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A high-density fermentation and Escherichia coli technology, applied in the field of fermentation engineering, can solve the problems of low enzyme production, difficult to increase product yield, and large material loss, and achieve the effects of good reproducibility, reduced production costs, and high stability
Active Publication Date: 2022-02-25
FOSHAN GOLDEN HEALTH TECH CO LTD +1
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Problems solved by technology
[0005] In terms of fermentation, the amount of enzyme produced by fermentation and cultivation by traditional fermentation methods is small, and the loss of materials is large, which makes it difficult to increase the output of the product, and at the same time, the cost of enzyme production remains high
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Embodiment 1
[0035] Construction of recombinant imine reductase Escherichia coli engineering bacteria:
[0036] Obtain imine reductase, the nucleotide sequence encoding imine reductase is shown in SEQ ID NO.1, and express it in Escherichia coli (E. On the vector pET-28a, the recombinant imine reductase Escherichia coli engineered strain was obtained, and the engineered strain was named BL21(DE3) / pET-28a-IRED.
Embodiment 2
[0038] Preparation before fermentation:
[0039] 1. Preparation of conventional reagents:
[0040]80% glucose solution: weigh 240g glucose, dilute to 300 mL with pure water, and sterilize under high temperature and high pressure; the sterilization condition is 115°C, 20 min;
[0041] 50% defoamer: take 50 mL of defoamer, add 50 mL of pure water, and sterilize under high temperature and high pressure; the sterilization condition is 115°C, 20 min;
[0042] 50% ammonia water: measure 100 mL of ammonia water, add 100 mL of sterilized water sterilized by high temperature and high pressure, and get it;
[0043] 1 mol / L isopropyl-β-D-thiogalactopyranoside (IPTG): After preparing according to the concentration, use a syringe to pass through the membrane to sterilize;
[0044] 100 mg / mL Kanamycin Sulfate: After preparing according to the concentration, use a syringe to pass through the membrane to sterilize.
[0045] 2. Preparation of seed medium and fermentation medium:
[0046] S...
Embodiment 3
[0049] Strain activation and seed liquid culture:
[0050] Streak the E.coil BL21(DE3) / pET-28a-IRED glycerol bacterium preserved at -20°C on LB solid medium, culture it upside down at 37°C for 16 hours; pick a single colony in 1 mL LB liquid medium, 37 ° C, 200 rpm shaking culture for 6 hours, to obtain activated strains.
[0051] All the activated strains were inoculated into 200 mL seed medium, and cultured with shaking at 200 rpm at 37°C for 12 hours to obtain seed liquid.
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Abstract
The invention discloses a rapid high-density fermentation method of recombinant imine reductase escherichia coli engineering bacteria, and relates to the technical field of fermentation engineering. The method comprises the following steps: constructing recombinant imine reductase Escherichia coli engineering bacteria, activating strains, culturing a seed solution and performing fed-batch high-density fermentation. The fed-batch high-density fermentation comprises the following steps: 1) inoculating a seed solution of recombinant imine reductase escherichia coli engineering bacteria obtained by seed solution culture into a fermentation medium for high-density fermentation, wherein the dissolved oxygen value of the fermentation culture medium is 30-35%, and the culture temperature is 32-37 DEG C; and (2) when the cell concentration OD600 of the recombinant imine reductase escherichia coli engineering bacteria in the fermentation culture medium is 12-15 and the culture temperature is 16-20 DEG C, adding an inducer, and keeping the dissolved oxygen content of the fermentation culture medium to be 30-35% until enzyme production is ended. By means of the fermentation method, the enzyme production amount and the enzyme production efficiency of the imine reductase can be remarkably improved.
Description
technical field [0001] The invention belongs to the technical field of fermentation engineering, in particular to a rapid high-density fermentation method of recombinant imine reductase Escherichia coli engineering bacteria. Background technique [0002] Chiral amines widely exist in clinical drugs, agricultural chemicals, natural products and surfactants, and are key synthetic intermediates of natural products, pharmaceuticals, chiral auxiliary agents and chiral resolving agents. In medicine, synthetic drugs containing chiral amine fragments have very significant biological activity. More than 70% of the drugs are chiral amines and their derivatives, including antihypertensive, neurological and cardiovascular drugs. In agriculture, chiral amine structural units are present in about 20% of agrochemicals. [0003] The main methods for the asymmetric synthesis of chiral amines are chemical methods and biological enzyme-catalyzed methods. The synthesis of chiral amines by che...
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