Fermentation method applied to industrialization of human superoxide gasification enzyme

Abstract

The invention provides a fermentation method applied to industrialization of human-derived superoxide dismutase, which comprises the following steps: inoculating an engineering strain for expressing the human-derived superoxide dismutase into a liquid culture medium, adding an inducer, and performing induced culture to recombine and express the human-derived superoxide dismutase, the amino acid sequence of the human-derived superoxide dismutase is SEQ ID NO: 1. Furthermore, according to the fermentation method, copper ions and zinc ions are also added into the culture medium. According to the method provided by the invention, the content and enzyme activity of the recombinant human SOD can be remarkably improved, large-scale production of the recombinant SOD is facilitated, and the method has very important significance on industrial fermentation of superoxide dismutase.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical products, and specifically relates to a fermentation method applied to the industrialization of human superoxide dismutase. Background Art

[0002] As one of the earliest and most mature expression systems, E. coli expression system is currently widely used in the expression of various recombinant proteins because of its fast growth rate and easy cultivation. It can obtain higher biomass and significantly increase the concentration of target gene expression products. In the process of industrial fermentation, there are many factors that affect biomass output, such as the composition of the culture medium (nutrients required for bacterial growth), fermentation tank technical parameters, fermentation culture conditions (such as inducers and induction conditions, inoculation amount, etc.).

[0003] Glucose is one of the common fast carbon sources, which can quickly participate in the growth metabolism of microorganisms, provide energy and carbon skeleton. However, Escherichia coli grows rapidly and may lead to the production of byproducts such as acetic acid, which will not only inhibit cell growth, but also affect the expression of exogenous proteins. In this scheme, the carbon sources of the overall fermentation system are mainly glycerol and lactose, replacing the traditional medium system based on glucose. First, the path of glycerol entering the tricarboxylic acid cycle is different from that of glucose. Choosing glycerol can effectively reduce the production of acetic acid, and glycerol as a carbon source will not grow too vigorously for Escherichia coli, which can effectively reduce production costs; second, lactose can be used as an inducer and can also be used by Escherichia coli as a carbon source. It is also non-toxic and inexpensive, which is of great significance to the industrial fermentation of superoxide dismutase.

[0004] Among them, the induction temperature, pH, inducer dosage, inoculation amount, etc. during the fermentation process are crucial as key parameters of the fermentation process. They are directly related to the amount of bacteria after the fermentation and the yield of the target product, that is, they affect the expression level of the recombinant protein. Summary of the invention

[0005] The purpose of the present invention is to provide a fermentation method for industrialization of human superoxide dismutase, which is to achieve the purpose of increasing the content and enzyme activity of recombinant human SOD by changing the fermentation conditions.

[0006] The present invention first provides a small-scale fermentation method for increasing the yield of human superoxide dismutase, which comprises inoculating an engineered strain expressing human superoxide dismutase into a liquid culture medium, adding an inducer, and inducing and cultivating to recombinantly express human superoxide dismutase; the amino acid sequence of the human superoxide dismutase is SEQ ID NO: 1;

[0007] The nucleotide sequence of a gene encoding the human superoxide dismutase is SEQ ID NO: 2.

[0008] Furthermore, in the fermentation method, copper ions and zinc ions are also added to the culture medium.

[0009] Preferably, the copper ion added concentration is 1600 μM and the zinc ion concentration is 40 μM.

[0010] The method, wherein the culture temperature is 30°C and the culture pH is 7.2;

[0011] Preferably, the lactose inducer is added in an amount of 18 g / L;

[0012] In the culture, the inoculation amount of the strain is 4%.

[0013] In another aspect, the present invention provides a fermentation method for industrialization of human superoxide dismutase, comprising the following steps:

[0014] 1) Seed activation and preparation: The constructed recombinant human SOD Escherichia coli seed culture liquid was inoculated into the primary seed culture medium at a 1% inoculation rate, and cultured at 37° C. with a shaking speed of 200 rpm / min for 12 h to 18 h to obtain the primary seed liquid;

[0015] The formula of the primary seed culture medium is as follows: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, kanamycin 50mg / L;

[0016] 2) The seed culture solution obtained in step 1) was transferred to a 5L secondary seed tank filled with a secondary seed culture medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 mg / L kanamycin) at a 1% inoculation rate to prepare secondary seeds, and the reaction temperature was controlled at 37° C., dissolved oxygen (DO) at 50-60%, a rotation speed at 200 rpm / min, pH 7.0 (adjusted with 25% ammonia water), a ventilation ratio of 1 VVM, and a fermentation tank pressure of 0.025 MPa;

[0017] 3) Large-scale fermentation: The medium composition was 8 g / L glycerol, 1 g / L glucose, 18 g / L lactose, 5 g / L yeast extract, 10 g / L tryptone, 0.05 M disodium hydrogen phosphate dodecahydrate, 0.05 M potassium dihydrogen phosphate, 0.05 M ammonium chloride, 0.025 M sodium sulfate, 2 mM magnesium sulfate heptahydrate; 50 mM FeCl 3 Ferric chloride hexahydrate, 0.12M concentrated hydrochloric acid, 2.94g / L calcium chloride dihydrate; 1.98g / L manganese chloride tetrahydrate, 2.88g / L zinc sulfate heptahydrate, 0.48g / L cobalt chloride hexahydrate, 0.34g / L cupric chloride dihydrate, 0.48g / L nickel chloride hexahydrate, 0.48g / L sodium molybdate dihydrate, 0.12g / L boric acid 80ml, kanamycin 50mg / L sterilize separately and add to the fermenter before inoculation;

[0018] The bacteria in the secondary seed tank grow for 4-6 hours, and the OD 600 =3.0, the tank pressure of the fermentation tank is 0.025Mpa, the stirring speed is controlled at 160rpm-400rpm, and linked with the dissolved oxygen to control the dissolved oxygen at 25%-35%, the ventilation ratio is 1VVM, and the pH of the fermentation tank is adjusted to 6.8 with ammonia water after inoculation. The temperature is controlled at 30°C, cultured under this condition for 6-8h, and then adjusted to 7.2 for fermentation for 18-22h.

[0019] The fermentation broth was purified using a Q column and hydrophobic chromatography.

[0020] The method provided by the present invention can significantly increase the content and enzyme activity of recombinant human SOD, is conducive to the large-scale production of recombinant SOD, and is of great significance to the industrial fermentation of superoxide dismutase. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Electrophoresis of recombinant expression of SOD protein;

[0022] Figure 2 : SDS-PAGE electrophoresis detection results, lanes 1-9 are samples, run-through, sodium chloride concentrations 80mM, 90mM, 100mM, 120mM, 180mM, 200mM, 2M;

[0023] Figure 3 :SDS-PAGE electrophoresis detection results, lanes 1-9 are samples, run-through, sodium chloride concentration 100mM front, middle and back, 500mM front and back peak, 1M;

[0024] Figure 4 : SDS-PAGE electrophoresis detection results, lanes 1-6 are samples, sodium chloride concentration 120mM, pass 1, pass 2, 2M NaCl, 0M NaCl; DETAILED DESCRIPTION

[0025] The present invention screened and obtained a human superoxide dismutase SOD with an amino acid sequence of SEQ ID NO: 1, and synthesized the coding gene of the above-mentioned recombinant human SOD (SEQ ID NO. 2), cloned into a pET-S1 vector after double restriction digestion and purification of the target fragment and the vector, extracted the plasmid after sequencing verification, transformed it into an Escherichia coli BL21 (DE3) competent cell, and obtained a positive recombinant after screening with 50ug / ml kanamycin.

[0026] Through experiments on skin fibroblasts, it was determined that the selected human superoxide dismutase SOD had no cytotoxicity and had a good oxidative damage repair effect. And through the detection of the expression of pro-inflammatory factors, it was confirmed that human SOD would not cause inflammatory response. The selected human superoxide dismutase SOD has good stability, no disease risk caused by mutation, will not cause immune rejection when used in the human body, and has good biosafety; compared with animal-derived SOD, it has the advantages of uniform molecular weight, high purity, no hidden dangers of virus transmission and low rejection reaction. It is a safe and effective biomedical material with high biological activity and is widely used in medical devices, cosmetics, food, biomedical materials and other fields.

[0027] In order to increase the yield of the screened human superoxide dismutase, the present invention constructs a fermentation method that can efficiently ferment and prepare human superoxide dismutase.

[0028] The present invention will be further described in the following embodiments, but the embodiments are only used to illustrate the present invention rather than to limit the present invention.

[0029] Example 1: Construction and expression of recombinant human SOD

[0030] The pUC-19 vector containing the human superoxide dismutase HuSOD gene fragment was synthesized at Sangon Company. The restriction sites at both ends were NcoI and BamHI, respectively. The target fragment HuSOD was obtained by double restriction digestion, and the vector pet-S1 vector was linearized using enzymes NcoI and BamHI, respectively. The target fragment SOD and the linearized vector pet-S1 were recovered by gel, and connected using T4-ligase at 37 degrees Celsius for 15 minutes. The connection system was transformed into 50uL competent DH5α, and PCR detection was used to verify the vector construction. The correct plasmid was extracted and tested, and transferred into the expression system Escherichia coli BL21 (DE3) competent cells, and a single clone was extracted for induced expression.

[0031] Pick a single clone from the transformation plate, inoculate it into 5 ml LB medium, and culture it at 37°C, 250 rpm overnight. Transfer it at 1:100, add lactose inducer and induce culture for 16-18 hours at 30°C in a shake flask, add copper chloride dihydrate (copper ions) and zinc sulfate heptahydrate (zinc ions) and continue to culture for 2 hours. Collect the wet cells by centrifugation and resuspend them in PBS buffer; repeat ultrasonic disruption 3 times (ultrasonication for 5s, stop for 8s, and cycle for 10min); heat denaturation treatment at 65°C for 10min; centrifuge at 10000rpm / min for 20min, and collect the supernatant. At this time, the solution contains a large amount of target human SOD protein, which is subjected to SDS-PAGE electrophoresis. Figure 1 As shown, the target band is visible, and the recombinant human SOD is expressed in a soluble manner.

[0032] Example 2: Optimization of fermentation process

[0033] 1. Screening of the optimal temperature

[0034] The optimum growth temperature of Escherichia coli is 37°C. The later induction expression temperature has a great influence on bacterial growth and protein expression. The SOD protein screened by the present invention is a soluble protein, and it is necessary to explore the appropriate expression temperature; at the same time, at a lower temperature, the specific growth rate and the nutrient intake rate of the bacteria are also reduced, and the production of harmful inhibitory products such as acetic acid is also reduced.

[0035] Each experiment was repeated 3 times, and the wet weight of the obtained bacteria and the total enzyme activity were measured. The results in Table 1 show that under 30℃, the SOD content and enzyme activity were the highest.

[0036] Table 1: Effect of temperature on SOD content and activity

[0037]

[0038]

[0039] 2. Screening of the optimal pH

[0040] Maintaining different pH values ​​at different stages of bacterial growth can significantly increase the yield of the target protein. Therefore, the test gradient is set to obtain the optimal pH in the expression stage and the growth conditions of the most suitable strain to achieve efficient expression of the target protein.

[0041] Each experiment was repeated 3 times, and the obtained wet weight of the bacteria and total enzyme activity were measured. As shown in Table 2, under the condition of pH 7.2, the SOD content and enzyme activity were the highest.

[0042] Table 2: Effect of pH on SOD content and activity

[0043]

[0044] 3. Effect of lactose inducer dosage on total enzyme activity

[0045] Lactose, as an inducer, is non-toxic, inexpensive and can be used as a carbon source by Escherichia coli, which is of great significance to industrial fermentation.

[0046] Each experiment was repeated 3 times, and the obtained wet weight of the bacteria and the total enzyme activity were measured. As shown in Table 3, the SOD content and enzyme activity were the highest under the condition of 18 g / L lactose concentration.

[0047] Table 3: Effect of inducer concentration on SOD content and activity

[0048]

[0049]

[0050] 4. Inoculation volume

[0051] The inoculum size refers to the volume ratio of the seed solution to the culture solution, which affects the growth of bacteria and the accumulation of metabolites. In the recombinant protein expression stage, the increase in the inoculum size can increase the specific synthesis rate of the engineered bacteria. If the inoculum size is too low, the specific growth rate of the bacteria will increase, and the logarithmic growth period will last longer; but when the inoculum size is too high, the bacteria will grow too fast, which will increase the consumption rate of nutrients and affect the later growth of bacteria.

[0052] Each experiment was repeated 3 times, and the obtained wet weight of the bacteria and the total enzyme activity were measured. The results in Table 4 show that under the condition of 4% inoculation amount, the SOD content and enzyme activity values ​​were the highest.

[0053] Table 4: Effect of inoculum concentration on SOD content and activity

[0054]

[0055] 5. Effect of metal ion concentration on SOD content and activity

[0056] Zinc and copper play a vital role in stabilizing the normal molecular conformation of SOD and improving the activity of SOD. Therefore, proper supplementation and adjustment of the concentration of copper and zinc is conducive to improving the activity and content of SOD.

[0057] Each experiment was repeated 3 times, and the obtained wet weight of the bacteria and the total enzyme activity were measured. The results in Table 5 and Table 6 show that the SOD content and enzyme activity were the highest under the conditions of copper ion concentration of 1600uM and zinc ion concentration of 40uM.

[0058] Table 5: Effect of copper ion concentration on SOD content and activity

[0059]

[0060]

[0061] Table 6: Effect of zinc ion concentration on SOD content and activity

[0062]

[0063] The optimal production process of recombinant human SOD was finally determined to be: 30℃, pH 7.2, inducer 18g / L, inoculation amount 4%, copper ion addition amount of 1600μM, and zinc ion 40μM.

[0064] Example 3: Small-scale fermentation and purification of recombinant human SOD

[0065] The constructed engineered bacteria expressing recombinant human SOD were fermented in a 5L fermenter to obtain fermentation sludge containing recombinant human SOD, thereby realizing the large-scale production of recombinant human SOD.

[0066] Primary seed solution: Take one tube of working bank cells and inoculate them at a 1% ratio into 50 ml LB culture medium containing 50 μg / mL Kana, and culture at 37°C, 200 rpm for 12 h as the primary seed.

[0067] Secondary seed solution: Inoculate 1% of the inoculum into two 1L shake flasks containing 200ml LB culture medium (containing 50μg / ml Kana), culture at 37°C, 200rpm, for about 6h as secondary seeds.

[0068] A 5L fermentation tank was used to prepare 2.9L fermentation medium (2.5L base material + 400ml secondary seed liquid). The fermentation base material and conditions are shown in Table 7. During the culture period, the dissolved oxygen was maintained at more than 30%, the rotation speed was 400-1000r / min, the dissolved oxygen was increased by adjusting the ventilation volume and the stirring speed, and the pH value was maintained at 6.8 by adding ammonia water. The foam was eliminated by adding a small amount of defoaming agent; the pH was adjusted to 7.2 after culturing at 30°C for about 6h, and the culture was continued for a total of 16-18h. Copper and zinc were added and the culture was continued for another 2h. The OD of the tank was 24-26, and the wet weight was 0.06g / ml.

[0069] Table 7: Base material formula information table during fermentation

[0070]

[0071] 3L of bacterial liquid was harvested from the tank and centrifuged at 4°C, 10,000 rpm, 30 min, and a total of 180.5 g of wet bacteria was harvested. The wet bacteria were resuspended in PBS buffer (bacteria: PBS = 1:5), and the APV cell disruptor was used for ice bath disruption twice; heat denaturation treatment was performed at 65°C, 10 min; centrifugation was performed at 10,000 rpm / min, 20 min, and the supernatant was collected.

[0072] Purify Cu and Zn-SOD proteins. Q column purification: Prepare 20mM Tris-HCl, pH 8.0, and the eluent is 20mM to 1M NaCl. Collect elution peaks of different concentrations for SDS-PAGE electrophoresis. The results are as follows Figure 2-3 . Hydrophobic chromatography: Mix the supernatants of the graded precipitations with saturated ammonium sulfate concentrations of 30%, 35%, 40%, 45%, 50% and 60% and perform hydrophobic chromatography. The equilibrium solution is 20mM Tris-HCl containing 4MNaCl, pH 7.0-8.0. The eluents range from 20mM Tris-HCl containing 20mMNaCl to 2MNaCl. The purity of the target protein is detected by SDS-PAGE electrophoresis. The results are as follows Figure 4.

[0073] The protein obtained after purification is desalted, concentrated and freeze-dried to obtain recombinant human SOD freeze-dried powder.

[0074] Example 4: Industrial large-scale fermentation to prepare SOD protein

[0075] (1) Seed activation and preparation: The constructed recombinant human SOD Escherichia coli seed culture liquid was inoculated into the primary seed culture medium at a rate of 1%, and cultured at 37° C. with a shaking speed of 200 rpm / min for 12 h to 18 h to obtain the primary seed liquid.

[0076] The formula of the first-level seed culture medium is: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, and kanamycin 50mg / L.

[0077] (2) The seed culture solution obtained in step (1) is transferred at a 1% inoculum amount to a 5L secondary seed tank (using 3L) filled with a secondary seed culture medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 mg / L kanamycin); the secondary seed tank is controlled to have a reaction temperature of 37°C, a dissolved oxygen (DO) of 50-60%, a rotation speed of 200 rpm / min, a pH of 7.0 (adjusted with 25% ammonia water), a ventilation ratio of 1VVM, and a fermentation tank pressure of 0.025 MPa for bacterial culture.

[0078] (3) 100 fermentation tank (usage volume is 80L): 8 g / L glycerol, 1 g / L glucose, 18 g / L lactose, 5 g / L yeast extract, 10 g / L tryptone, 0.05 M disodium hydrogen phosphate dodecahydrate, 0.05 M potassium dihydrogen phosphate, 0.05 M ammonium chloride, 0.025 M sodium sulfate, 2 mM magnesium sulfate heptahydrate were added to the fermentation tank, the volume was adjusted to 75 L, and sterilized at 115 °C for 30 min. The sterilized fermentation tank was placed at room temperature and cooled to 30 °C; trace elements (50 mM FeCl3 ferric chloride hexahydrate, 0.12M concentrated hydrochloric acid, 2.94g / L calcium chloride dihydrate; 1.98g / L manganese chloride tetrahydrate, 2.88g / L zinc sulfate heptahydrate, 0.48g / L cobalt chloride hexahydrate, 0.34g / L cupric chloride dihydrate, 0.48g / L nickel chloride hexahydrate, 0.48g / L sodium molybdate dihydrate, 0.12g / L boric acid 80ml, kanamycin 50mg / L are sterilized separately and added to the fermenter before inoculation.

[0079] The bacteria in the secondary seed tank grow for 4-6 hours, and the OD 600=3.0, all are connected to the fermentation tank sterilized in advance in step (3), the tank pressure of the fermentation tank is 0.025Mpa, the stirring speed is controlled at 160rpm-400rpm, and it is linked with dissolved oxygen to control dissolved oxygen at 25%-35%, the ventilation ratio is 1VVM, and after inoculation, the pH of the fermentation tank is adjusted to 6.8 with ammonia water, and the temperature is controlled at 30°C. Under this condition, it is cultured for 6-8h. This stage is the initial stage of fermentation. This stage is mainly to obtain a certain amount of pre-induction bacterial density, and this stage mainly consumes glucose. After 6-8h, Escherichia coli enters the logarithmic growth phase. In this stage, glucose has been exhausted and lactose has been utilized. After induction, the pH is adjusted to 7.2 for the expression of the target protein. During the fermentation process, the stable period after the logarithmic growth phase of Escherichia coli is 12-16h, and the fermentation is 18-22h. According to the ammonia supplement rate and the dissolved oxygen situation, that is, when the growth tends to saturation, 1600μM dihydrate copper chloride and 40μM heptahydrate zinc sulfate are added, stirred for 2h, and the fermentation is terminated to obtain the fermentation liquid.

[0080] The fermentation broth was purified by ammonium sulfate graded precipitation, and the specific steps were as follows:

[0081] Weigh 383.5g of ammonium sulfate powder into a 1L beaker, add 500mL of water, heat in a microwave oven to fully dissolve the ammonium sulfate, and filter with a single layer of filter paper. The next day, ammonium sulfate crystals will precipitate. Before use, adjust the pH of the saturated ammonium sulfate solution to 7.0-7.5 with ammonia water. Take a certain amount of sample and add ammonium sulfate to make the ammonium sulfate saturation 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%.

[0082] After mixing evenly, let it stand at room temperature for 2-5 hours, centrifuge at 12,000rpm and 4℃ for 20 minutes, and take the supernatant and precipitate for SDS-PAGE electrophoresis. The sample preparation method is 80μL sample + 20μL Loading Buffer, boil the sample at 100℃ for 10 minutes, take the supernatant and precipitate after centrifugation, and load them separately, with a loading volume of 10μL. After hydrophobic chromatography, SDS-PAGE electrophoresis is used to detect the purity of the target protein. The results are as follows Figure 4 .

[0083] The above results show that the method of the present invention can prepare a high-purity SOD protein that meets the industrial production capacity.

Claims

1. A small-scale fermentation method for increasing the yield of human superoxide dismutase, characterized in that: The method is to inoculate an engineered strain expressing human superoxide dismutase into a liquid culture medium, add an inducer, and induce and cultivate to recombinantly express human superoxide dismutase; the amino acid sequence of the human superoxide dismutase is SEQ ID NO:

1.

2. The method according to claim 1, characterized in that The nucleotide fragment of the gene encoding the human superoxide dismutase is SEQ ID NO:

2.

3. The method according to claim 1, characterized in that In the fermentation method, copper ions and zinc ions are also added to the culture medium.

4. The method according to claim 3, characterized in that The copper ion addition concentration is 1600 μM.

5. The method according to claim 3, characterized in that The added concentration of zinc ions is 40 μM.

6. The method according to claim 1, characterized in that The method, wherein the culture temperature is 30°C and the culture pH is 7.

2.

7. The method according to claim 1, characterized in that The added amount of the lactose inducer is 18 g / L.

8. The method according to claim 1, characterized in that The inoculation amount of the engineered strain is 4%.

9. A fermentation method for industrialization of human superoxide dismutase, characterized in that: The method described comprises the following steps: 1) Seed activation and preparation: The constructed recombinant human SOD Escherichia coli seed culture liquid was inoculated into the primary seed culture medium at a 1% inoculation rate, and cultured at 37° C. with a shaking speed of 200 rpm / min for 12 h to 18 h to obtain the primary seed liquid; The formula of the primary seed culture medium is as follows: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, kanamycin 50mg / L; 2) The seed culture solution obtained in step 1) is transferred to a secondary seed tank containing a secondary seed culture medium at a 1% inoculation rate to prepare secondary seeds, wherein the secondary seed culture medium contains 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride; the reaction temperature is controlled at 37° C., the dissolved oxygen is 50-60%, the rotation speed is 200 rpm / min, the pH is 7.0, the ventilation ratio is 1 VVM, and the fermentation tank pressure is 0.025 MPa; 3) Large-scale fermentation: the culture medium is composed of 8 g / L glycerol, 1 g / L glucose, 18 g / L lactose, 5 g / L yeast extract, 10 g / L tryptone, 0.05 M disodium hydrogen phosphate dodecahydrate, 0.05 M potassium dihydrogen phosphate, 0.05 M ammonium chloride, 0.025 M sodium sulfate, 2 mM magnesium sulfate heptahydrate; 50 mM FeCl3 ferric chloride hexahydrate, 0.12 M concentrated hydrochloric acid, 2.94 g / L calcium chloride dihydrate; 1.98 g / L manganese chloride tetrahydrate, 2.88 g / L zinc sulfate heptahydrate, 0.48 g / L cobalt chloride hexahydrate, 0.34 g / L copper chloride dihydrate, 0.48 g / L nickel chloride hexahydrate, 0.48 g / L sodium molybdate dihydrate, 0.12 g / L boric acid 80 ml, kanamycin 50 mg / L are sterilized separately and added to the fermenter before inoculation; The bacteria in the secondary seed tank grow for 4-6 hours, and the OD 600 =3.0, the tank pressure of the fermentation tank is 0.025Mpa, the stirring speed is controlled at 160rpm-400rpm, and linked with the dissolved oxygen to control the dissolved oxygen at 25%-35%, the ventilation ratio is 1VVM, and after inoculation, the pH of the fermentation tank is adjusted to 6.8 with ammonia water, and the temperature is controlled at 30°C. Under this condition, it is cultured for 6-8h, and then the pH is adjusted to 7.2 and fermented for 18-22h to obtain the fermentation liquid.

10. The industrial fermentation method of human superoxide dismutase according to claim 9, characterized in that: The fermentation broth is purified using a Q column and hydrophobic chromatography.

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