Batroxobin fermentation medium and fermentation method

Abstract

This invention provides a method for yeast fermentation of recombinant batroxobin and the culture medium used, the fermentation process mainly comprising primary seed culture, secondary seed culture, and fermentation culture. In the fermentation culture stage, the culture medium is optimized by adding multiple nutrients such as glycerol, casein amino acids, and PTM1 inorganic salts, and the pH of the fermentation medium is appropriately adjusted. The activity of the fermented recombinant protein is improved by methanol induction culture. The components of the optimized base fermentation medium are: K2SO4 0.91%, MgSO4 0.36%, CaSO4·2H2O 0.059%, 85% H3PO4 2.5% (V / V), KOH 0.206%, glycerol 4%, defoamer 0.05%~0.1%, PTM1 0.4% (V / V), casein amino acids 0.5%. The components of the optimized glycerol-added medium are: 50% glycerol + 12 mL / L PTM1 + 0.5% casein amino acids. The components of the optimized methanol induction medium are: (100% methanol + 12 mL / L PTM1) 95% + 4.5% sorbitol + 0.5% Casein is an amino acid.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the fermentation medium and fermentation method of batroxobin.

Background Art

[0002] In 1963, VanKlobusitzky et al. purified Batroxobin, an enzymatic hemostatic agent known as "batroxobin" in China, from the venom of Bothrops jararaca in Brazil. This is a single-chain glycoprotein with a total of 231 amino acids and a molecular weight of 39 - 43 kDa. Batroxobin belongs to the serine protease family, specifically acts on the Arg16 - Gly17 peptide bond at the N-terminus of the Αα chain of the fibrinogen molecule, releases fibrinopeptide A, generates unstable soluble fibrin I monomers, and further cross-link polymerizes into fibrin I polymers, thereby promoting platelet aggregation at the bleeding site to exert a hemostatic effect, activating vascular endothelium to release tissue-type plasminogen activator (t-PA), and is a single-chain glycoprotein that exhibits an antithrombotic effect.

[0003] With the increase in the number of patients with thromboembolic diseases, the research and production of antithrombotic drugs have become one of the hotspots. On the one hand, in order to obtain better thrombolytic drugs, molecular biology techniques are adopted to modify the molecular structure of conventional drugs. On the other hand, actively search for several natural-derived thrombolytic drugs with high safety, high therapeutic effect, and low side effects. Batroxobin has been widely applied clinically in the treatment of various diseases such as unstable angina pectoris and hyperviscosity syndrome, and the prevention of obstructive cardiovascular thromboembolic diseases, and it has been reported that the therapeutic effect is reliable and the adverse reactions are low.

[0004] Due to the limited large-scale production of batroxobin purified from snakes, methods for generating recombinant proteins have been intensively studied by many researchers.

[0005] The P. pastoris expression system has clear prokaryotic and eukaryotic genetic backgrounds, is easy to operate, and accurately induces glycosylation. Exogenous genes are stably integrated into the yeast genome, allowing for efficient expression of target proteins. Foreign proteins can perform post-translational modifications such as glycosylation, and the high secretion of target proteins into the extracellular space helps maintain the biological activity of the product and facilitates subsequent purification. This avoids the cumbersome steps of cell wall disruption associated with prokaryotic expression, thereby increasing yield. In 2004, You Weon-Kyoo from South Korea reported that when batroxobin was expressed in Pichia, the expression level reached 3.431 NIH / mL, and 7 mg could be purified from 1 L of fermentation broth.

[0006] Due to the characteristics of the enzyme structure of batroxobin itself, its activity tends to decrease during the fermentation production process using genetic engineering. The main reason for this is that proteases in the yeast host system biodegrade the batroxobin accumulated in the fermentation liquid, potentially affecting the yield and enzyme activity of batroxobin. While the expression of recombinant batroxobin in genetic engineering is generally carried out using Pichia systems, existing literature indicates that the expression levels are not high in any of these systems.

[0007] Therefore, conventional techniques have the problem that the activity of batroxobin prepared by fermentation is low, and enzymatic degradation is insufficient. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The present invention aims to provide a fermentation medium and fermentation method for batroxobin, addressing the problems of conventional technology, such as the low yield and low enzyme activity of purified batroxobin. [Means for solving the problem]

[0009] The present invention discloses a culture medium for the fermentation of recombinant batroxobin, comprising an optimized basal fermentation medium, an optimized glycerol supplemented medium, and an optimized methanol induction medium, wherein the components of the optimized basal fermentation medium are K2SO4 0.91%, MgSO4 0.36%, CaSO4·2H2O 0.059%, 85% H3PO4 2.5% (V / V), KOH 0.206%, glycerol 4%, antifoaming agent 0.05%~0.1%, PTM1 0.4% (V / V), and casein amino acids 0.5%, the components of the optimized glycerol supplemented medium are 50% glycerol + 12 mL / L PTM1 + 0.5% casein amino acids, and the components of the optimized methanol induction medium are (100% methanol + 12 mL / L PTM1) 95% + 4.5% sorbitol + 0.5% casein amino acids.

[0010] The present invention 1) A primary seed culture step is performed in which a preserved strain is inoculated into a primary seed medium, the formulation of which is 1.0% yeast powder, 2.0% polypeptone, and 2.0% glucose, and after being prepared with purified water, it is sterilized and ready for use. 2) Under sterile conditions, the primary seed solution is inoculated into a seed tank containing the secondary medium at an inoculation ratio of 5% to 11%. The formulation of the secondary medium is 1.0% yeast powder, 2.0% polypeptone, and 2.0% glucose. After being prepared with purified water, it is sterilized and ready for use. This is the secondary seed culture process. 3) When the OD600 of the secondary seed solution reaches 5-7, the optimized basal fermentation medium (4 mL / L) is added to the fermenter under sterile conditions, the secondary seed solution is transferred to the fermenter under sterile conditions, cultured for 4 hours, then the optimized glycerol supplement medium is added to the fermenter, the pH is adjusted, and methanol induction is performed in the fermentation culture process. 4) A step of collecting the supernatant and purifying it to obtain recombinant batroxobin, A method for fermenting batroxobin, including the method described herein, is disclosed.

[0011] Preferably, the components of the optimized basal fermentation medium are: K2SO4 0.91%, MgSO4 0.36%, CaSO4·2H2O 0.059%, 85% H3PO4 2.5% (V / V), KOH 0.206%, glycerol 4%, antifoaming agent 0.05%~0.1%, PTM1 0.4% (V / V), and casein amino acids 0.5%.

[0012] Preferably, the components of the optimized glycerol-supplemented medium are 50% glycerol + 12 mL / L PTM1 + 0.5% casein amino acids.

[0013] Preferably, during the methanol induction step, induction is carried out using an optimized methanol induction medium, the components of which are (100% methanol + 12 mL / L PTM1) 95% + 4.5% sorbitol + 0.5% casein amino acids.

[0014] Preferably, the pH range to be adjusted is 5.0 to 7.0.

[0015] Preferably, the components of PTM1 are 0.6% CuSO4·5H2O, 0.008% NaI, 0.3% MnSO4·H2O, 0.02% Na2MoO4·2H2O, 0.02% H3BO3, 0.05% CoCl2·6H2O, 6.5% FeSO4·7H2O, 2% ZnCl2, 0.02% biotin, and 0.0005% (V / V)H2SO4.

[0016] Preferably, the optimized basal fermentation medium, the optimized glycerol supplemented medium, and the optimized methanol induction medium are each prepared individually.

[0017] The present invention discloses a yeast fermentation method for recombinant batroxobin and the medium used, which includes an optimized basic fermentation medium, an optimized glycerol-added medium, and an optimized induction medium. The fermentation process mainly includes primary seed culture, secondary seed culture, and fermentation culture. In the fermentation culture stage, the pH of the fermentation medium is appropriately adjusted by adding a plurality of nutrient components such as glycerol, casein amino acids, and PTM1 inorganic salts to the medium, and the activity of the fermented recombinant protein is greatly improved by methanol induction culture. In the present invention, due to the inhibitory effect of the addition of casein amino acids on protease in the fermentation product, sorbitol and methanol improve the expression activity of the target protein as a shared carbon source, and by preparing a highly active batroxobin recombinant protein through subsequent purification, its activity can achieve an activity almost similar to that of commercially available natural batroxobin, which has the value of popularization and application.

Brief Description of the Drawings

[0018] [Figure 1] It is a diagram for screening and identifying transformed yeast colonies by PCR. [Figure 2] It is the measurement of the activity of recombinant batroxobin at different stages of methanol induction. [Figure 3] It is the influence of the components of the optimized basic medium on the activity of the fermentation protein. [Figure 4] It is the influence of the optimized methanol induction medium on the activity of the fermentation protein. [Figure 5] It is the influence of the addition of the glycerol-added medium on the activity of the fermentation protein. [Figure 6] It is the influence of the optimized glycerol-added medium on the activity of the fermentation protein. [Figure 7] It is the measurement of the activity of fermented recombinant batroxobin at different pH values of the fermentation medium. [Figure 8] It is a diagram of SDS electrophoresis measurement of the purified batroxobin protein.

Modes for Carrying Out the Invention

[0019] Hereinafter, specific embodiments of the present invention will be described in detail with reference to the drawings. It should be understood that the specific embodiments described herein are only for explaining and interpreting the present invention, and do not limit the present invention.

[0020] Example 1 Construction and Transformation of a Yeast Expression Vector Containing the Batroxobin Gene 1) Cloning of the Batroxobin-Encoding Gene Using the nucleic acid sequence of batroxobin (J02684.1) disclosed in GeneBank, the gene sequence was first artificially synthesized. Based on the information of the gene and the enzyme cleavage site information in the gene, a primer sequence for amplifying the batroxobin-encoding gene was designed, and Xhol and SacII enzyme cleavage site sequences were added to both ends of the primer. The designed amplification primer sequences are as follows.

[0021] Sense primer: 5’-aactcgaggtcattgga ggtgatgaat-3’ (SEQ ID NO: 1), Antisense primer: 5’-aaccgcggcgggcaagt cgcagttt-3’ (SEQ ID NO: 2).

[0022] Using the above amplification primers, PCR amplification was performed on the synthesized batroxobin gene J02684.1.

[0023] The conditions for the PCR amplification reaction were 95°C for 3 min, 95°C for 40 s, 53°C for 30 s, 72°C for 1 min, 35 cycles, 72°C for 15 min.

[0024] The obtained amplification product was stored at 4°C for future use.

[0025] 2) Construction and Transformation of the Yeast Expression Vector After purifying and recovering the PCR amplification product from step 1), the yeast expression vector pPICZαA was also subjected to double enzymatic digestion with Xhol and SacII. After recovering the respective products of the enzymatic digestion, they were ligated with T4 ligase, and the ligated products were then transformed into E. coli BL21. Recombinant strains were identified, recombinant plasmids were extracted, and positive clones were selected by double enzymatic digestion and PCR amplification of the target gene. The recombinant plasmid was named pPICZαA-Batr.

[0026] The pPICZαA-Batr plasmid was extracted, linearized with Sac I, and then transformed into Pichia X-33 using electrotransformation. All transformed cells were inoculated into YPD + Zeocin (300 μg / mL) medium and cultured at 30°C. Colonies were selected and identified by colony PCR, and further PCR identification was performed using the amplification primers described above. See Figure 1 for a diagram of yeast colony identification by PCR. The identified positive colony clones were saved for use.

[0027] Example 2: Fermentation expression of yeast process bacterium X-33 / pPICZαA-Batr 1) Initial screening of enzyme expression levels in positive colonies Positive clones were selected and first inoculated into glycerol complex medium (BMGY) and cultured overnight with shaking. The culture medium was removed by centrifugation, the precipitated cells were resuspended in BMGY, and the mixture was diluted until the OD600 was 1 to induce expression. Samples were taken approximately every 12 hours, and methanol was added until the final concentration was 1%. After 72 hours, the supernatant was collected by centrifugation. Thrombin activity was measured first by retaining the supernatant after each sample, and the measurement results are shown in Table 1. Colonies with high thrombin activity were obtained by initial selection and cloned for fermentation in a fermenter. The method for measuring thrombin activity is as follows.

[0028] 0.2 mL of human citrate-controlled plasma was taken and added to a 96-well plate. The plate was incubated at 37°C for 3 minutes. 0.2 mL of preheated sample solution (also at 37°C) was added, and the mixture was immediately shaken to ensure uniform mixing. The time was measured, and the plasma coagulation status was started at 40 seconds. The initial coagulation time was recorded, and three samples were measured simultaneously, with an error of <20 seconds. If the initial coagulation time was <40 seconds, the sample solution was diluted multiple times using the dilution formula, and the concentration of the test sample solution that coagulated within (60 ± 20) seconds was recorded. Under the above conditions, the amount of enzyme capable of coagulating 0.2 mL of human citrate-controlled plasma in 60 seconds was defined as one enzyme activity unit.

[0029] [Table 1]

[0030] Based on the measurement results, the batroxobin activity expressed in colony 2 was the highest, and the coagulation promotion time was the shortest. Therefore, the subsequent fermentation process was carried out using colony 2.

[0031] Example 3: Optimization of small-scale fermentation conditions for yeast X-33 / pPICZαA-Batr 1) Establishment of small-scale primary fermentation i) Primary seed culture: Take 1-2 working seed strains, open them under sterile conditions, and inoculate them into primary seed medium at 30±1℃ and 220rpm±10rpm with an inoculation ratio of 0.06%-0.2%. When the OD600 of the seed solution reaches 2-5, the culture is terminated, and the culture time is 30±8h.

[0032] [Table 2]

[0033] ii) Secondary seed culture Secondary seed inoculation: Under sterile conditions, the primary seed solution was inoculated into seed tanks containing secondary culture medium at an inoculation ratio of 5% to 11%.

[0034] The seed tank temperature was set to 30±1℃, the rotation speed to ≥100 rpm (initial rotation speed 100 rpm), and the tank pressure to <0.08 MPa. By controlling the rotation speed and aeration rate (initial aeration rate 20 L / min), the dissolved oxygen level at this stage was ensured to be >20%. After 4 hours of incubation, samples were taken once every 2 hours, examined under a microscope, the OD600 was measured, and each parameter was recorded. The incubation was terminated when the OD600 of the seed solution reached 5-7, with an incubation time of 10±4 hours.

[0035] [Table 3] iii) Fermentation culture When the OD600 of the secondary seed solution reached 5-7, the initial basal fermentation medium (4 mL / L) was added to the fermenter under sterile conditions, and the secondary seed solution was transferred to a 50 L fermenter under sterile conditions. The temperature was set to 30 ± 1 °C, the pH to 5.0 ± 0.2 (ammonia solution), the tank pressure to <0.08 MPa, and the rotation speed (initial rotation speed 100 rpm), aeration rate (initial aeration rate 100 L / min), and oxygen permeability were controlled to ensure that the dissolved oxygen at this stage was >20%. When the DO rose rapidly, this stage was terminated, at which point the wet weight of the microbial cells was 90-150 g / L, and the required time was approximately 16-24 hours. The components of the initial basic fermentation medium are: K2SO4 0.91%, MgSO4 0.36%, CaSO4·2H2O 0.059%, 85% H3PO4 2.5% (V / V), KOH 0.206%, glycerol 4%, defoaming agent 0.05%~0.1%, and PTM1 0.4% (V / V).

[0036] Methanol induction stage: The temperature was set to 20-30°C, the pH to 6.0±0.2 (ammonia solution), and the tank pressure to <0.08 MPa. By controlling the methanol addition rate, rotation speed, aeration rate, and oxygen permeability, it was ensured that the dissolved oxygen level at this stage was >20%. The initial methanol addition amount was not to exceed 0.5% of the initial fermentation volume. When the dissolved oxygen rapidly rose to its peak value (usually within 1 min), methanol was added at a constant rate, gradually increasing the flow rate, and ensuring that the maximum methanol flow rate did not exceed 12 mL / h·L (12 mL of auxiliary material added per hour per liter of fermentation liquid). If the DO could not be maintained above 20%, the addition was stopped, and the methanol solution was continued only after the DO reached its peak value. At this stage, the wet weight of the microbial cells was 350-600 g / L, and the methanol induction time was not less than 60 hours. The components of the aforementioned first methanol-inducing medium are 100% methanol + 12 mL / L PTM1.

[0037] The supernatant of the induction medium was taken at different stages after methanol induction, and the thrombin activity of the expressed batroxobin was measured using the method of Example 2. The fermentation results are shown in Figure 2. As can be seen from the measurement results in Figure 2, in small-scale fermentation, the methanol induction time was approximately 60 hours, and the activity of recombinant batroxobin in the medium was highest at this time. After 60 hours, the activity of batroxobin tended to stabilize, but it decreased slightly, suggesting that the biodegradation of the recombinant enzyme by impurity proteins in the medium may have been enhanced.

[0038] 2) Fermentation culture using the components of the optimized basic fermentation medium In the above "iii) Fermentation Culture," the components of the initial basic fermentation medium are optimized, that is, the secondary seed liquid is transferred to the fermenter and the optimized basic fermentation medium (4 mL / L) is added, but other fermentation conditions are the same as in 1) above. The components of the optimized basic fermentation medium are K2SO4 0.91%, MgSO4 0.36%, CaSO4·2H2O 0.059%, 85% H3PO4 2.5% (V / V), KOH 0.206%, glycerol 4%, antifoaming agent 0.05%~0.1%, PTM1 0.4% (V / V), and casein amino acids 0.5%.

[0039] The supernatant of the induction medium was taken at different stages after methanol induction, and the thrombin activity of the expressed batroxobin was measured using the method of Example 2. The fermentation results are shown in Figure 3. As can be seen from the measurement results in Figure 3, by optimizing the components of the basal fermentation medium, the activity of recombinant batroxobin in the medium was improved to some extent at each stage of methanol induction.

[0040] 3) Fermentation culture using the components of the optimized methanol-derived medium The fermentation induction method was carried out according to the method described in 2) above, except that the methanol induction step was performed using an optimized methanol induction medium consisting of (100% methanol + 12 mL / L PTM1) 95% + 4.5% sorbitol + 0.5% casein amino acids.

[0041] The supernatant of the induction medium was taken at different stages after methanol induction, and the thrombin activity of the expressed batroxobin was measured using the method of Example 2. The fermentation results are shown in Figure 4. As can be seen from the measurement results in Figure 4, by optimizing the components of the methanol induction medium, the activity of recombinant batroxobin in the medium was improved to some extent at each stage of induction.

[0042] 4) Effects of adding a glycerol step on the activity of fermented proteins At the start of microbial fermentation, the demand for carbon sources, nitrogen sources, and inorganic salts in the culture medium is very high. Therefore, by appropriately adding materials in the early stages of fermentation, the nutritional content in the culture medium is increased, and the expression level of recombinant proteins is enhanced.

[0043] Except for culturing for about 4 hours at the start of microbial fermentation at a temperature of 30±1℃, pH of 5.0±0.2, and tank pressure of <0.08MPa, the fermentation induction method was carried out according to 3) above. Different volumes (5mL, 10mL, 15mL, 20mL, 30mL) of the initial glycerol supplement medium were added to 1L of fermentation medium, and the components of the initial glycerol supplement medium were 50% glycerol + 12mL / L PTM1.

[0044] Approximately 60 hours after methanol induction, the supernatant of the induction medium was taken, and the thrombin activity of the expressed batroxobin was measured using the method of Example 2. The fermentation results are shown in Figure 5. As can be seen from the measurement results in Figure 5, the activity of all fermented proteins after methanol induction was improved to some extent by the glycerol addition step. Here, when the volume of glycerol in the initial glycerol addition medium was increased from 5 mL to 10 mL, the activity of batroxobin clearly improved, but the activity did not clearly improve even when the amount of glycerol was increased further.

[0045] 5) Effects of optimized glycerol supplementation medium on the activity of fermented proteins The fermentation induction method was carried out according to 4) above, except that the added glycerol medium was optimized and the supernatant was taken at different induction stages.

[0046] At the start of microbial fermentation, the temperature was set to 30±1℃, the pH to 5.0±0.2, and the tank pressure to <0.08MPa, and the culture was carried out for approximately 4 hours. 10 mL of optimized glycerol supplement medium was added to 1 mL of fermentation medium, and the components of the optimized glycerol supplement medium were 50% glycerol + 12 mL / L PTM1 + 0.5% casein amino acids.

[0047] The supernatant of the induction medium was taken at different stages after induction, and the thrombin activity of the expressed batroxobin was measured using the method of Example 2. The fermentation results are shown in Figure 6. As can be seen from the measurement results in Figure 6, the activity of all fermented proteins after methanol induction was improved to some extent by the step of optimizing the glycerol supplementation medium.

[0048] 6) Induction of fermentation media under different pH conditions Except for adjusting the starting pH of the fermentation medium, the fermentation induction method was carried out according to 5) above. The pH of the fermentation medium was adjusted to 5.0, 5.5, 6.0, 6.5, and 7.0, respectively, and methanol induction was performed for approximately 60 hours. After that, the activity of recombinant batroxobin in the medium was measured.

[0049] The supernatant of the induction medium was taken at different stages after methanol induction, and the thrombin activity of the expressed batroxobin was measured using the method of Example 2. The fermentation results are shown in Figure 7. As can be seen from the measurement results in Figure 7, adjusting the pH of the fermentation medium affects the activity of the fermented protein after methanol induction. Here, when the pH value is 6.5, the fermented recombinant protein shows high activity, while a lower pH has an inhibitory effect on the protein's expression activity.

[0050] Example 4: Fermentation and purification of yeast X-33 / pPICZαA-Batr-2 The fermentation under condition 6) in Example 3 (pH set to 6.5) was collected, and the supernatant of the culture medium was collected and purified according to the method described below.

[0051] 1) The supernatant of the batroxobin yeast fermentation solution was taken, and its pH and conductivity were adjusted. 2) Chromatography was performed by passing the solution through a cationic resin. Specifically, for elution, impurity proteins were first removed with a low-salt buffer, the dye was eluted and removed using PB with a buffer pH adjusted to 8.0, and finally eluted using Tris-HCl in a pH 9.0 buffer. 3) An elution buffer consisting of 50 m M Tris-HCl + 0.5 M NaCl was used, the pH was set to 9.0, and chromatography was performed by passing the solution through an anionic resin. 4) The solution was concentrated by single-pass tangential flow filtration. 5) Gel filtration chromatography was performed on the concentrated protein solution, and an elution buffer consisting of 20 m MPB + 0.15 M NaCl with a pH of 6.0 was used. 6) The target protein peak was collected by elution to obtain purified batroxobin. SDS electrophoresis was performed on the purified recombinant batroxobin to measure its purity, and the measurement results are shown in Figure 8.

[0052] Example 5: Measurement of enzyme activity of purified recombinant batroxobin and commercially available natural batroxobin Commercially available batroxobin was dissolved in buffer (20 m MPB + 0.15 M NaCl, pH 6.0), and the protein concentration, measured by a standard method, was consistent with that of purified recombinant batroxobin prepared by the method of Example 4.

[0053] Refer to the method in Example 2 for measuring enzyme activity. The method for measuring thrombin activity is as follows:

[0054] 0.2 mL of human citrate-controlled plasma was taken and added to a 96-well plate. The plate was incubated at 37°C for 3 minutes. 0.2 mL of preheated sample solution (also at 37°C) was added, and the mixture was immediately shaken to ensure uniform mixing. The time was measured, and the plasma coagulation status was started at 40 seconds. The initial coagulation time was recorded, and the three tubes were measured simultaneously, with an error of <20 seconds. If the initial coagulation time was <40 seconds, the sample solution was diluted multiple times using the dilution formula, and the concentration of the test sample solution that coagulated within (60 ± 20) seconds was recorded. Under the above conditions, the amount of enzyme capable of coagulating 0.2 mL of human citrate-controlled plasma in 60 seconds was defined as one enzyme activity unit. The measurement results are shown in Table 4.

[0055] [Table 4]

[0056] In this invention, the process method of the present invention is described by the above examples, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention cannot be carried out without relying on the above process steps. As those skilled in the art will see, any improvements to the present invention, equivalent substitutions of raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A culture medium for the fermentation of recombinant batroxobin, comprising an optimized basal fermentation medium, an optimized glycerol supplemented medium, and an optimized methanol induction medium, wherein the components of the optimized basal fermentation medium are K ２ SO ４ 0.91%, MgSO ４ 0.36%, CaSO ４ ・2H ２ O 0.059%, 85% H ３ PO ４ A culture medium for the fermentation of recombinant batroxobin, characterized in that the optimized glycerol supplement medium consists of 2.5% (V / V), 0.206% KOH, 4% glycerol, 0.05% to 0.1% defoaming agent, 0.4% (V / V) PTM1, and 0.5% casein amino acids, and the components of the optimized methanol-derived medium are 50% glycerol + 12 mL / L PTM1 + 0.5% casein amino acids, and the components of the optimized methanol-derived medium are 95% (100% methanol + 12 mL / L PTM1) + 4.5% sorbitol + 0.5% casein amino acids.

2. 1) A primary seed culture step is performed in which a preserved strain is inoculated into a primary seed medium, the formulation of which is 1.0% yeast powder, 2.0% polypeptone, and 2.0% glucose, and after being prepared with purified water, it is sterilized and ready for use. 2) A secondary seed culture step in which, under sterile conditions, primary seed solution is inoculated into a seed tank containing secondary medium at an inoculation ratio of 5% to 11%, the formulation of the secondary medium being 1.0% yeast powder, 2.0% polypeptone, and 2.0% glucose, and after being prepared with purified water, it is sterilized and prepared for use. 3) When the OD600 of the secondary seed solution reaches 5-7, the optimized basal fermentation medium (4 mL / L) is added to the fermenter under sterile conditions, the secondary seed solution is transferred to the fermenter under sterile conditions, cultured for 4 hours, then the optimized glycerol supplement medium is added to the fermenter, the pH is adjusted, and methanol induction is performed in the fermentation culture process. 4) A step of collecting the supernatant and purifying it to obtain recombinant batroxobin, A method for fermenting batroxobin, characterized by including the following:

3. The components of the aforementioned optimized basic fermentation medium are: K ２ SO ４ 0.91%, MgSO ４ 0.36%, CaSO ４ ·2H ２ O 0.059%, 85% H ３ PO ４ 2.5% (V / V), KOH 0.206%, glycerol 4%, antifoaming agent 0.05% to 0.1%, PTM1 0.4% (V / V), casein amino acid 0.5%, characterized in that it is the fermentation method according to claim 2.

4. The fermentation method according to claim 2, characterized in that the components of the optimized glycerol supplement medium are (100% methanol + 12 mL / L PTM1) 95% + 4.5% sorbitol + 0.5% casein amino acids.

5. The fermentation method according to claim 2, characterized in that the methanol induction step is performed using an optimized methanol induction medium, and the components of the optimized methanol induction medium are (100% methanol + 12 mL / L PTM1) 95% + 4.5% sorbitol + 0.5% casein amino acids.

6. The fermentation method according to claim 2, characterized in that the pH range to be adjusted is 5.0 to 7.

0.

7. The components of the aforementioned PTM1 are: 0.6% CuSO ４ ・5H ２ O, 0.008% NaI, 0.3% MnSO ４ ・H ２ O, 0.02% Na ２ MoO ４ ・2H ２ O, 0.02% H ３ BO ３ , 0.05% CoCl ２ 6H ２ O, 6.5% FeSO ４ 7H ２ 0, 2% ZnCl ２ 0.02% Biotin, 0.0005% (V / V) H ２ SO ４ The fermentation method according to claim 2, characterized in that...

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