A method for increasing the productivity of biosynthetic superoxide dismutase (SOD)

Abstract

The invention discloses a method for improving the productivity of biosynthetic superoxide dismutase (SOD). In the initial stage, a high yeast inoculation amount is used, and a carbon source, a nitrogen source and a nutrient salt required for biosynthesis are added, and the dissolved oxygen value in the culture medium is increased, and the optimum temperature and pH value are used, so that the content of yeast in the culture medium is rapidly increased in the middle stage of biosynthesis. After the middle stage of biosynthesis, a method of continuously adding a carbon source and a nitrogen source is adopted to continuously provide the biosynthetic superoxide dismutase (SOD) with energy substances required for yeast biosynthesis. In combination with increasing the dissolved oxygen value in the culture medium and continuously adding copper and zinc salts to a fermentation tank, yeast is stimulated to rapidly synthesize copper and zinc superoxide dismutase (SOD) in vivo. Through the above method, the yeast content and the superoxide dismutase (SOD) content in the yeast are increased, the overall biosynthesis time is shortened, and finally the productivity of biosynthetic superoxide dismutase (SOD) is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biosynthesis, and more specifically relates to a method for improving the productivity of biosynthetic superoxide dismutase (SOD). Background Art

[0002] Superoxide dismutase (SOD) is an important member of the antioxidant enzyme system in biological systems. It is widely present in microorganisms, plants and animals. It is an antioxidant metalloenzyme that can catalyze the dismutation of superoxide anion radicals to produce oxygen and hydrogen peroxide. It plays a vital role in the body's oxidation and antioxidant balance and is closely related to the occurrence and development of many diseases.

[0003] The main function of superoxide dismutase (SOD) is to catalyze the dismutation of superoxide anion free radicals into hydrogen peroxide and oxygen. Superoxide anion is a product of normal metabolism in organisms, but the accumulation of free radicals will cause lipid peroxidation in the cell membrane and cause membrane fission, leading to cell damage or even death. Superoxide dismutase (SOD) is the most important and best free radical scavenger in the cell body, maintaining the body's metabolic balance.

[0004] Superoxide dismutase is a class of metalloenzymes that can be divided into three types:

[0005] The first type contains copper and zinc, called copper-zinc superoxide dismutase, is blue-green in color, and is mainly found in the cytoplasm of eukaryotic cells;

[0006] The second type contains manganese, called manganese-containing superoxide dismutase, which is purple and mainly exists in the matrix of prokaryotic cells and eukaryotic cells (such as mitochondria, etc.);

[0007] The third type contains iron, called iron-containing superoxide dismutase, which is yellow-brown and mainly exists in prokaryotic cells and a few plant cells.

[0008] In eukaryotic organisms, including humans, the main superoxide dismutase is the first type of copper- and zinc-containing superoxide dismutase. The superoxide dismutase biosynthesized by the present invention belongs to the first type, namely, the copper- and zinc-containing superoxide dismutase.

[0009] Superoxide dismutase (SOD) can play an antioxidant and anti-aging role: Superoxide dismutase (SOD) can remove and reduce excessive free radicals in the human body and delay aging. It can continuously remove oxygen free radicals produced by excessive oxygen consumption of melanin due to light exposure, and prevent the formation of excessive melanin. At the same time, it can also continuously remove oxygen free radicals produced by lipofuscin and waxy fat to avoid excessive lipid peroxidation, and reduce the formation of lipofuscin and waxy fat, thereby playing an anti-aging role.

[0010] Superoxide dismutase (SOD) can play an anti-tumor role: in the human body, superoxide dismutase (SOD) can effectively inhibit cancer cells by removing superoxide anion free radicals. The superoxide dismutase (SOD) in the human body tends to decline gradually with age. Its decline leads to a decrease in the body's antioxidant capacity, and then a decrease in resistance. In severe cases, tumors may occur. Therefore, the majority of tumor patients are elderly people. From this perspective, detecting the level of superoxide dismutase (SOD) in a person's body can be used as an important indicator to determine whether a person has cancer and the aging index.

[0011] Anti-inflammatory effect: Superoxide dismutase (SOD) has a strong anti-inflammatory effect. Using superoxide dismutase (SOD) to treat pleurisy can reduce pleural effusion and the accumulation of white blood cells. Superoxide dismutase (SOD) can also inhibit arthritis, reduce the precipitation of IgG in the glomeruli, and interfere with the accumulation of white blood cells in the inflammatory site of the glomeruli. Superoxide dismutase (SOD) can inhibit the changes in organ permeability caused by acute tracheitis caused by xanthine / xanthine oxidase, and reduce the accumulation of polymorphonuclear leukocytes in the lungs. Superoxide dismutase (SOD) has a therapeutic and improving effect on idiopathic proctitis. Injecting an appropriate amount of superoxide dismutase (SOD) can improve the appearance of joints, improve joint mobility, and effectively treat osteoarthritis. It can be seen that superoxide dismutase (SOD) has a good application prospect for the treatment of inflammation.

[0012] Superoxide dismutase (SOD) application areas:

[0013] Medicine: Superoxide free radicals in the body can cause various diseases. Superoxide dismutase (SOD) is its natural scavenger and maintains a dynamic balance with it under normal circumstances. However, under pathological conditions, excessive superoxide free radicals are produced. The superoxide dismutase (SOD) produced by the body itself cannot completely remove these excessive superoxide free radicals, which are harmful to the body. Superoxide dismutase (SOD) can catalyze the dismutation reaction and alleviate the condition.

[0014] Food industry: Studies have shown that adding superoxide dismutase (SOD) to food as a natural antioxidant can be used as a preservative.

[0015] Clinical trials have shown that superoxide dismutase (SOD) is effective when taken orally and can be absorbed into the human body through the gastrointestinal tract. As a result, superoxide dismutase (SOD) chewing gum, superoxide dismutase (SOD) lozenges and oral liquid have appeared one after another, becoming one of the important ways to supplement superoxide dismutase (SOD) in vitro.

[0016] Daily chemical industry: The application of superoxide dismutase (SOD) in the daily chemical industry is mainly in skin care products and toothpaste.

[0017] Superoxide dismutase (SOD) has the following main functions in skin care products: First, as a cosmetic additive, it prevents skin aging, reduces wrinkles and removes spots, thus achieving skin care effects; second, it prevents and treats related skin diseases, such as dermatitis, acne, skin burns, etc.

[0018] Since superoxide dismutase (SOD) has anti-inflammatory effects, is purely natural, and has no toxic side effects, it plays an important role in oral and dental care.

[0019] Agriculture: Overexpression of superoxide dismutase (SOD) in transgenic plants can improve the resistance of plants to adverse environments to varying degrees. Increasing the expression of superoxide dismutase (SOD) in plants through genetic engineering can greatly enhance the stress resistance of plants.

[0020] From the above introduction, we can see that superoxide dismutase (SOD) is important to human health and life, so it is very important to find a high-content, low-cost production method of superoxide dismutase (SOD), especially the first production method of superoxide dismutase (SOD).

[0021] There are four main methods for producing superoxide dismutase (SOD):

[0022] 1. Using animal blood extraction method;

[0023] 2. Extracted from plants;

[0024] 3. Production by microbial biosynthesis;

[0025] 4. Superoxide dismutase (SOD) genetic engineering method.

[0026] Using animal blood extraction method: Due to the limitation of raw materials, it cannot be mass-produced. In addition, there are viruses in animal blood, which cannot be used directly on the human body. Moreover, the cost is too high. Due to the above shortcomings, large-scale industrial production cannot be achieved.

[0027] Extraction from plants: Due to the limitation of raw materials and the low content of superoxide dismutase (SOD) in natural plants, it is impossible to obtain high-purity superoxide dismutase (SOD), which limits the application of superoxide dismutase (SOD) in many fields.

[0028] Since the research on superoxide dismutase (SOD) genetic engineering has only begun in recent years, the existing technology can only produce small batches, and the cost is too high to meet the requirements of large-scale production.

[0029] Microbial biosynthesis is the biosynthesis method, and the microorganisms are mainly yeast and bacteria, which is the production method of the present invention. The advantage of this method is that there is no restriction on raw materials, and the biosynthesis process can be strictly controlled to perform aseptic operations, so the superoxide dismutase (SOD) produced does not contain any harmful pathogens such as viruses and bacteria.

[0030] In the prior art, the main reason that limits the industrial-scale production of superoxide dismutase (SOD) biosynthesized by microorganisms (mainly yeast) is that the productivity of superoxide dismutase biosynthesized by yeast is too low, that is, the biological activity (U) of superoxide dismutase biosynthesized per unit volume of fermenter (liter) per unit time (hour) is too low.

[0031] In the prior art, the main problems are that at the end of biosynthesis, the yeast content in the biosynthesis liquid is too low, the superoxide dismutase (SOD) content in the yeast is too low, and the biosynthesis time is too long, that is, the productivity is too low, resulting in excessive equipment investment, excessive consumption of raw and auxiliary materials, and excessively high production costs of superoxide dismutase (SOD). In addition, since the content of biosynthesized superoxide dismutase (SOD) in yeast is too low, the subsequent purification process is difficult and high-purity superoxide dismutase (SOD) cannot be obtained.

[0032] Therefore, finding a biosynthesis method with high superoxide dismutase (SOD) content, high yeast content and short time, that is, a biosynthesis method with high productivity, is the key to realizing large-scale industrial production of superoxide dismutase (SOD). Summary of the invention

[0033] The object of the present invention is to find a biosynthesis method with high superoxide dismutase (SOD) content, high yeast content and short time, that is, a biosynthesis method with high productivity.

[0034] The purpose of the present invention is achieved through the following technical solutions:

[0035] A method for improving the productivity of biosynthetic superoxide dismutase (SOD), comprising the following steps:

[0036] (1) Adding water to a fermentation tank, and then adding the carbon source, nitrogen source and nutrient salts required for yeast biosynthesis of superoxide dismutase (SOD) to obtain a basic culture medium;

[0037] (2) Use a high yeast inoculum in the basal medium;

[0038] (3) Setting the air flow rate, stirring speed, temperature and pH value of the fermenter to start the biosynthesis of superoxide dismutase (SOD);

[0039] (4) When the initial stage of biosynthesis is reached, carbon and nitrogen sources are started to be added to the fermenter;

[0040] (5) When the biosynthesis process reaches the middle stage, copper and zinc salts are added to the fermenter, and the air flow and rotation speed are increased.

[0041] (6) After the biosynthesis is completed, yeast with high yeast content and high superoxide dismutase (SOD) biological activity content is obtained in the biosynthesis liquid.

[0042] The yeast described in step (1) includes Saccharomyces cerevisiae, Pichia pastoris, etc.

[0043] In the high yeast inoculum described in step (2), the yeast inoculum amount is greater than 10 g / L.

[0044] The carbon sources described in steps (1) and (4) include glucose, sucrose and other carbon sources.

[0045] The nitrogen sources described in steps (1) and (4) include peptone, yeast powder, urea and ammonium sulfate.

[0046] The nutrient salts described in step (1) include copper sulfate, zinc sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride and sodium chloride.

[0047] The air flow rate in step (3) is 1-1.5 vvm (air flow per minute / volume of biosynthesis solution).

[0048] The stirring speed in step (3) is 200-500 rpm.

[0049] The temperature described in step (3) is 28-36°C.

[0050] The pH value described in step (3) is 4.5-7.5.

[0051] The initial biosynthesis period in step (4) is 2-6 hours.

[0052] The carbon source and nitrogen source described in step (4) are added until 5-10 hours before the end of the biosynthesis.

[0053] The copper- and zinc-containing salts described in step (5) include copper sulfate, zinc sulfate, and the like.

[0054] The time during the mid-biosynthesis phase described in step (5) is 20-24 hours.

[0055] When the biosynthesis in step (5) reaches the middle stage of biosynthesis, the wet yeast content in the biosynthesis liquid is not less than 170 g / L, and the dry matter content of the wet yeast is calculated as 33%.

[0056] The nutrient salt flow described in step (5) is added 3-6 hours before the end of the biosynthesis.

[0057] The air flow rate in step (5) is 1.5-2.5 vvm (air flow per minute / volume of biosynthesis solution).

[0058] The stirring speed in step (5) is 500-800 rpm.

[0059] The biosynthesis in step (6) is completed within 55 hours.

[0060] The yeast content in the biosynthetic liquid described in step (6) is not less than 200 g / L, calculated based on the content of wet yeast in each liter of biosynthetic liquid, and the dry matter content of the wet yeast is calculated as 33%.

[0061] The biological activity content of superoxide dismutase (SOD) described in step (6) is not less than 8000 U / g wet yeast, and the dry matter content of the wet yeast is calculated as 33%.

[0062] The biological activity of superoxide dismutase (SOD) described in step (6) was determined using the pyrogallol method.

[0063] The superoxide dismutase (SOD) described in step (6) is an intracellular enzyme.

[0064] The superoxide dismutase (SOD) described in step (6) is a copper- and zinc-containing superoxide dismutase (SOD).

[0065] The advantages and beneficial effects of the present invention are:

[0066] 1. The present invention relates to a biosynthesis technology. Biosynthesis is a multi-step, enzyme-catalyzed process in which substrates are converted into more complex substances within organelles. In the biosynthesis process, simple compounds are changed and converted into other compounds, or combined into macromolecular compounds. This process often includes metabolic pathways, which may occur in one organelle, and some may involve several organelles. The superoxide dismutase described in the present invention is synthesized in yeast cells in this way.

[0067] 2. The present invention adopts a high yeast inoculation amount at the initial stage of biosynthesis, and the high yeast inoculation amount is combined with the addition of appropriate carbon sources, nitrogen sources and nutrients required for biosynthesis, and the dissolved oxygen value in the culture medium is increased by high air intake and high stirring speed, and the optimum temperature and pH value are adopted, so that the yeast content in the culture medium reaches more than 170g / L in the middle stage of biosynthesis.

[0068] 3. In the middle stage of biosynthesis, that is, when the yeast content in the culture medium reaches 170 g / L or more, the present invention adopts a method of continuing to flow-add carbon source and nitrogen source, combined with a method of increasing air flow rate and rotation speed to further increase the dissolved oxygen value in the culture medium, to provide the required energy substances and oxygen for the continued growth of yeast, and ultimately increase the yeast content in the biosynthesis liquid, so that the yeast content in the final biosynthesis liquid exceeds 200 g / L.

[0069] 4. In the middle stage of biosynthesis, that is, when the yeast content in the culture medium reaches 170 g / L or more, the present invention adopts a method of continuously adding carbon source and nitrogen source to continuously provide energy substances required for yeast biosynthesis of superoxide dismutase (SOD), and combines increasing the air intake and rotation speed to further increase the dissolved oxygen value in the culture medium to provide oxygen required for yeast biosynthesis. At the same time, by continuously adding copper and zinc salts to the fermentation tank, the yeast is stimulated to quickly synthesize copper and zinc superoxide dismutase (SOD) in the body, and finally the biological activity content of superoxide dismutase (SOD) in the yeast in the biosynthesis liquid is increased, and the biological activity content of superoxide dismutase (SOD) exceeds 8000 U / g wet yeast.

[0070] 5. The present invention uses the above method to rapidly increase the yeast content in the culture medium, and the biological activity content of superoxide dismutase (SOD) in the yeast is also rapidly increased, ultimately shortening the overall biosynthesis time, so that the end time of biosynthesis does not exceed 55 hours.

[0071] 6. The present invention improves the productivity of biosynthesizing superoxide dismutase (SOD) by rapidly increasing the yeast content in the culture medium and rapidly increasing the biological activity content of superoxide dismutase (SOD) in the yeast, thereby shortening the overall biosynthesis time. DETAILED DESCRIPTION

[0072] The present invention will be further described below through specific embodiments.

[0073] Example 1

[0074] A 3-liter fully automatic fermentation tank is used, which has automatic control functions for temperature, pH and dissolved oxygen value (agitation speed and air intake), and a 6-way feeding automatic control system.

[0075] (1) Add 70 g / L of sterilized sugar, 1 g / L of peptone, 2 g / L of yeast powder, 1.3 g / L of urea, 0.03 g / L of copper sulfate pentahydrate, 0.03 g / L of zinc sulfate heptahydrate, 1.5 g / L of dipotassium hydrogen phosphate, 3 g / L of potassium dihydrogen phosphate, and 0.3 g / L of magnesium sulfate heptahydrate to the fermentation tank, and then add ultrapure water to 2.5 liters to obtain the basal culture medium.

[0076] (2) Angel brewer’s yeast (fresh or dry yeast) was used, and the yeast inoculation amount was 10 g / L.

[0077] (3) Set the air flow rate of the fermenter to 1 v / v / m (air flow per minute / biosynthesis tank volume), the stirring speed to 200 rpm, the temperature to 30 °C and the pH value to 6.5 to start the biosynthesis of superoxide dismutase (SOD).

[0078] (4) After 2 hours of biosynthesis, glucose solution at 7 ml / h (glucose content: 50%), yeast powder solution at 0.6 ml / h (yeast powder content: 20%), and urea solution at 0.4 ml / h (urea content: 20%) were added and the addition was stopped after 45 hours.

[0079] (5) After 20 hours of biosynthesis, start adding 0.01 ml / h of copper sulfate pentahydrate solution (copper sulfate content is 15%) and 0.01 ml / h of zinc sulfate heptahydrate solution (zinc sulfate content is 15%), increase the air flow rate to 1.5 vvm, increase the rotation speed to 500 rpm, and stop the addition after 45 hours.

[0080] (6) The biosynthesis was completed after 50 hours, and 2.6 L of biosynthesis liquid was obtained.

[0081] (7) After the biosynthesis reaches 20 hours, 50 ml of the biosynthesis liquid is taken and separated using a separator. The separator is set to 6000 rpm, the separation factor is 6439, and the separation time is 20 minutes. Wet yeast is obtained through separation. The obtained wet yeast is dissolved in 5 times the volume of water, stirred evenly, and separated again. The above steps are repeated three times to obtain pure wet yeast. The weight of the pure wet yeast is 8.6 g. The content of pure wet yeast in the biosynthesis liquid is calculated to be 17.2% (w / v).

[0082] (8) Take 500 ml of the biosynthesis liquid after the biosynthesis is completed, and separate the biosynthesis liquid using a separator. The separator is set to 6000 rpm, the separation factor is 6439, and the separation time is 20 minutes. After separation, wet yeast is obtained. The obtained wet yeast is dissolved in 5 times the volume of water, stirred evenly, and separated again. The above steps are repeated three times to obtain pure wet yeast. The weight of the pure wet yeast is 104 g. It is calculated that the content of pure wet yeast in the biosynthesis liquid is 20.8% (w / v).

[0083] (9) 100 g of the pure wet yeast obtained in step (8) was taken and then broken by a homogenizer. The pressure of the homogenizer was set to 140 MPa, the flow rate was set to 50 ml / min, and the number of cycles was 5 to obtain a yeast cell-breaking liquid. The yeast cell-breaking liquid was tested by the pyrogallol auto-oxidation method. The test showed that the superoxide dismutase (SOD) content in the wet yeast was 8200 U / g.

[0084] After calculation, the productivity of superoxide dismutase biosynthesized by yeast is 29564U / L / h {superoxide dismutase (U) biosynthesized per unit volume of fermenter (liter) per unit time (hour)}.

[0085] Example 2

[0086] A 3-liter fully automatic fermentation tank is used, which has automatic control functions for temperature, pH and dissolved oxygen value (agitation speed and air intake), and a 6-way feeding automatic control system.

[0087] (1) Add 75 g / L of sterilized sugar, 1.2 g / L of peptone, 2.3 g / L of yeast powder, 1.2 g / L of urea, 0.04 g / L of copper sulfate pentahydrate, 0.04 g / L of zinc sulfate heptahydrate, 1.7 g / L of dipotassium hydrogen phosphate, 4 g / L of potassium dihydrogen phosphate, and 0.4 g / L of magnesium sulfate heptahydrate to the fermentation tank, and then add ultrapure water to 2.5 liters to obtain a basal culture medium.

[0088] (2) Angel brewer’s yeast (fresh or dry yeast) was used, and the yeast inoculation amount was 12 g / L.

[0089] (3) Set the air flow rate of the fermenter to 1.2 v / v / m (air flow per minute / biosynthesis tank volume), the stirring speed to 300 rpm, the temperature to 31 °C and the pH value to 6.8 to start the biosynthesis of superoxide dismutase (SOD).

[0090] (4) After 3 hours of biosynthesis, glucose solution was added at a rate of 8 ml / h (glucose content was 50%), peptone solution at a rate of 0.3 ml / h (peptone content was 20%), yeast powder solution at a rate of 0.6 ml / h (yeast powder content was 20%), and urea solution at a rate of 0.3 ml / h (urea content was 20%). The addition was stopped after 45 hours.

[0091] (5) After 24 hours of biosynthesis, start adding 0.02 ml / h of copper sulfate pentahydrate solution (copper sulfate content is 15%) and 0.02 ml / h of zinc sulfate heptahydrate solution (zinc sulfate content is 15%), increase the air flow rate to 2 vvm, increase the rotation speed to 700 rpm, and stop the addition after 46 hours.

[0092] (6) The biosynthesis was completed after 55 hours, and 2.65 L of biosynthesis liquid was obtained.

[0093] (7) After the biosynthesis reaches 24 hours, 50 ml of the biosynthesis liquid is taken and separated using a separator. The separator is set to 6000 rpm, the separation factor is 6439, and the separation time is 20 minutes. Wet yeast is obtained through separation. The obtained wet yeast is dissolved in 5 times the volume of water, stirred evenly, and separated again. The above steps are repeated three times to obtain pure wet yeast. The weight of the pure wet yeast is 8.9 g. The content of pure wet yeast in the biosynthesis liquid is calculated to be 17.8% (w / v).

[0094] (8) Take 500 ml of the biosynthesis liquid after the biosynthesis is completed, and separate the biosynthesis liquid using a separator. The separator is set to 6000 rpm, the separation factor is 6439, and the separation time is 20 minutes. After separation, wet yeast is obtained. The obtained wet yeast is dissolved in 5 times the volume of water, stirred evenly, and separated again. The above steps are repeated three times to obtain pure wet yeast. The weight of the pure wet yeast is 112 g. The content of the pure wet yeast in the biosynthesis liquid is calculated to be 22.4% (w / v).

[0095] (9) 100 g of the pure wet yeast obtained in step (8) was taken and then broken by a homogenizer. The pressure of the homogenizer was set to 140 MPa, the flow rate was set to 50 ml / min, and the number of cycles was 5 to obtain a yeast cell-breaking liquid. The yeast cell-breaking liquid was tested by the pyrogallol auto-oxidation method. The test showed that the superoxide dismutase (SOD) content in the wet yeast was 9100 U / g.

[0096] After calculation, the productivity of superoxide dismutase biosynthesized by yeast is 32738U / L / h {superoxide dismutase (U) biosynthesized per unit volume of fermenter (liter) per unit time (hour)}.

[0097] Example 3

[0098] A 3-liter fully automatic fermentation tank is used, which has automatic control functions for temperature, pH and dissolved oxygen value (agitation speed and air intake), and a 6-way feeding automatic control system.

[0099] (1) Add 80 g / L of sterilized sugar, 1.5 g / L of peptone, 2.5 g / L of yeast powder, 1.25 g / L of urea, 0.05 g / L of copper sulfate pentahydrate, 0.05 g / L of zinc sulfate heptahydrate, 2 g / L of dipotassium hydrogen phosphate, 5 g / L of potassium dihydrogen phosphate, and 0.5 g / L of magnesium sulfate heptahydrate to the fermentation tank, and then add ultrapure water to 2.5 liters to obtain a basal culture medium.

[0100] (2) Angel brewer’s yeast (fresh or dry yeast) was used, and the yeast inoculation amount was 15 g / L.

[0101] (3) Set the air flow rate of the fermenter to 1.5 v / v / m (air flow per minute / biosynthesis tank volume), the stirring speed to 500 rpm, the temperature to 32 °C and the pH value to 7 to start the biosynthesis of superoxide dismutase (SOD).

[0102] (4) After 4 hours of biosynthesis, glucose solution was added at a rate of 10 ml / h (glucose content was 50%), peptone solution at a rate of 0.4 ml / h (peptone content was 20%), yeast powder solution at a rate of 0.7 ml / h (yeast powder content was 20%), and urea solution at a rate of 0.36 ml / h (urea content was 20%). The addition was stopped after 48 hours.

[0103] (5) After 22 hours of biosynthesis, start adding 0.03 ml / h of copper sulfate pentahydrate solution (copper sulfate content is 15%) and 0.03 ml / h of zinc sulfate heptahydrate solution (zinc sulfate content is 15%), increase the air flow rate to 2.5 vvm, increase the rotation speed to 800 rpm, and stop the addition after 50 hours.

[0104] (6) The biosynthesis was completed after 52 hours, and 2.7 L of biosynthesis liquid was obtained.

[0105] (7) After the biosynthesis reaches 22 hours, 50 ml of the biosynthesis liquid is taken and separated using a separator. The separator is set to 6000 rpm, the separation factor is 6439, and the separation time is 20 minutes. Wet yeast is obtained through separation. The obtained wet yeast is dissolved in 5 times the volume of water, stirred evenly, and separated again. The above steps are repeated three times to obtain pure wet yeast. The weight of the pure wet yeast is weighed on a balance and is 9.2 g. The content of the pure wet yeast in the biosynthesis liquid is calculated to be 18.4% (w / v).

[0106] (8) Take 500 ml of the biosynthesis liquid after the biosynthesis is completed, and separate the biosynthesis liquid using a separator. The separator is set to 6000 rpm, the separation factor is 6439, and the separation time is 20 minutes. After separation, wet yeast is obtained. The obtained wet yeast is dissolved in 5 times the volume of water, stirred evenly, and separated again. The above steps are repeated three times to obtain pure wet yeast. The weight of the pure wet yeast is 118 g. It is calculated that the content of pure wet yeast in the biosynthesis liquid is 23.6% (w / v).

[0107] (9) 100 g of the pure wet yeast obtained in step (8) was taken and then broken by a homogenizer. The pressure of the homogenizer was set to 140 MPa, the flow rate was set to 50 ml / min, and the number of cycles was 5 to obtain a yeast cell-breaking liquid. The yeast cell-breaking liquid was tested by pyrogallol auto-oxidation method. The test showed that the superoxide dismutase (SOD) content in the wet yeast was 9800 U / g.

[0108] After calculation, the productivity of superoxide dismutase biosynthesized by yeast is 40029U / L / h {superoxide dismutase (U) biosynthesized per unit volume of fermentation tank (liter) per unit time (hour)}.

Claims

1. A method for improving the productivity of biosynthetic superoxide dismutase (SOD), characterized in that: It includes the following steps: (1) Add 70 g / L of sterilized sugar, 1 g / L of peptone, 2 g / L of yeast powder, 1.3 g / L of urea, 0.03 g / L of copper sulfate pentahydrate, 0.03 g / L of zinc sulfate heptahydrate, 1.5 g / L of dipotassium hydrogen phosphate, 3 g / L of potassium dihydrogen phosphate, 0.3 g / L of magnesium sulfate heptahydrate, and then add ultrapure water to 2.5 liters to obtain a basal culture medium; (2) Using a high yeast inoculum in the basal medium, the inoculum amount is 10-15 g / L; (3) setting the air flow rate of the fermenter to 1-1.5 v / v / m (air flow per minute / biosynthesis tank volume), the stirring speed to 200-500 rpm, the temperature to 28-36° C., and the pH value to 4.5-7.5, and starting the biosynthesis of superoxide dismutase (SOD); (4) When the initial stage of biosynthesis is reached, the initial stage of synthesis time is 2-6 hours, and the carbon source is added to the fermenter: 7 ml / h of glucose solution, the glucose content of the glucose solution is 50%; and the nitrogen source: 0.6 ml / h of yeast powder solution, the yeast content of the yeast powder solution is 20% and 0.4 ml / h of urea solution, the urea content of the urea solution is 20%. The addition is stopped after 40-45 hours; (5) When the biosynthesis medium period is reached, which is 20-24 hours, 0.01 ml / h of copper sulfate pentahydrate solution with a copper sulfate content of 15% and 0.01 ml / h of zinc sulfate heptahydrate solution with a zinc sulfate content of 15% are started, the air flow rate is increased to 1.5 vvm, the rotation speed is increased to 500 rpm, and the addition is stopped after 44-47 hours; (6) After the biosynthesis is completed, the biosynthesis completion time is: 50-55 hours; a high yeast content of: 20-20.8% (w / v); and a high superoxide dismutase (SOD) biological activity content of: 8000-8200U / g of yeast are obtained in the biosynthesis liquid; the productivity of yeast biosynthesis of superoxide dismutase is: 29564U / L / h; the high yeast content in the biosynthesis liquid is calculated according to the weight ratio (w / v) of pure wet yeast in the volume of the biosynthesis liquid; the superoxide dismutase (SOD) biological activity content is calculated according to the superoxide dismutase (SOD) activity content in the wet yeast, the dry matter content in the wet yeast is calculated as 33%, and the determination method is based on the pyrogallol method; the productivity of yeast biosynthesis of superoxide dismutase is calculated as the superoxide dismutase (U) biosynthesized per unit volume of the fermentation tank (liter) per unit time (hour).

2. A method for increasing the productivity of biosynthetic superoxide dismutase (SOD) according to claim 1, characterized in that In step (1), the yeast includes Saccharomyces cerevisiae and Pichia pastoris.

3. A method for increasing the productivity of biosynthetic superoxide dismutase (SOD) according to claim 1, characterized in that In step (5), when the biosynthesis reaches the middle stage of biosynthesis, the wet yeast content in the biosynthesis liquid is not less than 170 g / L, and the dry matter content of the wet yeast is calculated as 33%.

4. A method for increasing the productivity of biosynthetic superoxide dismutase (SOD) according to claim 1, characterized in that In step (6), the superoxide dismutase (SOD) is an intracellular enzyme, and the superoxide dismutase (SOD) is a copper- and zinc-containing superoxide dismutase (SOD).

5. A method for improving the productivity of biosynthetic superoxide dismutase (SOD), characterized in that: It includes the following steps: (1) Add 75 g / L of sterilized sugar, 1.2 g / L of peptone, 2.3 g / L of yeast powder, 1.2 g / L of urea, 0.04 g / L of copper sulfate pentahydrate, 0.04 g / L of zinc sulfate heptahydrate, 1.7 g / L of dipotassium hydrogen phosphate, 4 g / L of potassium dihydrogen phosphate, 0.4 g / L of magnesium sulfate heptahydrate, and then add ultrapure water to 2.5 liters to obtain a basal culture medium; (2) Using a high yeast inoculum in the basal medium, the inoculum amount is 10-15 g / L; (3) setting the air flow rate of the fermenter to 1-1.5 v / v / m (air flow per minute / biosynthesis tank volume), the stirring speed to 200-500 rpm, the temperature to 28-36° C., and the pH value to 4.5-7.5, and starting the biosynthesis of superoxide dismutase (SOD); (4) When the initial stage of biosynthesis is reached, the initial stage of synthesis time is 2-6 hours, and the carbon source is added to the fermenter: 8 ml / h of glucose solution, the glucose content in the glucose solution is 50%; and the nitrogen source: 0.6 ml / h of yeast powder solution, the yeast content in the yeast powder solution is 20%, 0.3 ml / h of nitrogen peptone solution, the nitrogen peptone content in the nitrogen peptone solution is 20% and 0.3 ml / h of urea solution, the urea content in the urea solution is 20%, and the addition is stopped after 45-50 hours; (5) When the biosynthesis phase reaches the middle stage, which lasts for 20-24 hours, 0.02 ml / h of copper sulfate pentahydrate solution with a copper sulfate content of 15% and 0.02 ml / h of zinc sulfate heptahydrate solution with a zinc sulfate content of 15% are started, the air flow rate is increased to 2 vvm, the rotation speed is increased to 700 rpm, and the addition is stopped after 49-52 hours; (6) After the biosynthesis is completed, the biosynthesis completion time is: 55 hours; the high yeast content is: 20-22.4% (w / v); and the high superoxide dismutase (SOD) biological activity content is: 8000-9100U / g of yeast in the biosynthesis liquid; the productivity of yeast biosynthesis of superoxide dismutase is: 32738U / L / h; the high yeast content in the biosynthesis liquid is calculated according to the weight ratio (w / v) of pure wet yeast in the volume of the biosynthesis liquid; the superoxide dismutase (SOD) biological activity content is calculated according to the superoxide dismutase (SOD) activity content in the wet yeast, the dry matter content in the wet yeast is calculated as 33%, and the determination method is according to the pyrogallol method; the productivity of yeast biosynthesis of superoxide dismutase is calculated as the superoxide dismutase (U) biosynthesized per unit volume of the fermentation tank (liter) per unit time (hour).

6. A method for increasing the productivity of biosynthetic superoxide dismutase (SOD) according to claim 5, characterized in that: In step (1), the yeast includes Saccharomyces cerevisiae and Pichia pastoris.

7. A method for improving the productivity of biosynthetic superoxide dismutase (SOD) according to claim 5, characterized in that: In step (5), when the biosynthesis reaches the middle stage of biosynthesis, the wet yeast content in the biosynthesis liquid is not less than 170 g / L, and the dry matter content of the wet yeast is calculated as 33%.

8. A method for improving the productivity of biosynthetic superoxide dismutase (SOD) according to claim 5, characterized in that: In step (6), the superoxide dismutase (SOD) is an intracellular enzyme, and the superoxide dismutase (SOD) is a copper- and zinc-containing superoxide dismutase (SOD).

9. A method for increasing the productivity of biosynthetic superoxide dismutase (SOD), characterized in that: It includes the following steps: Add 80 g / L of sterilized sugar, 1.5 g / L of peptone, 2.5 g / L of yeast powder, 1.25 g / L of urea, 0.05 g / L of copper sulfate pentahydrate, 0.05 g / L of zinc sulfate heptahydrate, 2 g / L of dipotassium hydrogen phosphate, 5 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate heptahydrate into a fermenter, and then add ultrapure water up to 2.5 liters to obtain a basal culture medium; (2) Using a high yeast inoculum in the basal medium, the inoculum amount is 10-15 g / L; (3) setting the air flow rate of the fermenter to 1-1.5 v / v / m (air flow per minute / biosynthesis tank volume), the stirring speed to 200-500 rpm, the temperature to 28-36° C., and the pH value to 4.5-7.5, and starting the biosynthesis of superoxide dismutase (SOD); (4) When the initial stage of biosynthesis is reached, the initial stage of synthesis time is 2-6 hours, and the carbon source is added to the fermenter: 10 ml / h of glucose solution, the glucose content in the glucose solution is 50%; and the nitrogen source: 0.7 ml / h of yeast powder solution, the yeast content in the yeast powder solution is 20%, 0.4 ml / h of nitrogen peptone solution, the nitrogen peptone content in the nitrogen peptone solution is 20% and 0.36 ml / h of urea solution, the urea content in the urea solution is 20%, and the addition is stopped after 42-47 hours; (5) When the biosynthesis process reaches the middle stage, which lasts for 20-24 hours, 0.03 ml / h of copper sulfate pentahydrate solution with a copper sulfate content of 15% and 0.03 ml / h of zinc sulfate heptahydrate solution with a zinc sulfate content of 15% are started, the air flow rate is increased to 2.5 vvm, the rotation speed is increased to 800 rpm, and the addition is stopped after 46-49 hours; (6) After the biosynthesis is completed, the biosynthesis completion time is: 52-55 hours; a high yeast content of: 20-23.6% (w / v); and a high superoxide dismutase (SOD) biological activity content of: 8000-9800U / g of yeast are obtained in the biosynthesis liquid; the productivity of yeast biosynthesis of superoxide dismutase is: 40029U / L / h; the high yeast content in the biosynthesis liquid is calculated according to the weight ratio (w / v) of pure wet yeast in the volume of the biosynthesis liquid; the superoxide dismutase (SOD) biological activity content is calculated according to the superoxide dismutase (SOD) activity content in the wet yeast, the dry matter content in the wet yeast is calculated as 33%, and the determination method is based on the pyrogallol method; the productivity of yeast biosynthesis of superoxide dismutase is calculated as the superoxide dismutase (U) biosynthesized per unit volume of the fermentation tank (liter) per unit time (hour).

10. A method for increasing the productivity of biosynthetic superoxide dismutase (SOD) according to claim 9, characterized in that: In step (1), the yeast includes Saccharomyces cerevisiae and Pichia pastoris.

11. A method for increasing the productivity of biosynthetic superoxide dismutase (SOD) according to claim 9, characterized in that: In step (5), when the biosynthesis reaches the middle stage of biosynthesis, the wet yeast content in the biosynthesis liquid is not less than 170 g / L, and the dry matter content of the wet yeast is calculated as 33%.

12. A method for increasing the productivity of biosynthetic superoxide dismutase (SOD) according to claim 9, characterized in that In step (6), the superoxide dismutase (SOD) is an intracellular enzyme, and the superoxide dismutase (SOD) is a copper- and zinc-containing superoxide dismutase (SOD).