Glutamine transaminase solid-state fermentation method based on wheat bran and soybean meal

Abstract

The invention relates to a glutamine transaminase solid-state fermentation method based on wheat bran and soybean meal, and belongs to the technical field of microbial fermentation of glutamine transaminase (TG enzyme). At present, a deep liquid fermentation method is generally adopted to produce the TG enzyme industrially, and the problems of high cost of refined synthetic culture medium, large yield of organic wastewater in the fermentation process, high energy consumption, low oxygen transfer efficiency and the like exist, so that the invention aims to solve the problems, obviously reduce the production cost, have environmental friendliness and improve the yield of the TG enzyme. According to the technical scheme, agricultural waste wheat bran and soybean meal are used as main fermentation substrates, strain reviving, seed and solid culture medium preparation, 30-DEG C constant-temperature static culture are conducted for 7 days, deionized water is used as an extracting agent, and absolute ethyl alcohol purification is conducted. The TG enzyme activity of solid state fermentation is 5.19 U / gds. The method has the advantages that compared with TG from liquid fermentation, the TG obtained through solid fermentation has higher thermal stability and salt tolerance in enzymatic property, and in addition, the catalytic temperature and the catalytic pH range of the TG are larger, so that a better catalytic effect can be played in different use scenes; the method has good potential of being applied to a food processing process and other industries.

Description

I. Technical Field

[0001] This invention relates to the field of transglutaminase (TG enzyme) fermentation technology, specifically to solid-state fermentation using inexpensive wheat bran (agricultural waste) and soybean flour (easily available and inexpensive) as fermentation substrates. By screening fermentation substrate components, optimizing fermentation conditions, and improving crude enzyme extraction methods, a low-cost, high-yield TG enzyme was obtained from Streptomyces mobaraensis. The application characteristics of the TG enzyme obtained through solid-state fermentation were characterized. II. Background Technology

[0002] Transglutaminase (TG enzyme), an important protein cross-linking enzyme, can catalyze acyl transfer reactions to create covalent cross-links within and between proteins or peptides, thereby improving protein structure and function. Its unique and excellent properties make it promising for applications in the food, chemical, and pharmaceutical industries. However, its scarcity and high production costs limit its wider application. Currently, deep liquid fermentation is widely used in industry to produce TG enzyme. However, liquid fermentation technology inherently has many insurmountable drawbacks, such as high cost of refining and synthesizing fermentation substrates, large amounts of organic wastewater generated during fermentation, high energy consumption, and low oxygen transfer efficiency. Therefore, adopting new solid-state fermentation technologies is a viable solution.

[0003] Using inexpensive agricultural waste wheat bran and inexpensive soybean flour as substrates, solid-state fermentation technology was employed to produce TG enzymes from *Streptomyces mobaraensis*. Compared with traditional liquid fermentation, this method significantly reduces production costs and is environmentally friendly, making it significant for further optimization and large-scale industrial production of TG enzymes. Solid-state fermentation using polyurethane foam as an inert adsorbent carrier, as a novel solid-state fermentation method, overcomes the shortcomings of traditional solid-state fermentation while retaining the advantages of liquid fermentation. This fermentation strategy has high potential for further development and application value. TG enzymes obtained through solid-state fermentation differ in enzymatic properties from those derived from liquid fermentation, thus exhibiting better catalytic effects in different application scenarios and demonstrating good potential for application in food processing and other industries. III. Summary of the Invention

[0004] (I) Purpose of the Invention

[0005] This invention aims to develop an innovative fermentation method for producing TG enzymes from *S. mobaraensis* using agricultural waste wheat bran and inexpensive soybean flour as fermentation substrates. This method enables efficient TG enzyme production, significantly reducing production costs and demonstrating environmental friendliness compared to liquid fermentation. It holds practical significance for further optimization and scale-up cultivation to achieve large-scale industrial production of TG enzymes. Furthermore, TG enzymes obtained through solid-state fermentation differ in enzymatic properties from those derived from liquid fermentation, thus exhibiting better catalytic effects in various application scenarios. This method has excellent potential for application in food processing and other industries, which will be of great significance for energy conservation and emission reduction, technological upgrading, expanding domestic TG enzyme sources, and the development of TG enzyme and related industries in my country.

[0006] (II) Technical Solution

[0007] To address the above problems, this invention first provides a formulation for S. mobaraensis fermentation substrate. Furthermore, this invention also provides a cultivation method that, through screening fermentation substrate components, optimizing fermentation conditions, and improving crude enzyme extraction methods, fully utilizes the synergistic effect between various components to significantly increase the activity and yield of TG enzyme, thereby obtaining low-cost, high-yield TG enzyme, which is beneficial for improving economic efficiency.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] Based on the first aspect of the present invention, a fermentation substrate formulation is provided:

[0010] Subculture medium: The strains were subcultured using ISP4 medium. ISP4 medium composition (content / g·L) -1 The following mixture is prepared: 20g agar, 10g soluble starch, 1.0g K2HPO4, 1.0g MgSO4·7H2O, 1.0g NaCl, 1.0g (NH4)2SO4, 2.0g CaCO3, 2.0g FeSO4·7H2O, 0.001g MnCl2·7H2O. Dissolve in deionized water, adjust the pH to 7.2-7.4, sterilize at 121℃ for 30min, pour into plates while hot, and let the plates solidify before use.

[0011] Seed culture medium components (content / g·L) 1 ): 3.0g beef extract, 20g peptone, 30g soluble starch, 5.0g NaCl, 2.0g Na2HPO4, 2.0g KH2PO4, 2.0g MgSO4·7H2O, dissolved in deionized water and heated and stirred until the solid components are completely dissolved. Adjust the pH to 7.0 and sterilize for later use.

[0012] Solid-state fermentation substrate components for agricultural waste wheat bran and inexpensive soybean flour (materials calculated based on the addition amount for a 250mL conical flask): Add 5g each of coarse wheat bran and soybean flour to each flask, which are the carbon source and nitrogen source respectively. Add 3g of rice husk as a carrier. Moisten with 10mL of inorganic salt and trace element solution (composition: KH2PO4 5g / L, NH4NO3 5g / L, MgSO4·7H2O 1g / L, NaCl 1g / L, CoCl2·6H2O 1mg / L, MnSO4 0.8mg / L, ZnSO4·7H2O 1.7mg / L, FeSO4·7H2O 2.5mg / L), mix evenly, and sterilize for later use.

[0013] It is worth noting that the solid-state fermentation substrates of wheat bran and soybean flour were improved based on the original method. Wheat bran was selected as the initial substrate, and through one-way ANOVA, the effects of soybean flour addition, inert carrier addition, initial moisture content and inoculum size, and fermentation time on enzyme production were investigated in sequence. The extraction effects of different extractants on crude enzymes, the screening of optimal carbon and nitrogen sources, and the screening of optimal carriers were also investigated. Finally, the composition of the solid-state fermentation substrates of wheat bran and soybean flour, the culture conditions and extraction conditions were obtained, and the optimal process system for TG enzyme production by solid-state fermentation at a scale of 250 mL was determined.

[0014] The synergistic combination of the above formulations can significantly reduce the production cost of TG enzyme. Based on a second aspect of the invention, a solid-state fermentation method for transglutaminase using agricultural waste wheat bran and inexpensive soybean flour as fermentation substrates is also provided, comprising the following steps:

[0015] Step (1): Strain revival

[0016] The *S. mobaraensis* strain used in this invention was purchased from the China General Microbiological Culture Collection Center (CGMCC). The accession number is CGMCC4.1719, and the original accession number is DSM40847. The purchased strain (dry powder) was activated and cultured according to the instructions. The strain was then preserved in the laboratory. The activated *S. mobaraensis* was then propagated. The strain was identified using 16S rDNA. The purchased strain was confirmed to be correct.

[0017] Based on previous culture medium screening experiments in the laboratory, ISP4 medium was found to be optimal for the growth of the strain during subculturing. ISP4 medium was dissolved in deionized water, the pH was adjusted to 7.2-7.4, and the culture was sterilized at 121℃ for 30 minutes. The medium was then poured onto plates while still hot. After the plates solidified, the strain was subcultured using the streak method and incubated at 28℃. After approximately one week, the aerial mycelia gradually turned gray, indicating that the spores were beginning to mature. At this point, the microbial activity was high and the culture could be used for fermentation inoculation.

[0018] Step (2): Seed liquid preparation

[0019] Select a plate with good growth status, containing grayish-brown spores after 7 days of growth. Spores from half of the plate are scraped off using an inoculation spatula and inoculated into a 250mL Erlenmeyer flask containing 100mL of seed culture medium. Incubate at 30℃ and 150rpm in a constant temperature shaker for 48 hours to obtain the fermentation seed culture for subsequent solid-state fermentation inoculation.

[0020] Step (3): Solid-state fermentation of agricultural waste wheat bran and soybean flour

[0021] Through optimization of fermentation conditions, it was determined that the seed culture inoculation volume of 10 mL and the initial water content of 62.5% were most favorable for fermentation. The optimal fermentation time was controlled at 6-7 days, which could both increase enzyme yield and shorten the fermentation cycle. In the extraction of crude enzyme, deionized water had the best extraction effect compared with other buffer solutions and organic solvents, with a relative extraction rate of 112.73%.

[0022] The enzyme production effects of fermentation of different agricultural wastes vary greatly. Substrates with high nitrogen content are more conducive to the production of TG enzyme. Through comparative screening, the optimal fermentation medium consists of wheat bran and soybean flour. The addition of fermentation carrier can effectively improve the physical structure of the medium and thus improve enzyme activity. Comparative experiments showed that the enzyme production effect of nutrient carrier is better than that of inert carrier, and the optimal carrier is coarse wheat bran.

[0023] Based on the preliminary optimization results of the fermentation substrate composition, culture conditions, and extraction conditions from previous experiments, the optimal process system for solid-state fermentation to produce TG enzyme was determined (materials are calculated based on the addition amount of a 250mL Erlenmeyer flask): 5g each of coarse wheat bran and soybean flour were added to each flask as carbon and nitrogen sources, respectively, along with 3g of rice husk as a carrier. The mixture was moistened with 10mL of inorganic salt and trace element solution, thoroughly mixed, and autoclaved at 121℃ for 30min. After cooling, 10mL of seed culture and 5mL of deionized water were inoculated, stirred thoroughly, and then incubated at 30℃ for 7 days, manually mixed every 24 hours. After fermentation, 50mL of 4℃ deionized water was added to each flask for crude enzyme extraction. The mixture was shaken at 150rpm for 30min, filtered through gauze to remove large substrate particles, and centrifuged at 4℃ and 10000rpm. The supernatant was then collected to obtain the crude enzyme solution. The crude enzyme solution and anhydrous ethanol at 4℃ were mixed in equal volumes, allowed to stand for 15 minutes, and then centrifuged at 4℃, 10000 rpm for 10 minutes to obtain protein precipitate. The residual ethanol was washed 2-3 times with deionized water. After freezing at -20℃, the solution was placed in a vacuum freeze dryer for lyophilization to obtain dry powdered crude TG enzyme with an enzyme activity of 311.40 U / g.

[0024] (III) Beneficial Effects

[0025] A 1L glass bottle was used to simulate a fermenter, with 500g of coarse wheat bran and soybean flour added at one time for scale-up culture. Other parameters were scaled up proportionally. To ensure sufficient oxygen supply within the bottle, an air pump and a filter sterilizer were used. The final enzyme activity was 5.19 U / gds. Based on online purchasing prices (coarse wheat bran 6 yuan / kg, soybean flour 10 yuan / kg, rice husk 1.5 yuan / g), the raw material cost per unit of TG enzyme, without considering losses, is approximately 0.002 yuan. Under the same estimated conditions, the raw material cost per unit of TG enzyme for liquid fermentation is approximately 0.009 yuan (calculated at 1 U / mL). The raw material cost for solid-state fermentation TG enzyme is only 22.22% of that for liquid fermentation.

[0026] Most commercially available TG enzymes are obtained through liquid fermentation, but their high price limits their widespread application. To reduce the production cost of TG enzymes and establish an efficient solid-state fermentation system, this study used abundant and inexpensive agricultural waste, wheat bran and soybean meal, as substrates, and employed *S. mobaraensis* for solid-state fermentation to produce TG enzymes. Fermentation conditions, including inoculum size and fermentation time, were optimized, and nutrient substrates and carrier materials were screened. Finally, scale-up cultivation was conducted based on the optimized fermentation conditions and parameters.

[0027] Studies have found that different substrates exhibit significant differences in enzyme production, which may be related to their nutrient composition and pH buffering capacity. High-nitrogen substrates are more conducive to TG enzyme production. Through comparative screening, the optimal fermentation substrate consisted of wheat bran and soybean flour. Adding a carrier effectively improved the physical structure of the fermentation substrate, thereby increasing enzyme activity, with nutrient-rich carriers showing better results than inert carriers. The inoculum size and initial moisture content of the fermentation solution needed to be balanced. An optimal fermentation time of 6 days was deemed suitable. During crude enzyme extraction, deionized water showed better extraction performance compared to other buffers and organic solvents. Finally, a semi-purified extract with high TG enzyme activity was obtained through scale-up culture.

[0028] The results of this study indicate that solid-state fermentation of S. mobaraensis using agricultural waste wheat bran and soybean flour as substrates can efficiently produce TG enzymes. Compared with liquid fermentation, it can significantly reduce production costs and is environmentally friendly. It has practical significance for further optimization and scaling up of the culture, thereby realizing the large-scale industrial production of TG enzymes. IV. Description of the attached drawings

[0029] Figure 1 Determination and scale-up of fermentation system

[0030] Based on the preliminary optimization results of the culture medium composition, culture conditions, and extraction conditions from previous experiments, the optimal process system for solid-state fermentation to produce TG enzyme was determined as follows: 5g each of coarse wheat bran and soybean powder were added to each bottle as carbon and nitrogen sources, respectively, and 3g of rice husk was added as a carrier. The mixture was autoclaved at 121℃ for 30 minutes, cooled, and then inoculated with 10mL of seed culture and 5mL of deionized water. After stirring evenly, the mixture was incubated at 30℃ for 7 days, manually mixed every 24 hours. After fermentation, 50mL of 4℃ deionized water was added to each bottle for crude enzyme extraction. Large particles of substrate were removed by filtration through gauze to obtain the crude enzyme solution. The crude enzyme solution was mixed with anhydrous ethanol at 4℃ in equal volumes, and centrifuged to obtain protein precipitate. After freezing at -20℃, the precipitate was freeze-dried in a vacuum freeze dryer to obtain a dry powdered TG enzyme. A 1L glass bottle was used to simulate a fermenter, with 500g of coarse wheat bran and soybean powder added at a time for scale-up culture, with other parameters scaled up proportionally. To ensure sufficient oxygen in the bottle, an air pump and a filter sterilizer were used to supply oxygen. The final enzyme activity was 5.19 U / gds.

[0031] Figure 2 Effect of temperature on TG enzyme activity

[0032] Temperature significantly affects enzyme activity; enzymes exert their optimal catalytic effect only at their optimal temperature. As shown in the figure, compared to commercially available enzymes, this study demonstrates that solid-state fermentation-derived TG enzymes exhibit superior catalytic effects under both low and high temperature conditions. This may be because commercial enzymes are obtained through liquid fermentation, while the solid-state fermentation environment of TG enzymes better matches the natural habitat of microorganisms, resulting in higher quality secondary metabolites than those produced by deep liquid fermentation under high-density culture, and better enzyme resistance. Glodowsky et al. obtained a cold-active TG enzyme using Penicillium isolated from Antarctica through solid-state fermentation, exhibiting peak activity at 30°C and showing activity even at 4°C. This characteristic can be used to combine raw meat slices under refrigerated conditions to produce reconstituted meat products. In the actual industrial application of TG enzymes, there will inevitably be application scenarios at different temperatures, and enzymes can only exert their optimal catalytic effect at their optimal temperature. Therefore, developing TG enzymes suitable for application in different temperature ranges is of great necessity.

[0033] Different enzymes exhibit significant differences in their heat resistance. TG enzymes, widely used in the food and pharmaceutical industries, face various temperature challenges during storage and processing. Therefore, investigating the thermostability of TG enzymes produced through solid-state fermentation is crucial for better preserving their activity during production.

[0034] Figure 3 Effect of pH on TG enzyme activity

[0035] Both SSF-TG enzyme and the commercial enzyme maintained high enzyme activity within the pH range of 6.0-8.0, with relative activities both above 80%. Under acidic conditions, the relative activity of SSF-TG enzyme was consistently superior to that of the commercial enzyme, reaching 96.60% and 92.95% respectively at pH 6.0. The relative activity decreased more rapidly with increasing acidity. At pH 5.0, the enzyme activities decreased by 28.49% and 32.31% respectively compared to pH 6.0. At pH 4.0, the relative activity of SSF-TG enzyme was 37.31%, while that of the commercial enzyme was only 23.73%. This indicates that SSF-TG enzyme has better acid resistance than the commercial enzyme. Only at pH 8.0 did the relative activity of the commercial enzyme exceed that of SSF-TG enzyme, reaching 91.65% and 85.00% respectively. When pH > 8.0, the excessively alkaline environment causes a rapid decrease in enzyme activity, reaching only 26.92% and 20.44% at pH 10.0, respectively.

[0036] Figure 4 Effect of NaCl on the stability of TG enzyme

[0037] TG enzymes are a promising food additive in the food industry. However, they will inevitably encounter high-salt environments during production. Therefore, exploring their stability in salt solutions is of great practical significance. V. Detailed Implementation Methods

[0038] The present invention will be further illustrated below with specific embodiments. These embodiments are merely illustrative and do not limit the scope of the invention.

[0039] Unless otherwise specified, the raw materials and reagents used in the following embodiments of the present invention can be obtained commercially.

[0040] Subsequent embodiments of the present invention include:

[0041] 1. Determination of the optimal temperature for obtaining TG enzyme by solid-state fermentation in this invention;

[0042] 2. Determination of the thermal stability of TG enzyme obtained by solid-state fermentation in this invention;

[0043] 3. Determination of the optimal temperature for obtaining TG enzyme by solid-state fermentation in this invention;

[0044] 4. Determination of the thermal stability of TG enzyme obtained by solid-state fermentation in this invention.

[0045] Previous studies have demonstrated that TG enzymes from different microorganisms and fermentation methods differ in their molecular structure, physicochemical properties, and other enzymatic characteristics. After collecting and purifying the product enzymes at the end of fermentation, the molecular weight, optimal temperature, thermal stability, optimal pH, and stability in salt solution of the TG enzymes obtained by solid-state fermentation of agricultural waste wheat bran and soybean flour using S. mobaraensis were characterized.

[0046] Measurements revealed a distinct 37 kDa band in the SDS-PAGE electrophoresis results of the solid-state fermentation crude extract, with minimal contaminating proteins and no prozymogen band. The optimal catalytic temperature for this TG enzyme is 37 °C, exhibiting high thermal stability below 40 °C. Its catalytic performance at medium and low temperatures is significantly superior to commercially available enzymes derived from liquid fermentation. Furthermore, it maintains high catalytic activity within a pH range of 5.0–9.0 and in high-salt environments.

[0047] The results indicate that the TG enzyme obtained in this study differs from the TG enzyme derived from liquid fermentation in terms of enzymatic properties. Therefore, it can exert a better catalytic effect in different application scenarios and has good potential for application in food processing and other industries.

Claims

1. A method for solid state fermentation of transglutaminase based on wheat bran and soybean meal, characterized in that, The application relates to a solid-state fermentation method for producing transglutaminase (TGase) by using Streptomyces mobaraensis. The strain is activated by using ISP4 medium. The ISP4 medium is dissolved in deionized water, sterilized at 121 DEG C for 30 min, then poured into a plate while hot, and then the plate is placed in a 28 DEG C constant-temperature incubator for culture after the plate is solidified, and the aerial mycelium gradually turns gray after about one week; Seed liquid preparation: seed culture medium components: beef extract 3.0 g / L, peptone 20 g / L, soluble starch 30 g / L, NaCl 5.0 g / L, Na2HPO4 2.0 g / L, KH2PO4 2.0 g / L, MgSO4 7H2O 2.0 g / L, dissolved in deionized water, heated and stirred until the solid components are completely dissolved, pH is adjusted to 7.0, and sterilized for standby use. The plate with good growth state and gray-brown spores grown for 7 days is selected, 1 / 2 of the plate is scraped with an inoculation shovel to obtain spores, and the spores are inoculated into a 250mL conical flask containing 100mL seed liquid medium. The conical flask is placed in a 30 DEG C, 150rpm constant-temperature shaker for 48 hours of vibration culture as a fermentation seed liquid for subsequent solid-state fermentation inoculation; The optimal process system for solid-state fermentation for producing TGase is as follows: the material is calculated according to the adding amount of a 250m conical flask, 5g of coarse wheat bran and soybean powder are added as carbon source and nitrogen source respectively, 3g of rice husk is added as a carrier, 10mL of inorganic salt and trace element solution is used for wetting, and then the mixture is uniformly mixed, sterilized at 121 DEG C for 30min, and then inoculated with 10m seed liquid and 5mL deionized water after cooling, and then uniformly stirred and placed for constant-temperature culture at 30 DEG C for 7 days, and the mixture is manually mixed once every 24 hours; Product extraction: after the fermentation is completed, 50mL of 4 DEG C deionized water is added to each conical flask for extraction of the crude enzyme, the conical flask is shaken at 150rpm for 30min, then large-particle substrates are removed through filtration with gauze, and then the supernatant is obtained after centrifugation at 4 DEG C and 10000rpm. The crude enzyme liquid and 4 DEG C anhydrous ethanol are mixed in equal amounts, 4 DEG C centrifugation is conducted at 10000rpm for 10min after 15min of standing, and then the protein precipitate is washed with deionized water for 2-3 times. After being frozen at -20 DEG C, the protein precipitate is placed in a vacuum freeze dryer for freeze-drying treatment, and then dry powder TGase crude enzyme is obtained. The solid-state fermentation raw material cost is low, and is only 22.22% of the liquid-state fermentation cost.

2. The method of claim 1, wherein the agricultural waste wheat bran and the inexpensive soybean meal are used as a culture medium for the glutamine transaminase solid state fermentation. The Streptomyces mobaraensis strain is purchased from the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC4.1719 and the original number is DSM40847. The purchased strain (dry powder) is activated and cultured according to the culture conditions in the instruction manual, the strain is preserved in the laboratory, and the strain is identified through 16S rDNA to determine that the purchased strain is correct.

3. The method of claim 1, wherein the agricultural waste wheat bran and the inexpensive soybean meal are used as a culture medium for the glutamine transaminase solid state fermentation. ​ 4. The method of claim 1, wherein the method is a method of glutamine transaminase solid state fermentation using agricultural waste wheat bran and cheap soybean meal as a culture medium. The ISP4 culture medium is composed of soluble starch 10 g / L, K2HPO4 1.0 g / L, MgSO4·7H2O 1.0 g / L, NaCl 1.0 g / L, (NH4)2SO4 2.0 g / L, CaCO3 2.0 g / L, FeSO4·7H2O 0.001 g / L, MnCl2·7H2O 0.001 g / L, and agar 20 g / L.

5. The method of claim 1, wherein the agricultural waste of wheat bran and cheap soybean meal as a culture medium for glutamine transaminase solid state fermentation is characterized by, The soluble starch needs to be heated in a water bath for dissolution before being mixed with other ingredients, otherwise the seed liquid will be uneven.

6. The method of glutamine transaminase solid state fermentation using agricultural waste wheat bran and cheap soybean meal as medium according to claim 1, characterized in that, The seed liquid shaking flask rotation speed is 150 rpm, which can be appropriately increased or decreased according to the actual situation, but too low rotation speed will lead to insufficient dissolved oxygen, uneven mass transfer, cell aggregation, pH and metabolic deviation, and too high rotation speed will lead to impaired shear force, excessive dissolved oxygen, increased foam, abnormal metabolism, and increased energy consumption cost.

7. The method of glutamine transaminase solid state fermentation using agricultural waste wheat bran and cheap soybean meal as medium according to claim 1, characterized in that, The inorganic salt and trace element solution is composed of KH2PO4 5 g / L, NH4NO3 5 g / L, MgSO4·7H2O 1 g / L, NaCl 1 g / L, CoCl2·6H2O 1 mg / L, MnSO4 0.8 mg / L, ZnSO4·7H2O 1.7 mg / L, and FeSO4·7H2O 2.5 mg / L, and the pH is adjusted to 6.0, and then sterilized at 121 ℃ for 30 min.

8. The method of claim 1, wherein the method is a glutamine transaminase solid state fermentation method using agricultural waste wheat bran and cheap soybean meal as a culture medium. The soybean powder needs to be dried and then ground by a grinder to 35 mesh.

9. The method of claim 1, wherein the method is a glutamine transaminase solid state fermentation method using agricultural waste wheat bran and cheap soybean meal as a culture medium. The solid-state fermentation technology can efficiently produce TGase by fermenting S. mobaraensis, and compared with liquid-state fermentation, it can significantly reduce the production cost and has environmental friendliness, and has the practical significance of further optimizing and expanding the culture to realize large-scale industrial production of TGase.

10. The method of claim 1, wherein the agricultural waste wheat bran and cheap soybean meal are used as a medium for glutamine transaminase solid state fermentation, characterized by, The TGase obtained by solid-state fermentation has an optimal catalytic temperature of 37 ℃, and has high thermal stability below 40 ℃, and the catalytic effect at medium and low temperatures is significantly better than that of liquid-state fermentation-derived commercial enzyme; it can still maintain high catalytic activity in a pH range of 5.0-9.0 and a high-salt environment. There are differences in enzymatic properties between the TGase derived from liquid-state fermentation and the TGase derived from solid-state fermentation, so the TGase derived from solid-state fermentation can play a better catalytic effect in different application scenarios, and has good potential for application in food processing and other industries.

Images