CN110777096B - Streptomyces capable of producing trypsin with high yield and application thereof - Google Patents

Abstract

The invention belongs to the field of microbial or enzyme engineering, and particularly relates to a streptomycete capable of producing trypsin with high thermal stability and application thereof. The strain is Streptomyces sp LDE-79 with the preservation number of CGMCC No. 18646. 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

Streptomyces capable of producing trypsin with high yield and application thereof

The technical field is as follows:

the invention belongs to the field of microbial or enzyme engineering, and particularly relates to a streptomycete capable of producing trypsin with high thermal stability and application thereof.

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.

Example 1 mutagenesis of Streptomyces sp LDE-79

Inoculating original strain LDE-A7 to fresh solid culture medium, culturing at constant temperature of 30 deg.C to obtain colony, scraping spore with inoculating loop into triangular flask containing glass beads, shaking to disperse spore, and preparing spore suspension with spore concentration controlled at 10⁷-10⁸One per ml.

Starting an ARTP system, sterilizing and disinfecting the inside and outside of the operation room by alcohol, starting an ultraviolet lamp for irradiation sterilization, taking 10 mu L of bacterial suspension after sterilization is finished, dripping the bacterial suspension on the rough surface of the slide glass, and moving the slide glass into an operation room table under the aseptic condition. The valve is opened, nitrogen is introduced, and the air flow and the mutagenesis time are set for mutagenesis. The mutagenesis time was set to 20s, 30s, 40s, 50s, and 60s, respectively. After each mutagenesis, the mutagenized spores were transferred to an EP tube containing sterile physiological saline and collected by centrifugation.

EXAMPLE 2 screening of strains

The mutation breeding culture medium is as follows:

solid prescreening medium (g/L): skimmed milk powder 20, sodium chloride 1, magnesium sulfate 0.2, agar 20 and the balance of water, wherein the pH value is 7.5;

solid streaking medium (g/L): 10 parts of dried egg white, 20 parts of glucose, 1 part of sodium chloride, 20 parts of agar and the balance of water, wherein the pH value is 7.5;

shake flask rescreening culture medium: 12 parts of dried egg white, 5 parts of yeast extract, 0.8 part of sodium sulfate, 0.5 part of magnesium sulfate, 0.3 part of calcium oxide, 0.5 part of sodium hydrogen phosphate and the balance of water, wherein the pH value is 7.5, and the mixture is sterilized at 121 ℃ for 20 min.

Diluting the mutagenized spore by 10³-10⁷Doubling, spreading on a solid primary screening culture medium, and culturing in an incubator at 30 ℃. Primarily qualitatively screening single bacterial colony with typical characteristics of large transparent ring, shape and color by using a transparent ring method, carrying out streak separation and purification on a solid streak culture medium, repeating for 3 times, selecting typical single bacterial colony, streaking on a test tube inclined plane of the solid culture medium, culturing at 30 ℃ for 72h, and storing in a 4 ℃ refrigerator.

Shake flask enzyme production rescreening of strain: inoculating the strains qualitatively selected after mutagenesis into a shake flask re-screening culture medium, then carrying out shake flask fermentation re-screening, inoculating each strain into 5 bottles, inoculating into a 500mL triangular flask containing 100mL enzyme production culture medium, carrying out shake culture at 30 ℃, 260r/min for 62h, centrifuging at 5000r/min for 5min, and collecting supernatant to detect the enzyme activity.

5 mutant strains with higher fermentation enzyme activity are screened out again through a shake flask; the enzyme activities of the original strain and the mutant strain with higher enzyme activity are shown in the following table 1.

EXAMPLE 3 genetic stability test for LDE-79 Strain having highest enzymatic Activity

Continuously streaking LDE-79 strain on a flat plate for subculture, respectively inoculating seeds to a shake flask culture medium, culturing at 30 deg.C and 260r/mim for 72h, centrifuging at 5000r/min for 5min, collecting supernatant, and measuring enzyme activity at 25 deg.C and pH8.0. The shake flask fermentation activity of the strain after 10 passages is shown in the following table 2.

Example 4 Streptomyces sp LDE-79 enzyme production by fermentation

A fermentation enzyme production method of Streptomyces sp LDE-79 mainly comprises the following steps:

slant culture: selecting streptomyces with one-loop mutation LDE-79, inoculating to a solid slant culture medium, and culturing at constant temperature of 30 ℃ for 36 h;

and (3) shake flask culture: inoculating a strain cultured on a ring of inclined planes into a seed culture medium, and culturing for 36h at the constant temperature of 30 ℃ and the rotating speed of a shaking table of 260r/min to obtain a seed solution;

seed amplification culture: transferring the shake flask seeds into a seed tank according to the inoculation amount proportion of 5 percent of the volume ratio, and continuously culturing for 18 hours at the culture temperature of 30 ℃ and the rotation speed of 600 r/min;

fermentation culture: inoculating the seed liquid after the expanded culture into a fermentation culture medium according to the inoculum size proportion of 10% in volume ratio, and setting the ventilation rate at 30 ℃ and the rotation speed of 600 r/min: 0.4vvm for 0h to 6h, 0.8vvm for 6h to 15h, and 1.2vvm for 20h to the end of fermentation, wherein the pH of the fermentation broth is controlled by coordinately supplementing a feed medium, liquid ammonia and phosphoric acid in the fermentation process, so that the pH is maintained within the range of 7.5-7.8, the fermentation culture is performed until the enzyme activity is slowly increased or not increased, the fermentation is finished when the thalli are deformed and autolyzed, the fermentation period is 128h, and the trypsin enzyme activity in the fermentation broth reaches 260U/mL when the fermentation is finished.

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

The culture medium used in the above culture process is as follows:

solid slant medium (g/L): 200 parts of potato, 20 parts of glucose, 15 parts of agar and the balance of water, and sterilizing for 20min at 121 ℃ under natural pH;

seed medium (g/L): 12 parts of dried egg white, 5 parts of yeast extract, 0.8 part of sodium sulfate, 0.5 part of magnesium sulfate, 0.3 part of calcium oxide, 0.5 part of sodium hydrogen phosphate and the balance of water, wherein the pH value is 7.5, and the mixture is sterilized at 121 ℃ for 20 min;

seeding tank medium (g/L): bean cake powder 5, glucose 20, yeast extract 12, potassium dihydrogen phosphate 1.2, calcium oxide 0.3, and water in balance, pH7.5, sterilizing at 121 deg.C for 30 min;

fermentation medium (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;

feed medium (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.

The stabilizer is glycerol and sorbitol with the weight ratio of 2:1, and the preservative is prepared from the following components in a mass ratio of 1: 1 potassium sorbate and sodium diacetate.

Example 5 Streptomyces sp LDE-79 fermentation Performance validation

A50L fermentation tank verification experiment is carried out according to the Streptomyces sp LDE-79 fermentation enzyme production and the extraction and purification method of the trypsin produced by the same in example 4, the fermentation period is 120-128h, and the final fermentation liquid enzyme activity (at 25 ℃, pH 8.0) of the fermentation enzyme production test of 6 batches is shown in the following table 3. The data in table 3 show that the strain not only produces trypsin but also has significant stability in fermentation level.

TABLE 36 fermentation enzyme production of batches of high Trypsin producing strains

Fermentation batch	Fermentation period	Final fermentation broth vitality U/ml
			F1	123h	254
F2	125h	258
			F3	120h	253
F4	124h	259
			F5	128h	260
F6	126h	256

Example 6 method for measuring enzyme Activity of Trypsin in accordance with the present invention

Preparation of substrate solution: a certain amount of BAEE is weighed and dissolved in phosphate buffer solution with pH8.0 to prepare BAEE substrate with the concentration of 0.2mM for standby.

The specific determination method comprises the following steps: at 25 ℃, 3mL of substrate solution is added into a quartz cuvette with the optical path of 1cm, 0.2mL of enzyme solution is added, the mixture is immediately mixed, the mixture is put into a spectrophotometer, phosphate buffer solution with the pH of 8.0 is used as blank, the light absorption value is detected at 253nm, and the reading is carried out for 1 time at intervals of 30s for 5 min. And (5) drawing by taking the time (min) as an abscissa and the light absorption value as an ordinate, and performing linear fitting, wherein the slope of the linear fitting is the change rate of the light absorption value per minute. In the measuring process, the time of the linear relation between the time and the light absorption value is not less than 3min, if the time does not meet the requirement, the concentration of the enzyme solution is adjusted, and the measurement is carried out again.

Definition of enzyme activity: at a certain temperature and pH, the absorbance value at 253nm increased by 0.001 per minute by using BAEE as a substrate as an enzyme activity unit.

Wherein:

v is the volume (ml) of the enzyme solution

n is the dilution multiple

Example 7 optimal pH Range for fermentative Trypsin production

The trypsin produced by the invention is used for measuring the enzyme activity under different pH values (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5) respectively at 50 ℃, and a relative enzyme activity change curve is drawn by taking the pH8.0 as 100 percent of the enzyme activity, as shown in figure 1, the optimum action pH range of the enzyme is 7.5-9.0, and the optimum pH is 8.0.

EXAMPLE 8 optimal temperature Range for Trypsin production by fermentation

Taking trypsin with enzyme activity of 186U/mL as a determination object, determining the enzyme activity under the condition that the pH is 8.0 and at different temperatures (25, 30, 35, 40, 45, 50, 55, 60, 65 and 70 ℃), respectively, and drawing a relative enzyme activity change curve by taking the enzyme activity at 50 ℃ as 100 percent as shown in figure 2, wherein the optimal action temperature range of the enzyme is 35-60 ℃, and the optimal temperature is 50 ℃.

Example 9 Trypsin thermotolerance

Taking trypsin with the enzyme activity of 252U/mL as a determination object, carrying out heat preservation for 3h at 55 ℃ under the condition that the pH is 8.0, sampling every 30 minutes, detecting the enzyme activity under the conditions of 50 ℃ and pH8.0, drawing a relative enzyme activity change curve by taking the initial enzyme activity as 100%, and as shown in figure 3, the residual enzyme activity is 92% after heat preservation for 1h at 55 ℃, and the residual enzyme activity is 85% after heat preservation for 2h at 55 ℃, so that the trypsin heat resistance is good, the trypsin heat resistance test paper can be widely applied to high-temperature production, the application range of the trypsin is remarkably widened, and the application value of the trypsin is improved.

Claims (4)

1. A streptomycete with high trypsin yield is characterized in that the strain is streptomycete (Streptomyces:)Streptomyces sp.) LDE-79 with the preservation number of CGMCC No. 18646.

2. The Streptomyces strain (S) of claim 1Streptomyces sp.) Use of LDE-79 in the production of trypsin.

3. A culture medium comprising the Streptomyces (i), (ii) or (iii) of claim 1Streptomyces sp.) Trypsin produced by LDE-79.

4. The trypsin according to claim 3, characterized by the following enzymatic 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 ℃.

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