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Keratinase and application

A technology of keratinase and crude enzyme liquid, applied in the field of protease, can solve the problems of secondary pollution waste, difficult to handle, and less industrial application

Pending Publication Date: 2022-01-04
SHANDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The use of microbial degradation and other biotechnology methods to solve the decomposition and utilization of keratin can solve the problems of secondary pollution and difficult waste disposal in traditional chemical methods.
However, keratin has a large number of disulfide bonds, hydrogen bonds and hydrophobic interactions, and is not easily hydrolyzed by common proteases
Keratinase (Keratinase) is an enzyme that can specifically degrade keratin. It has been found that more than 30 microorganisms can secrete keratinase. The research on keratinase at home and abroad mainly focuses on dermatophytes and Candida albicans ( Candida albicans), Streptomyces and Thermomonas in actinomycetes and Bacillus licheniformis and Bacillus subtilis in bacteria, but most of them have substrates Due to problems such as narrow spectrum and insufficient enzyme activity, there are few industrial applications at present

Method used

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  • Keratinase and application
  • Keratinase and application
  • Keratinase and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The keratinase LbKer mentioned in the present invention has the amino acid sequence shown in the sequence table SEQ ID NO.1, and the keratinase LbKer is derived from Lysobacter sp., which was preserved in China Microorganisms on July 09, 2018 Center for General Microbiology of the Preservation Committee.

[0026] The substrate spectrum of the provided keratinase of the present invention:

[0027] The action substrate spectrum of LbKer has been studied in embodiment 1, select chicken feather, duck feather, goose feather, wool, cow hair and hoof horn as substrate, add 5mL LbKer crude enzyme liquid in 0.1g substrate solution, under stirring, let mixture The reaction was carried out at 45°C and pH 8.0 for 12 hours. Then the sample was boiled for 1 minute to inactivate the enzyme, and the degradation ability of different substrates was characterized by filtering and weighing the residual insoluble matter.

[0028] The results are shown in Table 1, LbKer has relatively high...

Embodiment 2

[0037] Regarding the enzymatic properties of the keratinases of the present invention

[0038] (1) In order to determine the optimal reaction temperature of the enzyme, the enzyme was reacted at different temperatures (30-80°C) and pH 7.5 for 60 minutes to detect the enzyme activity;

[0039] (2) In order to study the temperature stability of the enzyme, the enzyme was pre-incubated at different temperatures (30-80°C) for 30 minutes, pH 7.5, and reacted at 37°C for 60 minutes to detect the remaining enzyme activity of the enzyme;

[0040] (3) In order to determine the optimal pH value, the enzyme activity of the enzyme was measured for 60 minutes at 37°C under different pH conditions;

[0041] (4) In order to study the pH value stability of the enzyme, the enzyme was pre-incubated overnight in various pH5-12 buffers, and then reacted at pH7.5, 37°C for 60 minutes to detect the remaining enzyme activity;

[0042] The detection of the above enzymatic characteristics is to use k...

Embodiment 3

[0052] Application of LbKer 1

[0053] Weigh 1g of dried wool and add 50mL keratinase LbKer crude enzyme solution to react at 37°C for 12h, filter and wash the reaction solution with neutral filter paper, dry the residue to constant weight and weigh, calculate the degradation rate, wool degradation rate Reached 76.9%.

[0054] Application of LbKer 2

[0055]Weigh 0.5g of dried chicken feathers and add 25mL keratinase LbKer crude enzyme solution to react at 37°C for 12h, filter and wash the reaction solution with neutral filter paper, dry the residue to constant weight and weigh, calculate the degradation rate, chicken feather degradation The rate reached 84.1%.

[0056] Application of LbKer 3

[0057] Weigh 1g of dried hoof horn and add 50mL keratinase LbKer crude enzyme solution to react at 37°C for 12h, filter and wash the reaction solution with neutral filter paper, dry the residue to constant weight and weigh, calculate the degradation rate, hoof angle The degradation ...

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Abstract

The invention belongs to the technical field of protease, particularly relates to keratinase, and further relates to application of the keratinase in hydrolysis of animal cutin raw materials. The keratinase LbKer is derived from lysobacter CGMCC No.16072, the tolerance temperature of the keratinase LbKer is higher than 55 DEG C, the keratinase LbKer has enzyme activity in the pH range of 5.0-9.0, the animal keratin raw material degradation rate of crude enzyme liquid fermented by the keratinase for 12 h reaches 60% or above, the keratinase LbKer has an amino acid sequence shown as SEQ ID NO.1 in a sequence table, and the keratinase with the amino acid sequence is a novel protein sequence reported for the first time at present, and has low similarity with the reported keratinase protein sequence. The keratin provided by the invention has the greatest advantages that the keratin has the characteristics of wide substrate spectrum, high wool degradation efficiency and the like.

Description

technical field [0001] The invention belongs to the technical field of proteases, specifically relates to a keratinase, and also relates to the application of the above-mentioned keratinase. Background technique [0002] Livestock and poultry wastes are increasing year by year, which brings great troubles to environmental protection due to its refractory nature. Breeding pollution is listed as one of the pollutions that need to be dealt with mainly, and solid waste pollution from livestock and poultry farms is a major problem in the farming industry. Harmless treatment of livestock and poultry solid waste, resource utilization, prevention and elimination of pollution from livestock and poultry manure in farms are of great significance for protecting the ecological environment, promoting sustainable agricultural development and enhancing the market competitiveness of agricultural products. [0003] According to statistics, the world's annual output of poultry feathers and th...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N15/57C12P21/06
CPCC12N9/52C12P21/06
Inventor 马钦元高秀珍张祥鹤王笑笑张亚宁尹炎淼张春杨郑雪栾芳韩萍
Owner SHANDONG UNIV OF TECH
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