CN110527658B

CN110527658B - Culture medium for promoting rapid growth of streptomyces mobaraensis producing transglutaminase as well as preparation method and application of culture medium

Info

Publication number: CN110527658B
Application number: CN201810502575.1A
Authority: CN
Inventors: 金明飞; 高红亮; 田苏; 常忠义; 陈璐; 张玉如; 武世强; 陈中山
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2018-05-23
Filing date: 2018-05-23
Publication date: 2023-05-12
Anticipated expiration: 2038-05-23
Also published as: CN110527658A

Abstract

The invention discloses a culture medium for promoting rapid growth of Streptomyces mobaraensis producing transglutaminase, which comprises the following components in percentage by mass and volume: soluble starch 16-24 g/L, KNO ₃ 0.8～1.2g/L、MgSO ₄ ·7H ₂ O0.4～0.6g/L、K ₂ HPO ₄ ·3H ₂ O 0.4～0.6g/L、NaCl 0.4～0.6g/L、FeSO ₄ ·7H ₂ 0.008-0.012 g/L of O, 0.1-2 g/L of basic amino acid, 20g/L of agar and pH value of 6-8. The invention adopts the Streptomyces mobaraensis producing transglutaminase as an experimental strain, and the thickness of the lawn cultivated for four days is not obviously different from that of the lawn cultivated for seven days on a control medium without adding alkaline amino acid on the prepared culture medium. The four-day-grown lawn on the culture medium is subjected to seed and fermentation culture, and the yield of the obtained transglutaminase is not obviously different from that of a seven-day-cultured control group. The invention obviously reduces the growth time of the streptomyces mobaraensis producing the transglutaminase and greatly improves the industrial production efficiency.

Description

Culture medium for promoting rapid growth of streptomyces mobaraensis producing transglutaminase as well as preparation method and application of culture medium

Technical Field

The invention belongs to the field of microorganism breeding, and particularly relates to a culture medium for promoting rapid growth of Streptomyces mobaraensis producing transglutaminase, a preparation method and application thereof.

Background

Glutamine transferase (TGase, EC 2.3.2.13) catalyzes the intramolecular and intermolecular acyl transfer reactions of proteins to obtain good functional properties. Glutamine transglutaminase is approved by the FDA as a "generic safety certification (GRAS)" additive, widely used in the fields of food, textile, medicine, etc., and the presence of TGase was discovered successively in animals, plants, microorganisms, wherein TGas produced by microbial fermentatione because of not depending on Ca ²⁺ The method has the advantages of low cost and the like, and is applied to industrial production.

Streptomyces mobaraensis is the main production strain for industrial production of TGase, and MTG produced by the Streptomyces mobaraensis creates huge commercial value. However, the growth cycle of Streptomyces mobaraensis is relatively long, tian Shucui and the like report that after Streptomyces mobaraensis grows on a plate of Gao's No. one for seven days, the lawn is relatively thick and can be used for subsequent expansion culture (Tian Shucui, niu Yanning and the like, J.China bioengineering, 2016 (09): 47-53), 9.5 days are required for finally obtaining the transglutaminase, and the time cost in industrial production is relatively high.

Amino acid as a growth factor has great influence on the growth of microorganisms and the accumulation of target products, and many students can promote the growth of thalli and enzyme production by adding amino acid into a fermentation medium, but no report has been made so far that amino acid is added into a Gao's first plate to promote the growth of thalli so as to shorten the growth period.

Disclosure of Invention

The invention aims to provide a culture medium for promoting the rapid growth of Streptomyces mobaraensis producing transglutaminase, which shortens the growth time of Streptomyces mobaraensis, is beneficial to obviously shortening the culture period in the industrial production process, reducing the production cost and improving the culture efficiency.

The invention aims at providing a culture medium for promoting the rapid growth of Streptomyces mobaraensis (Streptomyces mobaraensis) producing transglutaminase, and a preparation method and a use method thereof.

In order to solve the technical problems, the invention provides the following technical scheme: the invention provides a culture medium for promoting rapid growth of Streptomyces mobaraensis producing transglutaminase, which comprises the following components in percentage by mass and volume: soluble starch 16-24 g/L, KNO ₃ 0.8～1.2g/L、MgSO ₄ ·7H ₂ O 0.4～0.6g/L、K ₂ HPO ₄ ·3H ₂ O 0.4～0.6g/L、NaCl 0.4～0.6g/L、FeSO ₄ ·7H ₂ 0.008-0.012 g/L of O, 0.1-2 g/L of basic amino acid, 20g/L of agar, and pH value: 6 to 8.

Preferably, it is a cocoaSoluble starch 20g/L, KNO ₃ 1g/L、MgSO ₄ ·7H ₂ O 0.5g/L、K ₂ HPO ₄ ·3H ₂ O 0.5g/L、NaCl 0.5g/L、FeSO ₄ ·7H ₂ 0.01g/L of O, 20g/L of agar, 0.1-2 g/L of basic amino acid and pH value of 7.4.

Wherein the amino acid is one or more of arginine, lysine and histidine; preferably, it is a complex amino acid of arginine, lysine, histidine.

Further preferably, the culture medium for promoting the rapid growth of the streptomyces mobaraensis producing the transglutaminase comprises the following components in percentage by mass and volume:

soluble starch 20g/L, KNO ₃ 1g/L、MgSO ₄ ·7H ₂ O 0.5g/L、K ₂ HPO ₄ ·3H ₂ O 0.5g/L、NaCl 0.5g/L、FeSO ₄ ·7H ₂ 0.01g/L of O, 20g/L of agar, 0.5g/L of arginine or lysine or histidine and 7.4 of pH value.

The invention also provides a preparation method of the culture medium (1L), which comprises the following specific steps:

(1) Weighing soluble starch and KNO ₃ 、MgSO ₄ ·7H ₂ O、K ₂ HPO ₄ ·3H ₂ O、NaCl、FeSO ₄ ·7H ₂ O, agar.

(2) After the volume is complemented to 1L by distilled water, the pH value is adjusted; sterilizing, putting into an oven for one hour at constant temperature, and adding the basic amino acid which is filtered and sterilized.

(3) After 1L of the prepared culture medium was mixed uniformly, the mixture was poured into each dish in a volume of 20 mL/dish and allowed to cool to room temperature.

Soluble starch and KNO in the 1L culture medium ₃ 、MgSO ₄ ·7H ₂ O、K ₂ HPO ₄ ·3H ₂ O、NaCl、FeSO4·7H ₂ The mass of O and agar is respectively 16-24 g of soluble starch and KNO ₃ 0.8～1.2g、MgSO ₄ ·7H ₂ O 0.4～0.6g、K ₂ HPO ₄ ·3H ₂ O 0.4～0.6g、NaCl 0.4～0.6g、FeSO ₄ ·7H ₂ O0.008-0.012 g, basic amino group0.1-2 g of acid and 20g of agar; preferably, it is 20g of soluble starch and KNO ₃ 1g，MgSO ₄ ·7H ₂ O 0.5g，K ₂ HPO ₄ ·3H ₂ O 0.5g，NaCl 0.5g，FeSO ₄ ·7H ₂ O0.01 g and agar 20g.

In the step (2), the pH value is 6-8; preferably 7.4.

In the step (2), the sterilization condition is that the high temperature and high pressure sterilization is carried out for 20min at the temperature of 121 ℃.

In the step (2), the temperature of the oven is 65-75 ℃; preferably 65 ℃.

In the step (2), the basic amino acid is one or a combination of more of arginine, lysine and histidine; preferably, it is a complex amino acid of arginine, lysine, histidine.

The content of the basic amino acid in the 1L culture medium is 0.1-2 g/L; preferably 0.5g/L.

In the culture medium prepared by the method, the Streptomyces mobaraensis reaches the optimal state after the Streptomyces mobaraensis grows to the fourth day, and the thickness of the lawn of the Streptomyces mobaraensis after four days of culture is not much different from that of the lawn of the Streptomyces mobaraensis cultured for seven days on a control culture medium without adding basic amino acid; in addition, the enzyme activity of the streptomyces mobaraensis after four days of culture is not obviously different from that of the streptomyces mobaraensis after seven days of culture in a control group.

The invention also provides a culture medium prepared by the preparation method.

The invention also provides a fungus lawn of the streptomyces mobaraensis for culturing transglutaminase.

Amino acid is used as an important biological small molecule, plays a key role in the growth of thalli in the microbial fermentation process, and the type and the content of the amino acid also influence the growth rate of the thalli. The three amino acids used in the present invention function as follows: arginine participates in the synthesis of various nutrient substances (protein, pyrimidine and the like) in the growth process of thalli, so that the biomass of the thalli is improved; lysine promotes cell growth and promotes Fe in the medium ²⁺ Can also cooperate with other nutrientsThe effects of the nutrient components are better exerted; histidine is involved in the composition of protein structure and function during cell growth.

The culture medium provided by the invention is used for culturing the Streptomyces mobaraensis, the growth period of the Streptomyces mobaraensis on the culture medium is only four days, and compared with the growth period (seven days) of the Streptomyces mobaraensis on the culture medium in the prior art, the culture medium provided by the invention has the beneficial effects that the growth period of the Streptomyces mobaraensis is effectively shortened, namely, nearly half of the growth period is shortened; according to the normal culture flow, the total time required from the plate culture to the seed culture and the fermentation culture of the streptomyces mobaraensis is 9.5 days, and the time for obtaining the transglutaminase is shortened to 6.5 days by adopting the plate culture medium, if the culture medium is applied to industrial production, the production period of the transglutaminase can be obviously shortened, the time cost is reduced, the product competitiveness is increased, and thus, the higher economic value is realized.

Drawings

FIG. 1 is a graph showing the results of verification of the growth state of Streptomyces mobaraensis on a plate of the corresponding amino acid in each example, and FIG. 1a is a graph showing the growth state of Streptomyces mobaraensis cultured on a control plate for four days; FIG. 1b shows the growth of Streptomyces mobaraensis grown on control plates for seven days; FIG. 1c shows the growth of Streptomyces mobaraensis grown on arginine (0.5 g/L) supplemented plates for four days; FIG. 1d shows the growth of Streptomyces mobaraensis grown on lysine (1 g/L) plates for four days; FIG. 1e shows the growth of Streptomyces mobaraensis grown on histidine (2 g/L) plus plates for four days; FIG. 1f shows the growth of Streptomyces mobaraensis grown on plates with addition of complex amino acids (arginine, lysine 0.25g/L each) for four days; FIG. 1g shows the growth state of Streptomyces mobaraensis cultivated on plates with addition of complex amino acids (lysine, histidine each 0.5 g/L) for four days; FIG. 1h shows the growth of Streptomyces mobaraensis grown on plates with addition of complex amino acids (arginine, lysine, histidine each at 0.1 g/L) for four days.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, to which the present invention is not limited. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims. The procedures, conditions, reagents, test methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for those specifically mentioned below, and the present invention is not particularly limited.

The preservation, bacterial seed and fermentation culture and enzyme activity detection methods of Streptomyces mobaraensis in the following examples are as follows:

preservation of Streptomyces mobaraensis

The Streptomyces mobaraensis (CGMCC No. 10804) bacterial liquid is coated on a first Gaoshi plate and cultured for seven days at 28 ℃. Adding proper amount of sterile water on the flat plate, scraping spores on the flat plate by using a gun head, mixing spore suspension with 40% glycerol at a ratio of 1:1, and placing into a glycerol pipe for preservation at-80 ℃.

Seed liquid acquisition

Taking out the lawn plate (control plate culture for four days and seven days, plate culture with alkaline amino acid added) cultured on the plate culture medium, scraping off the lawn, adding 2mL of sterile water to prepare a bacterial suspension, sucking 1mL of the bacterial suspension into a 250mL triangular flask filled with 30mL of seed culture medium, and culturing at 30 ℃ for 24h at 200r/min to obtain a seed solution.

Shaking flask fermentation culture

The seed solution was transferred to a 250mL Erlenmeyer flask containing 30mL of fermentation medium at an inoculum size of 10%, and cultured at 30℃for 36 hours at 200r/min, whereby the enzyme activity was measured.

Determination of the enzymatic Activity of Glutamine Transaminase

Preparation of solution A (500 mL): 3.475g of hydroxylamine hydrochloride, 11.22g of Tirs, 5.06g of N-alpha-CBZ-Gln-Gly and 1.536g of reduced glutathione are weighed, dissolved in 400mL of distilled water, pH value is regulated to 6.0 by using 12mol/L of concentrated hydrochloric acid, the volume is regulated to 500mL, and the mixture is stored in a brown bottle and stored at 4 ℃.

Preparation of liquid B (3L): (1) 250mL of concentrated hydrochloric acid with the concentration of 12mol/L is measured, and distilled water is used for constant volume to 1L; (2) 50g FeCl was weighed out ₂ ·6H ₂ O is dissolved in 800mL distilled water, and using distilled water to fix volume to 1L; (3) 120g of trichloroacetic acid was weighed out and dissolved in 800mL of distilled water, and the volume was set to 1L with distilled water. The reagents in (1), 2) and (3) were mixed in equal volumes, filtered and stored in a brown bottle at room temperature.

1mL of fermentation broth is sucked into a 2mL plastic centrifuge tube, and centrifuged at 6000r/min for 5min, and the supernatant is collected. Meanwhile, the solution A is placed in a water bath kettle at 37 ℃ for preheating for 5min, 100 mu L of supernatant is sucked into a 10mL test tube, the test tube is placed in a test tube rack, 2mL of preheated solution A is added every 10s in the water bath kettle, 2mL of solution A is also added in a reference tube for reaction for 10min, 2mL of solution B is added every 10s according to the sequence of adding the solution A, and 2mLB of solution is also added in the reference tube. Determination of absorbance A at 525nm Using a Spectrophotometer ₅₂₅ . The enzyme activity was calculated by taking the following equation.

Where k and b are the slope and intercept of the standard curve, V is the amount of enzyme added (mL), and T is the reaction time (min).

One enzyme activity unit is defined as the amount of enzyme required to produce L mol of L-glutamic acid-Y-monohydroxy hydroxamic acid after reaction at 37℃for 1 min.

EXAMPLE 1 control group

The plate culture medium No.1 of the control example without adding basic amino acid is prepared according to the following components in percentage by volume:

soluble starch 20g/L, KNO ₃ 1g/L、MgSO ₄ ·7H ₂ O 0.5g/L、K ₂ HPO ₄ ·3H ₂ O 0.5g/L、NaCl 0.5g/L、FeSO ₄ ·7H ₂ O0.01 g/L, agar 20g/L, pH:7.4.

the method for preparing 1L of the culture medium comprises the following steps:

1. weighing 20g of soluble starch and KNO ₃ 1g、MgSO ₄ ·7H ₂ O 0.5g、K ₂ HPO ₄ ·3H ₂ O 0.5g、NaCl 0.5g、FeSO ₄ ·7H ₂ O0.01 g and agar 20g.

2. The above components were added and dissolved in 900mL of distilled water, and the volume was made up to 1L with distilled water, and the pH was adjusted to 7.4. Sterilizing at 121deg.C under high temperature and high pressure for 20min.

3. After the prepared culture medium was mixed uniformly, the mixture was poured into each culture dish in a volume of 20 mL/dish and allowed to cool to room temperature.

4. The freeze-dried liquid of the streptomyces mobaraensis stored in the glycerol pipe is coated on a flat plate, the flat plate prepared in the step 3 is adopted for culturing for four days at 28 ℃, and the growth states of the streptomyces mobaraensis are respectively shown in figure 1 a.

5. The bacterial cells cultured on the plate for four days are scraped off, seed and fermentation culture are respectively carried out according to a seed culture method and a shake flask fermentation culture method, and enzyme activity is measured according to a transglutaminase enzyme activity measuring method.

EXAMPLE 2 control group

4. The freeze-dried liquid of the streptomyces mobaraensis stored in the glycerol pipe is coated on a flat plate, the flat plate prepared in the step 3 is adopted for culturing for seven days at 28 ℃, and the growth states of the streptomyces mobaraensis are respectively shown in figure 1 b.

5. The cells cultured on the plate for seven days were scraped off, respectively, and seed and fermentation culture were performed according to the seed culture method and the shake flask fermentation culture method, respectively, and the enzyme activity was measured according to the transglutaminase enzyme activity measurement method.

Example 3 preferred embodiment

The invention prepares a No. 3 culture medium containing arginine for promoting the rapid growth of streptomyces mobaraensis producing transglutaminase, which comprises the following components in percentage by mass and volume:

soluble starch 20g/L, KNO ₃ 1g/L、MgSO ₄ ·7H ₂ O 0.5g/L、K ₂ HPO ₄ ·3H ₂ O 0.5g/L、NaCl 0.5g/L、FeSO ₄ ·7H ₂ O0.01 g/L, agar 20g/L, arginine 0.5g/L, pH:7.4.

2. The above components were added and dissolved in 950mL of distilled water, respectively, and the pH was adjusted to 7.4. Sterilizing at 121deg.C under high temperature and high pressure for 20min, and placing into a 65deg.C oven for one hour.

3. 100mL of arginine solution at a concentration of 10g/L was prepared and sterilized by filtration through a 0.22 μm microporous filter.

4. 50mL of arginine mother liquor is sucked and added into the constant temperature culture medium, and after the prepared culture medium is uniformly mixed, the mixture is poured into each culture dish in a volume of 20 mL/dish to be cooled to room temperature.

5. The freeze-dried solution of the streptomyces mobaraensis stored in the glycerol pipe is coated on an arginine flat plate, the flat plate prepared in the step 4 is adopted, and the flat plate is cultivated for four days at the temperature of 28 ℃, and the growth state of the streptomyces mobaraensis is shown in a figure 1 c.

6. The bacterial cells cultured on the arginine plate for four days were scraped, seed and fermentation culture were performed according to a seed culture method and a shake flask fermentation culture method, respectively, and the enzyme activity was measured according to a transglutaminase enzyme activity measurement method, and the enzyme activity results are shown in Table 1.

Example 4 preferred embodiment

The preparation method of the culture medium No. 4 containing lysine for promoting the rapid growth of streptomyces mobaraensis producing transglutaminase comprises the following components in percentage by mass and volume:

soluble starch 20g/L, KNO ₃ 1g/L、MgSO ₄ ·7H ₂ O 0.5g/L、K ₂ HPO ₄ ·3H ₂ O 0.5g/L、NaCl 0.5g/L、FeSO ₄ ·7H ₂ 0.01g/L of O, 20g/L of agar and 1g/L of lysine, and adjusting the pH to 7.4.

3. 50mL of a lysine solution having a concentration of 20g/L was prepared and sterilized by filtration through a 0.22 μm microporous filter.

4. 50mL of lysine mother liquor is sucked and added into the culture medium with constant temperature, and after the prepared culture medium is uniformly mixed, the mixture is poured into each culture dish with the volume of 20 mL/dish to be cooled to room temperature.

5. The freeze-dried solution of the streptomyces mobaraensis stored in the glycerol pipe is coated on a lysine flat plate, the flat plate prepared in the step 4 is adopted, and the flat plate is cultivated for four days at the temperature of 28 ℃, and the growth state of the streptomyces mobaraensis is shown in a figure 1 d.

6. The bacterial cells cultured on the lysine plate for four days were scraped, seed and fermentation culture were performed according to a seed culture method and a shake flask fermentation culture method, respectively, and the enzyme activity was measured according to a transglutaminase enzyme activity measurement method, and the enzyme activity results are shown in Table 1.

Example 5 preferred embodiment

The preparation method of the culture medium No. 5 containing histidine for promoting the rapid growth of streptomyces mobaraensis producing transglutaminase comprises the following components in percentage by mass and volume:

soluble starch 20g/L, KNO ₃ 1g/L、MgSO ₄ ·7H ₂ O 0.5g/L、K ₂ HPO ₄ ·3H ₂ O 0.5g/L、NaCl 0.5g/L、FeSO ₄ ·7H ₂ O0.01 g/L, agar 20g/L, histidine 2g/L, pH:7.4.

3. 50mL of histidine solution at a concentration of 40g/L was prepared and sterilized by filtration through a 0.22 μm microporous filter.

4. 50mL of histidine mother liquor is added into the culture medium with constant temperature, and after the prepared culture medium is uniformly mixed, the mixture is poured into each culture dish with the volume of 20 mL/dish to be cooled to room temperature.

5. The freeze-dried liquid of Streptomyces mobaraensis stored in the glycerol pipe is coated on a histidine plate, the plate is prepared by the step 4, and the plate is cultivated for four days at 28 ℃, and the growth state of the Streptomyces mobaraensis is shown in figure 1 e.

6. The bacterial cells cultured on the histidine plate for four days were scraped, seed and fermentation culture were performed respectively according to a seed culture method and a shake flask fermentation culture method, and the enzyme activity was measured according to a transglutaminase enzyme activity measurement method, and the enzyme activity results are shown in Table 1.

Example 6 preferred embodiment

The preparation method of the culture medium No. 6 containing arginine and lysine for promoting the rapid growth of streptomyces mobaraensis producing transglutaminase comprises the following components in percentage by mass and volume:

soluble starch 20g/L, KNO ₃ 1g/L、MgSO ₄ ·7H ₂ O 0.5g/L、K ₂ HPO ₄ ·3H ₂ O 0.5g/L、NaCl 0.5g/L、FeSO ₄ ·7H ₂ 0.01g/L of O, 20g/L of agar, 0.25g/L of arginine, 0.25g/L of lysine, pH:7.4.

1. weighing 20g of soluble starch and KNO ₃ 1g、MgSO ₄ ·7H ₂ O 0.5g、K ₂ HPO ₄ ·3H ₂ O 0.5g、NaCl 0.5g、FeSO ₄ ·7H ₂ 0.01g of O and 20g of agar strips.

3. 50mL of arginine and lysine solutions were prepared and sterilized by filtration through a 0.22 μm microporous filter.

4. 50mL of the amino acid mother solution is sucked and added into the culture medium with constant temperature, and after the prepared culture medium is uniformly mixed, the mixture is poured into each culture dish with the volume of 20 mL/dish for cooling to room temperature.

5. The freeze-dried liquid of Streptomyces mobaraensis stored in the glycerol pipe is coated on the composite amino acid plate, the plate prepared in the step 4 is adopted, and the plate is cultivated for four days at 28 ℃, and the growth state of the Streptomyces mobaraensis is shown in figure 1 f.

6. The bacterial cells cultured on the composite amino acid flat plate for four days are scraped, seed and fermentation culture are respectively carried out according to a seed culture method and a shake flask fermentation culture method, and the enzyme activity is measured according to a transglutaminase enzyme activity measuring method, and the enzyme activity results are shown in Table 1.

Example 7 preferred embodiment

The preparation method of the culture medium No. 7 containing lysine and histidine for promoting the rapid growth of streptomyces mobaraensis producing transglutaminase comprises the following components in percentage by mass and volume:

soluble starch 20g/L, KNO ₃ 1g/L、MgSO ₄ ·7H ₂ O 0.5g/L、K ₂ HPO ₄ ·3H ₂ O 0.5g/L、NaCl 0.5g/L、FeSO ₄ ·7H ₂ 0.01g/L of O, 20g/L of agar, 0.5g/L of lysine, 0.5g/L of histidine, pH:7.4.

3. 50mL of a solution of lysine and histidine, each 10g/L, was prepared and sterilized by filtration through a 0.22 μm microporous filter.

5. The freeze-dried liquid of the streptomyces mobaraensis stored in the glycerol pipe is coated on the composite amino acid flat plate, the flat plate prepared in the step 4 is adopted, and the flat plate is cultivated for four days at the temperature of 28 ℃, and the growth state of the streptomyces mobaraensis is shown as figure 1 g.

Example 8 preferred embodiment

The preparation method of the No. 8 culture medium containing arginine, lysine and histidine for promoting the rapid growth of the streptomyces mobaraensis producing the transglutaminase comprises the following components in percentage by mass and volume:

soluble starch 20g/L, KNO ₃ 1g/L、MgSO ₄ ·7H ₂ O 0.5g/L、K ₂ HPO ₄ ·3H ₂ O 0.5g/L、NaCl 0.5g/L、FeSO ₄ ·7H ₂ 0.01g/L of O, 20g/L of agar, 0.1g/L of arginine, 0.1g/L of lysine and 0.1g/L of histidine, pH:7.4.

1. weighing soluble starch20g、KNO ₃ 1g、MgSO ₄ ·7H ₂ O 0.5g、K ₂ HPO ₄ ·3H ₂ O 0.5g、NaCl 0.5g、FeSO ₄ ·7H ₂ 0.01g of O and 20g of agar strips.

3. 50mL of arginine, lysine and histidine solutions were prepared, each at 2g/L, and filtered through a 0.22 μm microporous filter.

5. The freeze-dried liquid of the streptomyces mobaraensis stored in the glycerol pipe is coated on a composite amino acid flat plate, the flat plate prepared in the step 4 is adopted, and the flat plate is cultivated for four days at the temperature of 28 ℃, and the growth state of the streptomyces mobaraensis is shown in a figure 1 h.

The thickness and the enzyme activities of the lawn of the Streptomyces mobaraensis obtained in examples 1 to 8 above are shown in Table 1 below:

table 1 results of measurement of lawn thickness and enzyme Activity (U/mL) in examples 1 to 8

Note that: "-" means thinner, "+" means thick, and "++" means very thick

Seven days after culturing Streptomyces mobaraensis producing transglutaminase in the medium of comparative example 2, seed and fermentation were performed, and enzyme activity was measured. Four days after culturing Streptomyces mobaraensis producing transglutaminase in the culture medium of example 1 and the culture medium of examples 3 to 7, the enzyme activities were measured after seed and fermentation, and are shown in Table 1. As can be seen from Table 1, the enzyme activities of comparative example 2 were not significantly different from those of examples 3 to 7 of the present invention, and the thickness of the Streptomyces mobaraensis after four days of culture in examples 4 to 6 of the present invention was not much different from that of the Streptomyces mobaraensis after seven days of culture in the control medium without addition of basic amino acids, but the thickness of the Streptomyces mobaraensis after four days of culture in examples 3,7 and 8 of the present invention was significantly higher than that of the Streptomyces mobaraensis after seven days of culture in the control medium without addition of basic amino acids. In addition, the thickness of the Streptomyces mobaraensis lawn after four days of culture in examples 3-8 of the present invention was significantly higher than that of the lawn after four days of culture on the control medium without the addition of basic amino acids. The culture medium can obviously shorten the growth period of the streptomyces mobaraensis producing the transglutaminase; the time cost in production is reduced.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.

Claims

1. A culture medium for promoting rapid growth of streptomyces mobaraensis producing transglutaminase in an activation stage is characterized by comprising the following components in percentage by mass and volume:

soluble starch 16-24 g/L, KNO ₃ 0.8~1.2 g/L、MgSO ₄ ·7H ₂ O 0.4~0.6 g/L、 K ₂ HPO ₄ ·3H ₂ O 0.4~0.6 g/L、NaCl 0.4~0.6 g/L、FeSO ₄ ·7H ₂ 0.008-0.012 g/L of O, 0.3-2 g/L of basic amino acid, 20-g/L of agar and pH value of 7.4; the basic amino acid is one or a combination of more of arginine, lysine and histidine;

the preservation number of the streptomyces mobaraensis is CGMCC No.10804.

2. The culture medium according to claim 1, wherein the culture medium comprises the following components in percentage by mass and volume:

solubility ofStarch 20g/L, KNO ₃ 1 g/L、MgSO ₄ ·7H ₂ O 0.5 g/L、K ₂ HPO ₄ ·3H ₂ O 0.5 g/L、NaCl 0.5 g/L、FeSO ₄ ·7H ₂ O0.01 g/L, basic amino acid 0.5g/L, agar 20g/L and pH 7.4.

3. The method for preparing the culture medium according to claim 1 or 2, comprising the following specific steps:

(1) Weighing soluble starch and KNO ₃ 、MgSO ₄ ·7H ₂ O、K ₂ HPO ₄ ·3H ₂ O、NaCl、FeSO4·7H ₂ O, agar;

(2) After the volume is complemented to 950mL by distilled water, the pH value is adjusted; sterilizing, putting the mixture into a baking oven for one hour at constant temperature, and adding basic amino acid which is filtered and sterilized; the basic amino acid is one or a combination of more of arginine, lysine and histidine;

(3) After 1L of the prepared culture medium was mixed, the mixture was poured into each dish at a volume of 20 mL/dish and allowed to cool to room temperature.

4. The preparation method according to claim 3, wherein the 1L of the medium comprises soluble starch and KNO ₃ 、MgSO ₄ ·7H ₂ O、K ₂ HPO ₄ ·3H ₂ O、NaCl、FeSO ₄ ·7H ₂ The mass of O and agar is 16-24 g of soluble starch and KNO respectively ₃ 0.8~1.2 g、MgSO ₄ ·7H ₂ O 0.4~0.6 g、 K ₂ HPO ₄ ·3H ₂ O 0.4~0.6 g、NaCl 0.4~0.6 g、FeSO ₄ ·7H ₂ 0.008-0.012 g of O, 0.3-2 g of basic amino acid and 20-g g of agar.

5. A formulation method according to claim 3, wherein in step (2), the pH is 7.4; the sterilization condition is that the high temperature and high pressure sterilization is carried out for 20min at the temperature of 121 ℃; the temperature of the oven is 65-75 ℃.

6. The preparation method of claim 3, wherein in the step (3), the mass of the basic amino acid in the 1L medium is 0.3-2 g.

7. The medium according to claim 1 or 2 for culturing a lawn of Streptomyces mobaraensis producing transglutaminase.