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A method for producing keratinase

A keratinase content technology, applied in the field of microorganisms and fermentation, can solve the problems of low fermentation enzyme activity and high fermentation cost, and achieve the effects of good fermentation repeatability, increased keratinase production, and low cost

Active Publication Date: 2022-05-24
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to solve the problem of low fermentation enzyme activity and high fermentation cost of keratinase production strains

Method used

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  • A method for producing keratinase
  • A method for producing keratinase
  • A method for producing keratinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Strain culture and fermentation

[0046] 1. Bacterial culture method

[0047] Strain activation: Take out the keratinase-producing Bacillus subtilis WB600-pP43NMK-ker from the -80°C refrigerator (the strains are described in "Biotransformation of keratin waste to aminoacids and active peptides based on cell-free catalysis" by Zheng Peng et al.) Glycerol tubes were streaked in solid LB medium and incubated at 37°C for 14-16h.

[0048]First-class seed liquid culture: pick a single colony from the plate and inoculate it in 3 mL of LB medium, and cultivate overnight at 37°C and 220 rpm.

[0049] Secondary seed liquid culture: transfer the primary seed liquid to a 250 mL Erlenmeyer flask containing 50 mL of LB medium at 2% inoculum, and cultivate at 37°C and 220 rpm for 4-6 hours.

[0050] Shake flask fermentation culture: the OD 600 The secondary seed liquid of 0.8 was transferred to a 250 mL conical flask containing 50 mL of fermentation medium at an inoculat...

Embodiment 2

[0051] Example 2: Preparation of keratinase using media containing different carbon sources

[0052] (1) Selection of the optimal carbon source

[0053] Initial fermentation medium components (g / L): glucose 20, yeast powder 10, peptone 20, Na 2 HPO 4 ·12H 2 O 6, KH 2 PO 4 3. MgSO 4 ·7H 2 O 0.3.

[0054] Keep other components of the fermentation medium unchanged, replace glucose with galactose, glycerol, sucrose, maltose, dextrin and starch as carbon sources, and the addition amount is 20g / L; according to Example 1, carry out shake flask fermentation culture for 24h, and measure The enzyme activity of the fermentation broth determines that the best carbon source is sucrose ( figure 1 ).

[0055] (2) Selection of carbon source concentration

[0056] The specific embodiment is the same as the above-mentioned step (1), the difference is that the carbon source in the fermentation medium is replaced with sucrose (10, 15, 20, 25, 30, 35, 40g / L) of different concentrations;...

Embodiment 3

[0057] Example 3: Preparation of keratinase using media containing different nitrogen sources

[0058] (1) Selection of the first nitrogen source

[0059] After determining the optimal carbon source, the yeast powder and tryptone in the fermentation medium were replaced with a single nitrogen source. The single nitrogen source was peptone, yeast extract, bran, soybean meal, corn steep liquor meal, ammonium sulfate and ammonium chloride. Any one, the addition amount is 30g / L; According to embodiment 1, carry out shaking flask culture 24h, measure the enzyme activity of fermentation broth, determine that the best nitrogen source is soybean meal ( image 3 ).

[0060] (2) Selection of the concentration of the first nitrogen source

[0061] The specific embodiment is the same as the above step (1), the difference is that the yeast powder and tryptone in the culture medium are replaced with soybean meal of different concentrations (20, 25, 30, 35, 40, 45, 50g / L); When the enzyme...

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Abstract

The invention discloses a method for producing keratinase, which belongs to the technical field of microorganisms and fermentation. The present invention proceeds from two aspects of fermentation medium components (carbon source, nitrogen source, metal ion, phosphate) and fermentation conditions (temperature, pH, inoculum size), and utilizes Bacillus subtilis to ferment and produce the technique of keratinase. optimization. Utilizing the method of the invention to ferment and produce keratinase can increase the yield of keratinase by 3.3 times compared with the control, and the enzyme is produced in a 3L fermenter, and the enzyme activity reaches 704400U / mL. Therefore, the method of the present invention has broad application prospects in the preparation of keratinase in feed, tanning and the like.

Description

technical field [0001] The invention relates to a method for producing keratinase, belonging to the technical field of microorganisms and fermentation. Background technique [0002] Keratinase is a protease that can specifically degrade keratin-like substrates. The microorganisms that produce keratinase are mainly bacteria, fungi and actinomycetes. At present, the research on the optimization of keratinase fermentation mainly focuses on the screening of wild bacteria, the optimization of fermentation medium components and the optimization of fermentation conditions. In the mining of keratinase-producing strain resources, the most common bacteria are bacteria, which have the advantages of short fermentation cycle, enzyme With the characteristics of high activity and good production safety, keratinase has good application prospects in the fields of feed, fertilizer, detergent, leather, textile, cosmetic industry and medical treatment. With the deepening of people's understan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/54C12N1/20C12P21/06C12R1/125
CPCC12N9/54C12N1/20C12P21/06Y02W30/40
Inventor 张娟冒鑫哲万云蕾陈坚郭荣邓小华李江华周冠宇
Owner JIANGNAN UNIV
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