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Re -reorganized amine restoration enzyme E. coli engineering fungus of the rapid high -density fermentation method

A high-density fermentation and Escherichia coli technology, which is applied in the field of fermentation engineering, can solve the problems of low enzyme production, difficulty in increasing product output, and large material loss, achieving high stability, good reproducibility, and reduced production costs.

Active Publication Date: 2022-05-17
FOSHAN GOLDEN HEALTH TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

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  • Re -reorganized amine restoration enzyme E. coli engineering fungus of the rapid high -density fermentation method
  • Re -reorganized amine restoration enzyme E. coli engineering fungus of the rapid high -density fermentation method
  • Re -reorganized amine restoration enzyme E. coli engineering fungus of the rapid high -density fermentation method

Examples

Experimental program
Comparison scheme
Effect test

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, a recombinant imine reductase Escherichia coli engineering bacterium was obtained, and the engineering bacterium 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

A rapid high-density fermentation method for recombinant imine reductase Escherichia coli engineering bacteria, involving the technical field of fermentation engineering; including: construction of recombinant imine reductase Escherichia coli engineering bacteria, strain activation, seed liquid culture and fed-batch high-density fermentation The step of the fed-batch high-density fermentation comprises: 1) inoculating the seed liquid of the recombinant imine reductase Escherichia coli engineering bacterium obtained by culturing the seed liquid into the fermentation medium for high-density fermentation; The oxygen value is 30-35%, and the culture temperature is 32-37°C; 2) When the cell concentration OD of recombinant imine reductase Escherichia coli engineering bacteria in the fermentation medium 600 When =12-15, the culture temperature is 16-20°C, and an inducer is added to keep the dissolved oxygen of the fermentation medium at 30-35% until the end of enzyme production. The fermentation method of the invention can significantly improve the enzyme production amount and enzyme production efficiency of the imine reductase.

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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/38C12N1/21C12N9/06C12N15/53C12R1/19
CPCC12N1/38C12N9/0028Y02A50/30
Inventor 吴子蓥李慧灵胡浩轩黄佳俊江瑞冰黎韵琳周金林卢宇靖
Owner FOSHAN GOLDEN HEALTH TECH CO LTD
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