阅读说明：本技术 一种生产胰蛋白酶的方法 (Method for producing trypsin ) 是由 王兴吉 郭庆文 王克芬 伏圣秘 刘胜利 刘文龙 张�杰 于 2019-11-15 设计创作，主要内容包括：本发明属于微生物或酶工程领域,具体涉及一种生产胰蛋白酶的方法。该方法使用的发酵菌株为链霉菌(Streptomyces sp.)LDE-79,保藏号为CGMCC No.18646,发酵液中酶活达到252U/mL至260U/mL之间,通过提取、精制获得的胰蛋白酶液体酶制剂的酶活达到1000U/mL以上。得到的胰蛋白酶最适pH范围为7.5-9.0,最适作用温度范围为35℃-60℃,并且在55℃保温1h后剩余酶活为92％,保温2h后剩余酶活为85％,具有显著的耐热性,可广泛应用到高温工业生产中,大大地拓宽了胰蛋白酶的应用范围,增加了胰蛋白酶的应用价值。(The invention belongs to the field of microorganism or enzyme engineering, and particularly relates to a method for producing trypsin. The fermentation strain used in the method is Streptomyces sp LDE-79 with the preservation number of CGMCC No.18646, the enzyme activity in the fermentation broth reaches between 252U/mL and 260U/mL, and the enzyme activity of the trypsin liquid enzyme preparation obtained by extraction and refining reaches over 1000U/mL. The obtained trypsin has the optimum pH range of 7.5-9.0, the optimum action temperature range of 35-60 ℃, the residual enzyme activity of 92% after heat preservation for 1h at 55 ℃ and 85% after heat preservation for 2h, has obvious heat resistance, can be widely applied to high-temperature industrial production, greatly widens the application range of the trypsin, and increases the application value of the trypsin.)

1. A method for producing trypsin by fermentation is characterized in that a fermentation strain is streptomyces sp LDE-79 with the preservation number of CGMCC No. 18646.

2. The method of fermentative production of trypsin according to claim 1, wherein the fermentation process is as follows: inoculating the seed liquid into a fermentation culture medium according to an inoculation proportion of 10-15% of the volume ratio, setting the air volume at 30-32 ℃, the rotating speed of 400-: 0.4-0.5vvm from 0h to 6h, 0.6-0.8vvm from 6h to 15h, 1.0-1.2vvm from 20h to the end of fermentation, pH 7.5-7.8, fermenting and culturing until the enzyme activity is slowly increased or not increased, finishing fermentation when the thalli is deformed and autolyzed, and the fermentation period is 120-128 h.

3. The method for producing trypsin by fermentation according to claim 2, wherein the pH of the fermentation broth is controlled to be maintained in the range of 7.5 to 7.8 by the coordinated addition of a feed medium, or liquid ammonia, or phosphoric acid during the fermentation;

the feed medium comprises the following components: 40g/L of corn steep liquor, 120g/L of corn flour liquefied liquid, 1g/L of monopotassium phosphate, 0.3g/L of calcium oxide and the balance of water, and sterilizing for 30min at the temperature of 123 ℃ with the pH value of 7.5 and 121-.

4. The method of fermentative production of trypsin according to claim 2, wherein said fermentation medium consists of: 20g/L of bean cake powder, 20g/L of glucose, 20g/L of corn steep liquor, 1g/L of magnesium sulfate, 1g/L of monopotassium phosphate and the balance of water, and sterilizing at the temperature of 121 ℃ for 30min at the pH value of 7.5.

5. The method for producing trypsin by fermentation according to claim 2, wherein the final fermentation broth is subjected to extraction and refining processes to obtain a finished enzyme preparation of trypsin, which comprises the following steps:

adding 0.1% of sodium diacetate preservative into the final fermentation liquor, adding 5% of perlite filter aid, and performing filter pressing to obtain clarified filter-pressed enzyme liquor;

carrying out ultrafiltration concentration on the clarified filter-pressed enzyme liquid by using a 10000 molecular weight ultrafiltration membrane to obtain a concentrated solution;

and adding 30 mass percent of stabilizer and 0.3 mass percent of preservative into the concentrated solution, adjusting the pH to 7.5, and then performing sterilization by using a sterilization membrane to obtain the trypsin finished product liquid enzyme preparation.

6. The method of fermentative production of trypsin according to claim 5, wherein said stabilizer is glycerol and sorbitol in a weight ratio of 2: 1; the preservative is prepared from the following raw materials in a mass ratio of 1: 1 potassium sorbate and sodium diacetate.

7. A trypsin produced by the method of any one of claims 1-6.

The technical field is as follows:

the invention belongs to the field of microorganism or enzyme engineering, and particularly relates to a method for producing trypsin.

Background art:

trypsin (Trypsin, EC 3.4.21.4) belongs to the class of serine proteases, which are typically characterized by a catalytic triad consisting of histidine, aspartic acid and serine amino acid residues. Trypsin, in turn, degrades polypeptide chains by specifically cleaving the carboxy-terminal peptide bond of arginine or lysine and activates other proenzymes in the pancreas. Trypsin is an alkaline protease because of its optimum catalytic pH above 7.0. Trypsin is a commonly used protease in industry, and plays an important role in the fields of medicine, leather processing, agricultural production, food processing and the like.

At present, most of the widely used commercial trypsin comes from animal pancreas, but a plurality of defects or limitations restrict the application of the trypsin. The extraction of trypsin from animal pancreas is a byproduct of the extraction of insulin from pancreas, but with the genetic engineering production of insulin, the cost of simply extracting trypsin from pancreas is greatly increased.

In addition, the pancreas contains a relatively complicated digestive system, inevitably reducing the purity of trypsin (e.g., chymotrypsin) or requiring purification by increasing the cost. Animal-derived trypsin has the hidden danger of carrying animal viruses, serious peculiar smell, religion and other restrictive problems, thereby hindering the application of the animal-derived trypsin in commercialization to a certain extent. Compared with animal-derived trypsin, the microbial trypsin has incomparable advantages because the microorganisms grow rapidly and the culture cost is low, and a large amount of target products can be synthesized in a relatively short time. In addition, the microbial trypsin also has diversity in properties, and the application field of the trypsin is widened. And the yield and catalytic property of the enzyme can be improved on a molecular scale through a genetic engineering technology.

However, the existing trypsin derived from microorganisms is not well applied in industry due to the limitation factors of low catalytic activity, low fermentation level and the like. Therefore, the breeding of the bacterial strain with high trypsin yield and the fermentation method have great significance for the good heat resistance of the fermentation product, the wide range of the catalytic action pH, the low production cost, the large-scale mechanical production and the like.

The invention content is as follows:

the invention aims to provide a Streptomyces sp LDE-79 strain with high yield of thermostable trypsin and a fermentation enzyme production method thereof, and the trypsin has excellent heat-resistant characteristic while ensuring the stability of the Streptomyces sp LDE-79 strain for high yield of trypsin.

The purpose of the invention is realized by the following technical scheme:

a streptomycete with high trypsin yield is specifically streptomycete (Streptomyces sp.) LDE-79, and is deposited in China general microbiological culture Collection center in 09 months 10 and 2019 at the address of: no. 3 of Xilu No.1 of Beijing Kogyo of Chaoyang, China, zip code 100101, the preservation number is CGMCC No. 18646.

The Streptomyces sp LDE-79 is a mutant strain LDE-79 of high-yield thermostable trypsin obtained by mutagenesis breeding of an original strain LDE-A7 through plasma at normal temperature and normal pressure; and (3) colony morphology characteristics: the bacterial colony is round, the bacterial colony is thick, and the mycelium is grey white and wrinkled; convex in the center and powdery.

The invention also aims to provide a method for producing trypsin by fermenting the Streptomyces sp LDE-79, which mainly comprises the following steps:

fermentation culture: inoculating the seed liquid after the expanded culture into a fermentation culture medium according to an inoculation proportion of 10-15% of the volume ratio, at the temperature of 30-32 ℃, the rotating speed of 400-: 0.4-0.5vvm from 0h to 6h, 0.6-0.8vvm from 6h to 15h, 1.0-1.2vvm from 20h to the end of fermentation, controlling the pH of the fermentation liquid by coordinately adding a supplementary culture medium, or liquid ammonia, or phosphoric acid in the fermentation process, keeping the pH of the fermentation liquid within the range of 7.5-7.8, ending the fermentation when the enzyme activity is slowly increased or not increased and the thalli are deformed and autolyzed, wherein the fermentation period is 128 h; when the fermentation is finished, the enzyme activity of trypsin in the fermentation liquor reaches 252U/mL to 260U/mL;

and (4) carrying out extraction and refining processes on the final fermentation liquor to prepare the trypsin finished enzyme preparation.

The fermentation medium consists of (g/L): 20 parts of bean cake powder, 20 parts of glucose, 20 parts of corn steep liquor, 1 part of magnesium sulfate, 1 part of monopotassium phosphate and the balance of water, wherein the pH value is 7.5, and the soybean cake is sterilized at 121 ℃ for 30 min;

the feed medium consists of (g/L): 40 parts of corn steep liquor, 120 parts of corn powder liquefied liquid, 1 part of monopotassium phosphate, 0.3 part of calcium oxide and the balance of water, and sterilizing for 30min at the temperature of 123 ℃ under the conditions of pH7.5 and 121-;

the extraction and refining method of the trypsin comprises the following steps:

adding 0.1% of sodium diacetate preservative into the final fermentation liquor, adding 5% of perlite filter aid, and performing filter pressing to obtain clarified filter-pressed enzyme liquor;

carrying out ultrafiltration concentration on the clarified filter-pressed enzyme liquid by using a 10000 molecular weight ultrafiltration membrane to obtain a concentrated solution;

and adding 30 percent (m/v) of stabilizer and 0.3 percent (m/v) of preservative in percentage by mass into the concentrated solution, adjusting the pH to 7.5, and then performing sterilization by a sterilization membrane to obtain the finished liquid trypsin enzyme preparation.

Preferably, the stabilizer is glycerol and sorbitol in a weight ratio of 2:1, and the preservative is glycerol and sorbitol in a mass ratio of 1: 1 potassium sorbate and sodium diacetate.

The trypsin produced by the invention has the following enzymological properties:

(1) the optimum action pH range is 7.5-9.0, and the optimum pH is 8.0;

(2) the optimal action temperature range is 35-60 ℃, and the optimal temperature is 50 ℃;

(3) temperature stability: the residual enzyme activity is 92% after heat preservation for 1h at 55 ℃, and 85% after heat preservation for 2h at 55 ℃.

Has the advantages that:

according to the invention, a mutant strain Streptomyces sp LDE-79 of high-yield thermostable trypsin is bred by carrying out normal-temperature normal-pressure plasma mutagenesis on an original strain LDE-A7, and the feeding condition of a 50L fermentation tank is optimized in the fermentation process of Streptomyces sp LDE-79, so that the enzyme activity in the final fermentation liquid reaches between 252U/mL and 260U/mL, and the enzyme activity of the trypsin liquid enzyme preparation obtained by extraction and refining reaches over 1000U/mL. And the highest enzyme activity in the final fermentation broth is 120U/mL by feeding and fermenting the streptomycete LDE-79 original strain LDE-A7 through a 50L fermentation tank. Therefore, the streptomyces LDE-79 is obviously higher than the enzyme activity of the original strain of the streptomyces LDE-A7.

The culture medium adopted in the fermentation method of the bacterial strain belongs to raw materials with lower cost, so that the fermentation cost is greatly reduced while the single-batch fermentation level and the production efficiency are improved, and the method has a great promotion effect on industrial large-scale production.

The trypsin obtained by fermenting streptomycete LDE-79 has the optimum pH range of 7.5-9.0, the optimum pH of 8.0, the optimum action temperature range of 35-60 ℃ and the optimum temperature of 50 ℃. And the residual enzyme activity after heat preservation for 1h at 55 ℃ is 92%, and the residual enzyme activity after heat preservation for 2h is 85%, so that the heat resistance is remarkable, the trypsin can be widely applied to high-temperature industrial production, the application range of the trypsin is greatly expanded, and the application value of the trypsin is increased.

Description of the drawings:

FIG. 1: relative enzyme activity at different pH:

FIG. 2: relative enzyme activity at different temperatures:

FIG. 3: relative enzyme activity after heat preservation for 0-3h at 55 ℃.

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples. Unless otherwise specified, the technical means used in the present invention are conventional methods known to those skilled in the art;

in the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

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.