阅读说明：本技术 一种生产角蛋白酶的方法 (Method for producing keratinase ) 是由 王克芬 张�杰 刘文龙 钱娟娟 王兴吉 刘胜利 贾仁洁 于 2019-11-18 设计创作，主要内容包括：本发明属于生物工程技术领域,特别涉及一种生产角蛋白酶的方法。该方法所采用的生产菌株为地衣芽孢杆菌(Bucillus licheniformis)JR-101,保藏编号为CGMCC No.18643。该菌株经过液态发酵生产的角蛋白酶发酵液酶活力平均达到27703U/mL,所生产的角蛋白酶具有耐热、酶活力高、pH作用范围广泛、性能稳定而且价格较低的特点,应用范围广泛。(The invention belongs to the technical field of bioengineering, and particularly relates to a method for producing keratinase. The production strain adopted by the method is bacillus licheniformis (Bucillus licheniformis) JR-101, and the preservation number is CGMCC No. 18643. The enzyme activity of the keratinase fermentation liquor produced by the strain through liquid fermentation is 27703U/mL on average, and the produced keratinase has the characteristics of heat resistance, high enzyme activity, wide pH action range, stable performance, lower price and wide application range.)

1. A method for producing keratinase is characterized in that a strain adopted for producing the keratinase by fermentation is Bacillus licheniformis (JR-101) with the preservation number of CGMCC No. 18643.

2. The method for producing keratinase as claimed in claim 1, wherein the seed solution is inoculated into the fermentation medium at an inoculum size of 5%, the pressure in the tank is 0.05-0.08MPa, the cultivation temperature is 37 ℃, the rotation speed is 300-800r/min, when the dissolved oxygen is reduced to the minimum and then increased to 20%, feeding is started, the dissolved oxygen is controlled at 20% -30%, and the tank is placed after 96-110 h.

3. The method for producing keratinase according to claim 2, wherein the feed medium comprises: 25% of corn starch, 5.2% of bean cake powder, 2.3% of corn steep liquor, 1.5% of feather meal, 0.5% of ammonium sulfate, 0.2% of potassium dihydrogen phosphate and pH 6.5.

4. The method of producing keratinase of claim 2, wherein the fermentation medium comprises: 2.5 percent of corn starch, 3.5 percent of corn flour, 1.5 percent of feather meal, 3.5 percent of soybean meal, 3 percent of corn steep liquor, 3.2 percent of cane sugar, 1.7 percent of monopotassium phosphate, 0.27 percent of magnesium sulfate and pH 6.5.

5. The method for producing keratinase according to claim 2, wherein the extraction and purification of keratinase are as follows:

after fermentation is finished, 40% of water and 0.3% of calcium chloride are added according to the volume of fermentation liquor, 3% of perlite filter aid is added, filter pressing is carried out, and after ultrafiltration concentration, alcohol elution is carried out on filtrate to obtain a solid enzyme preparation, or 20% of glycerol is added into concentrated solution to be used as a stabilizer to obtain a liquid enzyme preparation.

The technical field is as follows:

the invention belongs to the technical field of bioengineering, and particularly relates to a method for producing keratinase.

Background art:

keratin is widely found in nature, mainly derived from animal ectodermal cells, including skin and skin derivatives such as feathers, nails, hair, etc., and is a hard protein that is difficult to degrade. The chemical structure of keratin is stable, insoluble in water and difficult to be hydrolyzed by protease such as pepsin, trypsin and the like. Keratin is a hard protein with extremely strong resistance and difficult biodegradation, animals are difficult to directly utilize, and the traditional physical and chemical processing methods (such as acid-base treatment, high temperature and high pressure and the like) not only consume a large amount of energy, but also cause serious environmental pollution problems.

Keratinase is a novel proteolytic enzyme capable of hydrolyzing keratin into free amino acids or polypeptides. The characteristic of the keratinase can be used for treating waste animal feathers, hair and the like, can reduce the pollution of the waste such as feathers and the like to the environment, can be used as an excellent protein resource, and plays an important role in relieving the situation of short supply of the protein resource. The substrate which can be hydrolyzed by keratinase is more, such as collagen, keratin, elastin and the like, the hydrolyzing capability of the substrate to the protein is higher than that of common protease, and the substrate can be widely applied to the aspects of animal feed, leather industry, medical raw materials and environmental management.

There are more than 30 kinds of microorganisms that can degrade keratin, among which are bacteria, fungi and actinomycetes. The industrial application of keratinase is seriously influenced by the keratinase with poor thermal stability, so that the improvement of the thermal stability of the keratinase is beneficial to the industrial application.

The invention content is as follows:

in order to solve the technical problems, the invention provides a bacillus licheniformis strain of keratinase with high heat production stability, which is obtained by NTG mutagenesis, in particular to bacillus licheniformis (Bucillus licheniformis) JR-101, and the strain is preserved in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms in 2019, 10 and 9 days, with the address: no. 3 of Xilu No.1 of Beijing Korean Yang district in China, the preservation number is CGMCC No. 18643.

The invention also aims to provide a liquid microbial fermentation production method of keratinase, which has high fermentation enzyme activity, high extraction yield and low manufacturing cost. The object of the invention can be achieved by the following measures:

1. liquid fermentation for producing keratinase

The culture medium comprises the following components in percentage by mass and volume:

a. fermentation tank culture medium: 2.5% of corn starch, 3.5% of corn flour, 1.5% of feather meal, 3.5% of soybean meal, 3% of corn steep liquor, 3.2% of cane sugar, 1.7% of monopotassium phosphate, 0.27% of magnesium sulfate and 6.5% of pH;

b. the fermentation tank sterilization process conditions are as follows: sterilizing at 121-124 deg.C and 0.11-0.12MPa for 35 min.

c. Fermentation process conditions of the fermentation tank are as follows: inoculating 5%, maintaining at 37 deg.c and 300 deg.c at 5-0.08MPa, feeding material while controlling the dissolved oxygen content in 20-30% and fermenting to obtain thallus with serious autolysis and no obvious raised enzyme activity;

2. feed supplement

a. A supplemented medium: 25% of corn starch, 5.2% of bean cake powder, 2.3% of corn steep liquor, 1.5% of feather meal, 0.5% of ammonium sulfate, 0.2% of potassium dihydrogen phosphate and pH 6.5.

b. The material supplementing method comprises the following steps: when the dissolved oxygen is reduced to the minimum and then increased to 20 percent, feeding is started, and the dissolved oxygen is controlled to be 20 to 30 percent.

3. Can for placing food

Culturing for 96-110h, slowly increasing enzyme activity, and placing the thallus into a tank when the thallus begins to autolyze partially. When the fermentation is finished, the enzyme activity of the fermentation liquor averagely reaches 27703U/mL.

4. Extraction and purification of keratinase

After fermentation, adding 40% of water and 0.3% of calcium chloride according to the volume of the fermentation liquor, adding 3% of perlite filter aid, carrying out filter pressing, carrying out ultrafiltration concentration on the filtrate, and then carrying out alcohol elution to obtain a solid enzyme preparation, or adding 20% of glycerol as a stabilizer into the concentrated solution to obtain a liquid enzyme preparation.

The keratinase prepared by the invention has the following enzymological characteristics:

(1) the optimal reaction temperature is 75 ℃, the enzyme activity is kept unchanged after 2 hours of heat preservation at the temperature of 80 ℃, the enzyme activity of more than 87 percent can still be kept after 2 hours of heat preservation at the temperature of 95 ℃, the enzyme activity is reduced to about 65 percent after 2 hours of heat preservation under the boiling condition, and the thermal stability is good;

(2) the optimum reaction pH is 7.5, and the relative enzyme activity is still kept above 80% after the treatment for 2h under the condition of pH 3.0-11.0.

Has the advantages that:

compared with the prior art, the technical scheme disclosed by the keratinase and the production method thereof has prominent substantive characteristics and remarkable progress, and can produce the following positive effects:

1. the invention provides a bacillus licheniformis obtained by mutagenesis, the keratinase produced by the strain through liquid fermentation has the characteristics of heat resistance, high enzyme activity, wide pH action range, stable performance, lower price and wide application range.

2. The invention provides a production method of keratinase liquid state biological fermentation, which has higher fermentation activity, higher extraction yield and lower manufacturing cost.

The attached drawings of the specification:

FIG. 1 is a graph of the optimum reaction temperature;

FIG. 2 thermal stability graph;

FIG. 3 is a graph of optimum reaction pH;

figure 4pH stability graph.

The specific implementation mode is as follows:

the invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.