A high-density fermentation method of recombinant nitrile hydratase Escherichia coli genetically engineered bacteria

A technology of genetically engineered bacteria and high-density fermentation, which is applied in the field of high-density fermentation of recombinant nitrile hydratase Escherichia coli genetically engineered bacteria, can solve the problems of large equipment investment, low efficiency and high cost, and achieves simplified fermentation equipment and improved production. Efficiency, the effect of reducing the amount of three wastes

Active Publication Date: 2018-09-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the microorganisms producing nitrile hydratase in industrial applications are all obtained through low-density fermentation, and the general cell density (in terms of dry weight) is below 10g / L, which leads to large investment and low efficiency for the production of nitrile hydratase , high cost

Method used

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  • A high-density fermentation method of recombinant nitrile hydratase Escherichia coli genetically engineered bacteria
  • A high-density fermentation method of recombinant nitrile hydratase Escherichia coli genetically engineered bacteria
  • A high-density fermentation method of recombinant nitrile hydratase Escherichia coli genetically engineered bacteria

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Experimental program
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Effect test

Embodiment 1

[0038] The mensuration of embodiment 1 bacterial classification and enzymatic activity

[0039] (1) Strain construction

[0040] The strain used in this example is: E. coli BL21(DE3) / pET-30a(+)-NHaseP, a genetically engineered strain expressing nitrile hydratase derived from Bordetella petrii DSM12804. For the specific method of genetically engineered bacteria construction, please refer to the invention patent application document with the application publication number CN104498466A entitled "Nitrile Hydratase and Its Application". The base sequence of the nitrile hydratase gene in the genetically engineered bacteria is shown in SEQ ID NO.1.

[0041] Genetically engineered bacteria E.coli BL21(DE3) / pET-30a(+)-pENHase-1229 expressing nitrile hydratase derived from Aurantimonas manganoxydans SI859A (Aurantimonas manganoxydans SI859A), the base of nitrile hydratase gene in genetically engineered bacteria The base sequence is shown in SEQ ID NO.2. The construction method of gen...

Embodiment 2

[0062] 10L fermentation of embodiment 2 genetically engineered bacteria E.coli BL21(DE3) / pET-30a(+)-NHaseP

[0063] (1) Strain activation: use an inoculation loop to take the genetically engineered bacteria E.coli BL21(DE3) / Streak the pET-30a(+)-NHaseP strain on the surface of LB-Kan-agar solid medium (plate or eggplant bottle), place the plate upside down in a constant temperature incubator at 37°C, and cultivate for 12 hours;

[0064] (2) Seed cultivation: re-inoculate the activated strains into 40mL of primary seed culture medium, shake and culture at 37°C for 12 hours to obtain primary seed liquid; then, transfer the primary seed liquid into 400mL of secondary seed culture medium with shaking at 35°C for 4 hours to obtain a secondary seed liquid;

[0065] Among them, the primary seed medium is LB medium, 10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, 5M sodium hydroxide solution to adjust the pH value to 7.0, sterilized at 121°C for 20min, and inoculated Befo...

Embodiment 3

[0076] 15L fermentation of embodiment 3 genetically engineered bacteria E.coli BL21(DE3) / pET-30a(+)-pENHase-1229

[0077] (1) Strain activation: use an inoculation loop to take the genetically engineered strain E.coli BL21 (DE3) expressing nitrile hydratase derived from Aurantimonas manganoxydans SI859A (Aurantimonas manganoxydans SI859A) preserved in a -80°C glycerol tube in the strain tube / pET-30a(+)-pENHase-1229 strains were drawn on the surface of LB-Kan-agar solid medium (plate or eggplant bottle), and the plate was placed upside down in a constant temperature incubator at 37°C for 12 hours;

[0078] (2) Seed cultivation: re-inoculate the activated strains into 50 mL of primary seed culture medium, shake and culture at 37°C for 12 hours to obtain primary seed liquid; then, transfer the primary seed liquid into 500 mL of secondary seed culture medium with shaking at 35°C for 4 hours to obtain a secondary seed liquid;

[0079] Among them, the primary seed medium is LB med...

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Abstract

The invention discloses a high-density fermentation process for recombinant nitrile hydratase Escherichia coli genetically engineered bacteria. Fed-batch fermentation is adopted in the process, and the procedures comprise: a cell rapid propagation stage, inoculating culture into a basal fermentation medium, wherein the temperature is 30-37 DEG C, the dissolved oxygen is 5-75%, the pH value is 6.8-7.2, and a specific growth rate of the engineering bacteria is 0.2-0.5; and an enzyme production stage: adding lactose induced engineering bacteria to produce enzymes until fermentation is ended whenthe cell concentration OD600 reaches 70-90, wherein the culture temperature is 15-25 DEG C, the dissolved oxygen is 5-75%, the pH value is 6.8-7.2, and the specific growth rate of the engineering bacteria is 0.01-0.1. According to the process disclosed by the invention, the fed-batch fermentation is manually divided into two stages, and culture conditions at each stage are controlled, so that high-density fermentation of the recombinant nitrile hydratase Escherichia coli genetically engineered bacteria is realized. The bacteria concentration of the engineering bacteria of producing nitrile hydratase is improved, and the enzyme activity of the nitrile hydratase is improved.

Description

technical field [0001] The invention belongs to the technical field of fermentation engineering, in particular to a high-density fermentation method of recombinant nitrile hydratase Escherichia coli genetically engineered bacteria. Background technique [0002] Nitrile hydratase (EC 4.2.1.84) is a class of microbial enzymes widely present in nature (almost all of the reported nitrile hydratases come from bacteria), which catalyzes nitrile compounds to generate corresponding amides. The discovery and application of nitrile hydratase are One of the classic masterpieces in the field of industrial biotechnology, it has incomparable advantages over chemical catalysts, so it is widely used in the synthesis of amide chemicals. Nitrile hydratase was first used to catalyze the production of acrylamide from acrylonitrile. Japan is the earliest practice and owner of biosynthetic acrylamide technology, and its production technology is also the most advanced. After three strain innovati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12R1/19
CPCC12N9/88C12Y402/01084
Inventor 杨立荣周海胜张红玉吴坚平徐刚
Owner ZHEJIANG UNIV
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