogenase mutant and its application", specifically: the glufosinate-ammonium dehydrogenase gene (amino acid sequence shown in SEQ ID NO.2, nuclear The nucleotide sequence is shown in SEQID NO.1) to construct the expression vector pETDuet-1-lvPDH, transform Escherichia coli, and obtain the starting strain recombinant glufosinate-ammonium dehydrogenase engineering bacteria E.coli BL21(DE3) / pETDuet-1-lvPDH.

Embodiment 2

[0043] Embodiment 2, the method for the high-density fermentation of recombinant glufosinate-ammonium dehydrogenase engineered bacteria controlled pH by glycerol (partially serving as carbon source) under phosphorus limitation

[0044] 1. Slant culture: inoculate the recombinant glufosinate-ammonium dehydrogenase engineering bacteria E.coli BL21(DE3) / pETDuet-1-lvPDH constructed in Example 1 into LB medium containing 50 μg / mL ampicillin, and culture overnight at 37°C (about 10h), obtain slant thalline; described LB medium composition: 10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, agar powder 20g / L, solvent is distilled water, pH 7.4.

[0045] 2. Seed culture: pick the slant bacteria and inoculate them into 100 mL of LB medium containing 50 μg / mL ampicillin, and culture overnight at 37° C. (about 10 h) to obtain seed liquid for later use. The composition of the LB medium: 10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, distilled water as solvent, pH 7.4.

...

Embodiment 3

[0054] Embodiment 3, the determination of the stability of the recombinant glufosinate-ammonium dehydrogenase controlled pH by glycerol (partially serving as carbon source) and the recombinant glufosinate-ammonium dehydrogenase controlled pH by phosphoric acid

[0055] The glufosinate-ammonium dehydrogenase cells obtained from high-density fermentation using glycerol to adjust the pH value in Example 2 were resuspended with pH 7 phosphate buffer, and then placed at 35°C, 40°C, 45°C, 50°C, and 55°C respectively. After being incubated with 60°C water bath for 30min, the activity of the enzyme was measured again, and the relative enzyme activity of the group with the highest enzyme activity was defined as 100%. The results showed that the thermal stability of glufosinate-ammonium dehydrogenase was the best at 35 and 40°C. After incubation for 30 minutes, the activity remained above 99%, and then slowly decreased; and after incubation at 60°C for 30 minutes, the relative activity r...

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Abstract

The invention discloses a high-density fermentation method of recombinant phosphinothricin dehydrogenase engineering bacteria under phosphorus emission limitation. The high-density fermentation methodcomprises the following steps of: inoculating the recombinant phosphinothricin dehydrogenase engineering bacteria into a fermentation culture medium, carrying out fermentation culture at 37 DEG C under the conditions that the initial ventilation capacity is 5 L / min, the initial stirring speed is 300 rpm and the initial dissolved oxygen is 100% until the OD<600> value is 8-10, adding lactose withthe final concentration of 5-15 g / L into fermented liquid, adjusting the pH value through a supplemented culture medium and glycerin, carrying out fermentation culture until the OD<600> value is 100-120, and centrifuging the fermented liquid to obtain a wet thallus containing recombinant phosphinothricin dehydrogenase. According to the high-density fermentation method disclosed by the invention, phosphoric acid does not need to be added to adjust the pH value; environmental pollution is small; wastewater treatment is easier; the phosphorus concentration in the fermented liquid is 1.15*10<3> mg / L; the OD<600> value reaches 100-120; and the stability of the thallus at 60 DEG C is improved by 1.6 times compared with that of phosphoric acid for adjusting the pH value.

Description

[0001] (1) Technical field [0002] The invention relates to a high-density fermentation method of recombinant glufosinate-ammonium dehydrogenase Escherichia coli genetically engineered bacteria under phosphorus limitation. [0003] (2) Background technology [0004] The chemical name of glufosinate (phosphinothricin, also called glufosinate) is 2-amino-4-[hydroxy(methyl)phosphono]-butyric acid, which is the second most resistant herbicide in genetically modified crops in the world. (now owned by Bayer after several mergers) developed and produced, also known as glufosinate ammonium salt, Basta, Buster, etc., are phosphonic acid herbicides, non-selective (killing) contact herbicides are glutamine synthetase inhibitors agent. Since July 2016, the domestic paraquat aqueous solution has been completely banned, and the domestic substitution space alone has reached 20,000 tons, which has greatly increased the demand for glufosinate-ammonium as a substitute. As early as 2018, the wo...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12R1/19
CPCC12N9/0016
Inventor 薛亚平程峰李举谋曹成浩徐建妙沈其邹树平郑裕国
Owner ZHEJIANG UNIV OF TECH
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