Fermentation method for efficiently producing keratinase through recombinant escherichia coli

A technology of keratinase and fermentation method, which is applied in the field of fermentation engineering, can solve the problems of low keratinase production, unstable activity, and reduce the development and application of keratinase, and achieve the effect of good application prospects

Active Publication Date: 2016-02-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although keratinase has great application and research value, the production of keratinase from wild bacter

Method used

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  • Fermentation method for efficiently producing keratinase through recombinant escherichia coli
  • Fermentation method for efficiently producing keratinase through recombinant escherichia coli
  • Fermentation method for efficiently producing keratinase through recombinant escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of recombinant Escherichia coli capable of efficiently secreting keratinase

[0037](1) A gene sequence template SEQ ID NO. 1 of a highly active keratinase mutant obtained according to multiple molecular transformations in this experiment, or a template obtained by chemical synthesis, and subjected to PCR amplification. The reaction conditions are: pre-denaturation at 95°C for 5 minutes, followed by a cycle: denaturation at 98°C for 10 seconds, annealing at 55°C for 10 seconds, extension at 72°C for 7 minutes and 50 seconds, and 30 cycles; extension at 72°C for 1 minute and 50 seconds, and then cooling down to 12°C to obtain the final reaction solution. The DNA amplification enzyme used was PrimerSTAR from TaKaRa Company, and the formula was used according to the product manual.

[0038] (2) The amplified PCR product and the pET22b(+) plasmid were treated with NcoI and XhoI endonucleases to obtain DNA sequences at cohesive ends, and then ligated o...

Embodiment 2

[0043] Fermentation optimization of embodiment 2 recombinant escherichia coli on the shake flask medium

[0044] The resulting genetically engineered bacteria were cultured overnight at 37°C in a 50mL medium containing 100μg / L ampicillin, and then inserted into a 50mL liquid medium containing 100μg / L ampicillin and cultured at 37°C until OD 600 =0.6, lower the temperature to 20°C and culture, add the inducer IPTG with a final concentration of 0.1mM to induce culture, centrifuge at 72h to obtain the supernatant enzyme liquid, which is the crude enzyme liquid, and measure the enzyme activity.

[0045] Five kinds of basal media were selected to compare the efficiency and yield of Escherichia coli fermented to produce keratinase in shake flasks. These five media are LB, 2×YT, SOC, TB, SB. The main differences of these five media are the presence or absence of carbon source and the concentration of different nitrogen sources or differences in salt ions.

[0046] LB (g / L): peptone...

Embodiment 3

[0061] Embodiment 3 recombinant escherichia coli produces keratinase on 3L fermenter

[0062] 3L fermenter fermentation conditions: initial culture temperature 37°C, inoculum size 2%, ampicillin content 100 μg / l, stirring speed 400-800rpm, initial liquid volume 1L, ventilation volume 1vvm, using BioFlo / CelliGen115 fermenter from New Brunswick Company.

[0063] On the basis of 2×YT medium, carbon source medium (g / l, glycerol 500, yeast powder 15, peptone 30, MgSO 4 15), and feed the dissolved oxygen when it rebounds for the first time, and the flow speed is 0.8ml / min. When the cell concentration reached 10OD600, IPTG was added to a final concentration of 200mM for low-temperature induction at 20°C, during which the pH of the medium was controlled by adding ammonia water to not less than 7.0. The experimental results show that (such as figure 2 ), using fed-batch strategy, Escherichia coli can indeed reach 12.6OD in 16h 600 , but 6 hours after the induction, the bacterial ce...

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Abstract

The invention discloses a fermentation method for efficiently producing keratinase through recombinant escherichia coli, and belongs to the field of fermentation engineering. According to the fermentation method, the recombinant escherichia coli which can efficiently secrete the high-activity keratinase is built, and the keratinase is produced and prepared in a shake flask or a fermentation tank. A keratinase preparation can be efficiently prepared by optimizing a culture medium and improving a fermentation strategy, and the activity of the fermented keratinase is increased to 4500 U/mL from 412 U/mL (the fermentation time is 48 h). The keratinase preparation has a good application prospect in the fields of washing, textile, feed digestion, medicine and the like.

Description

technical field [0001] The invention relates to a fermentation method for efficiently producing keratinase by using recombinant Escherichia coli, belonging to the field of fermentation engineering. Background technique [0002] Keratinase is an enzyme that can specifically degrade keratin, which is produced by various microorganisms such as fungi, actinomycetes and bacteria. Keratinase is widely used in industries such as food, medicine, feed, refining and tanning. It has the functions of tenderizing meat, producing advanced nutrition, immune preparations and feed additives, as well as beautifying and softening leather. It can also cause mad cow disease and human Degradation of Prion in Creutzfeldt-Jakob disease. [0003] The keratinase gene reported so far mainly comes from the kerA gene of a foreign strain of Bacillus licheniformis. Although keratinase has great application and research value, the production of keratinase from wild bacteria is low and the activity is uns...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N1/21C12R1/19
CPCC12N9/52
Inventor 张娟陈坚方真堵国成李光磊隆明星
Owner JIANGNAN UNIV
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