A method for synthesizing coenzyme A by combining magnetic immobilized enzyme with weak electric field

Abstract

The invention relates to the technical field of enzyme synthesis, and discloses a method for synthesizing coenzyme A by combining magnetic immobilized enzyme with weak electric field, comprising the following steps: S1, bacterial seed culture; S2, fermentation culture; S3, bacterial collection and preservation; S4, preparation of magnetic immobilized carrier and immobilization of intracellular enzyme protein of Brevibacterium ammoniagenes; S5, synthesis of coenzyme A by magnetic immobilized coenzyme A synthetase. Coenzyme A is synthesized by combining magnetic immobilized enzyme with weak electric field, with high unit titer, short synthesis time, reduced oxidation of reduced coenzyme A, and no need to add mercaptoethanol for reduction in the later stage. Brevibacterium ammoniagenes is broken to free several enzyme proteins required for coenzyme A synthesis, and the purified coenzyme A synthetase is immobilized by using activated hydroxy resin carrier, with mild immobilization conditions, high protein fixation rate, less enzyme inactivation, good stability of magnetic immobilized enzyme, and one-time catalysis of pantothenic acid, adenosine triphosphate and cysteine ​​to synthesize coenzyme A at a fast speed.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme synthesis, and more specifically discloses a method for synthesizing coenzyme A by combining magnetic immobilized enzyme with weak electric field. Background Art

[0002] Coenzyme A is an important coenzyme, often abbreviated as CoA. It plays a vital role in the energy metabolism of cells and can transport and transform a variety of metabolites. The main properties of coenzyme A include: it is composed of pantothenic acid; adenine; ribose; cysteamine and phosphate. It has high solubility and is soluble in water and organic solvents. It exists in an active form in cells and participates in metabolic regulation by combining with other enzymes. The main uses of coenzyme A are: in fatty acid metabolism, coenzyme A combines with fatty acids to form coenzyme A fatty acid esters, thereby promoting the oxidative metabolism of fatty acids under enzyme catalysis. In glucose metabolism, coenzyme A participates in multiple reaction steps of the glycolysis pathway to convert glucose into energy. In cholesterol metabolism, coenzyme A participates in multiple reaction steps of cholesterol synthesis. The preparation method of coenzyme A is mainly obtained by microbial fermentation or by chemical synthesis. Microbial fermentation uses organisms to synthesize coenzyme A, while chemical synthesis is prepared by organic synthesis of compounds.

[0003] Current methods for producing Coenzyme A include:

[0004] 1. Resin adsorption method using pig liver as raw material: He Zhaoxiong introduced the preparation of coenzyme A from pig liver. Each kg of pig liver only contained 2 × 104 units of coenzyme A acetone powder, with a potency of 120 units / mg, a purity of 50%, and a yellowish color. The yield of the GMA-LD-601 combined process was 30%.

[0005] 2. Extraction method using yeast as raw material: The activity loss is 20~30%. Calculated based on freshly squeezed yeast, the yield is only 3000 units / kg.

[0006] 3. Microbial biosynthesis: Brevibacterium ammoniagenes ferments and synthesizes coenzyme A, using pantothenic acid, cysteic acid, and adenosine triphosphate to synthesize coenzyme A. Patent CN200810049249.6 requires multiple feedings and air flow control. The coenzyme A synthesis titer is 300-350 units / ml after 20 hours of reaction; Patent CN201110225924.8 requires air flow control, and the coenzyme A synthesis titer is 500-600 units / ml after 22 hours of reaction; Patent CN202211578695.2 requires air flow control, monitoring and adjusting aerobic and anaerobic synthesis, and the coenzyme A synthesis titer is 564 units / ml after 48 hours of reaction. Luan Yihong used immobilized Brevibacterium ammoniagenes to produce coenzyme A in "Study on the Fermentation of Brevibacterium ammoniagenes and the Synthesis of Coenzyme A by Immobilized Brevibacterium ammoniagenes", and the synthesis rate was only 273 units / ml. In the study of "Study on the preparation of immobilized Brevibacterium ammoniagenes for the synthesis of coenzyme A by different carriers", Wang Haiying et al. used polyacrylamide, polyvinyl alcohol and carrageenan as carriers to encapsulate Brevibacterium ammoniagenes cells to synthesize coenzyme A, with the highest synthesis yield reaching 293 units / ml.

[0007] However, the current methods for producing coenzyme A have the following disadvantages: the yield of coenzyme A synthesized by existing yeast, pig liver, extraction or fermentation of Brevibacterium ammoniagenes is low. The operation steps for synthesizing coenzyme A by fermentation of Brevibacterium ammoniagenes are cumbersome, there are too many influencing factors, there are multiple additions of precursor substances, staged reactions, and the need to monitor aerobic and anaerobic conditions, which places high demands on equipment and production environment. The synthesis time is long, and the risk of fermentation contamination by foreign bacteria increases, resulting in product consumption and increased costs. The unit titer of immobilized Brevibacterium ammoniagenes cells is low, with a maximum of 293 units / ml, which is about 50% lower than the 500~600 units / ml synthesized by bacterial cells. The reaction time is long, the processing time is long, and in addition to the reduced coenzyme A, a large part is oxidized to oxidized coenzyme A, which needs to be reduced by adding mercaptoethanol in the later stage. Summary of the invention

[0008] The present invention mainly provides a method for synthesizing coenzyme A by combining magnetic immobilized enzyme with weak electric field, which can solve the problems of low yield, high cost and long reaction time of current coenzyme A production method.

[0009] To solve the above technical problems, according to one aspect of the present invention, more specifically, a method for synthesizing coenzyme A by combining a magnetic immobilized enzyme with a weak electric field comprises the following steps:

[0010] S1. Bacteria seed culture:

[0011] Brevibacterium ammoniagenes was inoculated on liquid culture medium and cultured at 30-40°C and 220 rpm for 24 h;

[0012] S2. Fermentation culture:

[0013] The qualified shake flask seeds in step S1 were inoculated on the seed tank medium at an inoculum of 10%, and cultured at 30-40°C and 220 rpm for 24 h;

[0014] S3. Collecting and preserving bacteria:

[0015] The liquid obtained by fermentation culture in step S2 is centrifuged to separate the bacterial cells and the supernatant to obtain bacterial cells, and then stored at -20°C;

[0016] S4. Preparation of magnetic immobilized carrier and immobilization of intracellular enzyme protein of Brevibacterium ammoniagenes:

[0017] Add polyvinyl alcohol, superparamagnetic Fe3O4 nanoparticles, sodium chloride and deionized water into a container to fully dissolve them, then add mixed hydroxyethyl methacrylate, divinylbenzene and mixed porogen n-heptane and ethyl acetate, stir at 200-300rpm, heat to 75°C for reaction for 2 hours, then heat to 85°C for reaction for 3 hours, and finally heat to 95°C for curing for 6 hours; wash the obtained resin carrier with hot water, extract with acetone for 8 hours, and dry in vacuum at 50-60°C; after drying, use a rubidium magnet to screen the magnetic carrier, retain the adsorbed ones, and exclude the immobilized carrier without magnetism;

[0018] The bacterial cells of Brevibacterium ammoniagenes were taken out and thawed, dissolved in 500mM imidazole-50mM PBS-200mM NaCl at a concentration of 15%, and broken by a high-pressure homogenizer to release the intracellular Coenzyme A synthetase. The Coenzyme A synthetase solution was obtained by centrifugation at 4000-5000rpm. The Coenzyme A synthetase solution was stirred for 4 hours or more on an activated magnetic hydroxy resin carrier at a rate of 30-60mg protein / 1g, so that the iminocarbonic acid groups on the carrier reacted with the amino groups on the enzyme molecules, and the magnetic immobilized Coenzyme A synthetase was obtained, and the enzyme protein immobilization rate reached 90%;

[0019] S5. Synthesis of Coenzyme A by Magnetic Immobilized Coenzyme A Synthetase:

[0020] Add magnetic immobilized enzyme A synthetase to a weak electric field reaction tank, wash the magnetic immobilized enzyme A synthetase with purified water, filter and set aside, the mass volume ratio of magnetic immobilized coenzyme A synthetase to substrate reaction liquid is 1:10-5:10; pump the reaction liquid to the weak electric field reaction tank, set the voltage of the weak electric field controller to 12V and the current to 2A, and generate coenzyme A by enzyme-catalyzed stirring reaction at a temperature of 32-39°C.

[0021] Furthermore, the liquid culture medium comprises the following components by weight percentage: 1-3% glucose, 1-3% peptone, 1-3% yeast extract, 0.2-0.5% magnesium sulfate, and 0.15-0.45% sodium chloride.

[0022] Furthermore, in step S1, the pH of the liquid culture medium is adjusted to 6.0-7.5 before sterilization, and then sterilized at 121° C. for 20 minutes.

[0023] Furthermore, the seed tank culture medium includes the following components by weight percentage: sucrose 3-8%, peptone 1-3%, magnesium sulfate 0.5-2.0%, KH2PO4 1-3%, K2HPO4 1-3%, and urea 0.5-1.0%.

[0024] Furthermore, in step S2, the sucrose in the seed tank culture medium is 3-8%, the peptone is 1-3%, and the magnesium sulfate is 0.5-2.0%, the pH is adjusted to 7.0-8.0 before sterilization, and then sterilized at 115°C for 20 minutes; the KH2PO4 in the seed tank culture medium is 1-3%, K2HPO4 is 1-3%, and the urea is 0.5-1.0%, and sterilized at 105°C for 15 minutes.

[0025] Furthermore, in S4, the activation method of the magnetic hydroxy resin carrier is: adding the carrier to a 1% cyanogen bromide solution and stirring for 4 hours or more to activate the hydroxyl groups on the carrier to produce imidocarbonic acid derivatives.

[0026] Furthermore, the weak electric field reaction tank includes: a jacketed tank body, a stirring paddle, a stirring motor and a weak electric field controller; the jacketed tank body accessories include an enzyme-fixing partition, a product discharge port, an enzyme-fixing discharge port and internally fixed weak electric field positive and negative plates; the working output voltage of the weak electric field controller is 6~24V, and the controllable current range is 0.5~20A.

[0027] The weak electric field reaction tank is preferably controlled at 12V, 2A to provide active electrons for the reaction, and the enzyme activity is better. At the same time, water molecules are electrolyzed under the action of the weak electric field to provide hydrogen ions to prevent the oxidation of the synthesized reduced coenzyme A. At the same time, the magnetic immobilized coenzyme A synthetase moves in a more orderly manner under the action of the weak electric field and magnetic field, which promotes the reaction.

[0028] Furthermore, in step S5, the substrate reaction solution is prepared in a preparation tank, and the substrate reaction solution includes the following raw materials: 10-30 mmol / L calcium pantothenate, 50-150 mmol / L adenosine triphosphate, 20-60 mmol / L magnesium chloride, and 15-35 mmol / L cysteine; then the pH of the substrate reaction solution is adjusted to 6.5-8.5, and the temperature is raised to 35-39° C. for insulation.

[0029] The beneficial effects of the method for synthesizing coenzyme A by combining a magnetic immobilized enzyme with a weak electric field are as follows: the purpose of the present invention is to synthesize coenzyme A by combining a magnetic immobilized enzyme with a weak electric field, with high unit titer, short synthesis time, reduced coenzyme A oxidation reduction, and no need to add mercaptoethanol for reduction in the later stage. The ammonia-producing bacillus is broken to free several enzyme proteins required for the synthesis of coenzyme A, and the purified coenzyme A synthetase is immobilized using an activated hydroxy resin carrier, the immobilization conditions are mild, the protein fixation rate is high, the enzyme inactivation is small, the magnetic immobilized enzyme has good stability, and the catalytic synthesis of pantothenic acid, adenosine triphosphate, and cysteine ​​by one time is fast, and the reaction can be completed in 8 to 10 hours, the coenzyme A synthesis rate is high, the effective rate is more than 1700 units / ml, the oxidized coenzyme A accounts for a small proportion, which is 2.83 times that of the existing bacterial synthesis of coenzyme A; the synthesis capacity does not decrease after repeated use for more than 30 times, the separation operation of the reaction solution and the immobilized enzyme is simple, the utilization rate of the production equipment is improved, and it is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention is further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0031] Figure 1 This is the 8-hour spectrum of the first reaction of the embodiment;

[0032] Figure 2 This is the 8-hour spectrum of the fifteenth reaction of Example;

[0033] Figure 3 This is the 8-hour spectrum of the 30th reaction of Example;

[0034] Figure 4 This is a schematic diagram of the structure of a weak electric field reaction tank. DETAILED DESCRIPTION

[0035] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that the embodiments and features in the embodiments of the present application can be combined with each other without conflict.

[0036] According to one aspect of the present invention, a method for synthesizing coenzyme A by combining a magnetically immobilized enzyme with a weak electric field is provided, comprising the following steps:

[0037] 1. Bacteria seed culture:

[0038] Glucose 1~3%, peptone 1~3%, yeast extract 1~3%, magnesium sulfate 0.2~0.5%, sodium chloride 0.15~0.45%, pH adjusted to 6.0-7.5 before sterilization, then sterilized at 121℃ for 20min. After inoculation of the preferred ammonia-producing Brevibacterium, culture at 30~40℃, 220rpm for 24h;

[0039] 2. Fermentation culture:

[0040] Sucrose 3~8%, peptone 1~3%, magnesium sulfate 0.5~2.0%, pH adjusted to 7.0-8.0 before sterilization, sterilized at 115℃ for 20min; KH2PO4 1~3%, K2HPO4 1~3%, urea 0.5~1.0%, sterilized at 105℃ for 15min. Inoculate the above bacterial seeds with 10% inoculation amount, culture at 30~40℃, 220rpm for 24h;

[0041] 3. Collecting and preserving bacteria:

[0042] The liquid obtained by fermentation culture in step S2 is centrifuged to separate the bacterial cells and the supernatant to obtain bacterial cells, and then stored at -20°C;

[0043] 4. Preparation of magnetic immobilized carrier and immobilization of intracellular enzyme protein of Brevibacterium ammoniagenes:

[0044] Add polyvinyl alcohol, superparamagnetic Fe3O4 nanoparticles, sodium chloride and deionized water into a container to fully dissolve them, then add mixed hydroxyethyl methacrylate, divinylbenzene and mixed porogen n-heptane and ethyl acetate, stir at 200-300rpm, heat to 75°C for reaction for 2 hours, then heat to 85°C for reaction for 3 hours, and finally heat to 95°C for curing for 6 hours; wash the obtained resin carrier with hot water, extract with acetone for 8 hours, and dry in vacuum at 50-60°C; after drying, use a rubidium magnet to screen the magnetic carrier, retain the adsorbed ones, and exclude the immobilized carrier without magnetism;

[0045] The activation method of the magnetic hydroxy resin carrier is as follows: adding the carrier to a 1% cyanogen bromide solution and stirring for 4 hours or more to activate the hydroxyl groups on the carrier to produce iminocarbonic acid derivatives;

[0046] The bacterial cells of Brevibacterium ammoniagenes were taken out and thawed, dissolved in 500mM imidazole-50mM PBS-200mM NaCl at a concentration of 15%, and broken by a high-pressure homogenizer to release the intracellular Coenzyme A synthetase. The Coenzyme A synthetase solution was obtained by centrifugation at 4000-5000rpm. The Coenzyme A synthetase solution was stirred for 4 hours or more on an activated magnetic hydroxy resin carrier at a rate of 30-60mg protein / 1g, so that the iminocarbonic acid groups on the carrier reacted with the amino groups on the enzyme molecules, and the magnetic immobilized Coenzyme A synthetase was obtained, and the enzyme protein immobilization rate reached 90%.

[0047] 5. Synthesis of Coenzyme A by Magnetic Immobilized Coenzyme A Synthetase:

[0048] Add magnetic immobilized enzyme A synthetase to a weak electric field reaction tank, wash the magnetic immobilized enzyme A synthetase with purified water, filter and set aside, the mass volume ratio of magnetic immobilized coenzyme A synthetase to substrate reaction liquid is 1:10-5:10; mix in a mixing tank, the substrate reaction liquid includes the following raw materials: 10-30 mmol / L calcium pantothenate, 50-150 mmol / L adenosine triphosphate, 20-60mmol / L magnesium chloride, 15-35mmol / L cysteine; then adjust the pH of the substrate reaction liquid to 6.5-8.5, heat to 35-39°C and keep warm; pump the reaction liquid to a weak electric field reaction tank, set the voltage of the weak electric field controller to 12V and the current to 2A, and generate coenzyme A by enzyme-catalyzed stirring reaction at a temperature of 32-39°C. The yield of synthesized coenzyme A is above 1700 units / ml. The immobilized enzyme and the reaction solution can be reused after simple solid-liquid separation. The activity of the immobilized enzyme will not decrease significantly after being reused 30 times.

[0049] The weak electric field reaction tank includes: a jacketed tank, a stirring paddle, a stirring motor and a weak electric field controller; the jacketed tank accessories include an enzyme-fixing partition, a product discharge port, an enzyme-fixing discharge port and internally fixed weak electric field positive and negative plates, such as Figure 4 As shown; the working output voltage of the weak electric field controller is 6~24V, and the controllable current range is 0.5~20A.

[0050] The weak electric field reaction tank is preferably controlled at 12V, 2A to provide active electrons for the reaction, and the enzyme activity is better. At the same time, water molecules are electrolyzed under the action of the weak electric field to provide hydrogen ions to prevent the oxidation of the synthesized reduced coenzyme A. At the same time, the magnetic immobilized coenzyme A synthetase moves in a more orderly manner under the action of the weak electric field and magnetic field, which promotes the reaction.

[0051] Example

[0052] Fermentation of bacteria: Take one of the preferred ammonia-producing Brevibacterium seeds and inoculate it in a sterilized liquid culture medium containing 2% glucose, 2% peptone, 2% yeast extract, 0.35% magnesium sulfate, and 0.35% sodium chloride. Incubate it in a shaking incubator at 200rpm and 37℃ for 20 hours. If the OD600 is 4.3 and there are no foreign bacteria under microscopic examination, it is a qualified shake flask seed. Inoculate the shake flask seeds in a first-level seed tank culture medium containing 50L 4.5% sucrose, 5% peptone, 1.5% magnesium sulfate, 2% KH2PO4, 2% K2HPO4, and 0.75% urea. Incubate it in a rotating speed of 220rpm and aerate for 1.3~1.5m 3 / h, temperature 31~32℃, pressure 0.05Mpa, culture for 5 hours, after OD600 is greater than 4, transfer to 500L secondary seed tank culture medium, speed 200rpm, ventilation 10~12m 3 / h, temperature 31 ~ 34 ℃, pressure 0.05Mpa, culture for 6 hours, OD600 greater than 4, the secondary seed liquid into the fermentation tank sucrose 8%, peptone 3%, magnesium sulfate 2%, KH2PO43%, K2HPO4 3%, urea 1% culture medium, speed 120 ~ 150rpm, ventilation 40 ~ 80m 3 / h, temperature 31~35℃, continue to feed fermentation. Ferment for 24 hours, until the feed is finished or the OD600 and protein amount do not increase significantly, then stop the fermentation. Collect the bacterial cells by centrifugation. The bacterial cells can be stored at -20℃ when not in use.

[0053] Preparation of magnetic hydroxy resin carrier: Add polyvinyl alcohol, superparamagnetic Fe3O4 nanoparticles, sodium chloride, and deionized water to a three-necked flask equipped with a condenser, stirring, and a thermometer to fully dissolve them, then add the mixed hydroxyethyl methacrylate, divinylbenzene, and mixed porogens n-heptane and ethyl acetate, slowly start stirring and adjust to 200rpm, then heat to 75℃ for two hours, then heat to 85℃ for three hours, and finally heat to 95℃ for six hours of boiling. Pour the reactants into a filter bag, rinse with hot water until the effluent is colorless, then extract with acetone for 8 hours, dry, and vacuum dry at 55℃. After drying, use a rubidium magnet to screen the magnetic carrier, retain those that can be adsorbed, and exclude the immobilized carrier without magnetism.

[0054] Activation of magnetic hydroxy resin carrier: 200 kg of hydroxy resin carrier was added into 1000 L of 5% cyanogen bromide solution, stirred for 4 hours, and filtered.

[0055] Enzyme protein extraction: 75 kg of Brevibacterium ammoniagenes cells were taken and dissolved in 500 L of 500 mM imidazole-50 mM PBS-200 mM NaCl to obtain a bacterial solution. The bacterial solution was crushed by a 550 bar high-pressure homogenizer and centrifuged at 4500 rpm to obtain 500 L of crude enzyme solution. The protein content in the enzyme solution was detected to be 7.02 kg. 200 kg of activated magnetic hydroxy resin carrier was weighed and added to the crude enzyme. The enzyme was fixed by stirring for 4 hours and filtered. The protein residue in the filtrate was determined and the protein immobilization rate was calculated to be 96%. The immobilized protein content per unit weight of the hydroxy resin carrier was 32.70 mg / g. The immobilized coenzyme A synthetase was washed 3 times with 300 L of water for standby use.

[0056] 100 kg of magnetic immobilized CoA synthetase was weighed and added to 1000 L of substrate reaction solution at pH 7.5. The temperature was raised to 37°C for reaction for 8 hours. The CoA synthesis titer was measured. The immobilized enzyme was reused 30 times, and the CoA synthesis yield titer was maintained above 1700 units / ml.

[0057] The first reaction 8 hours spectrum is as follows Figure 1As shown, the peak area of ​​coenzyme A is 75.6254 mAU*min, which is substituted into the standard curve. Potency = (0.0079*peak area+0.0065)*dilution factor*standard labeled potency 298.

[0058] The yield of coenzyme A synthesis was 1799.743 units / ml, and the ratio of reduced coenzyme A to oxidized coenzyme A was 99.96:0.04.

[0059] The spectrum of the fifteenth reaction after 8 hours is as follows Figure 2 As shown, the peak area of ​​coenzyme A is 76.6349 mAU*min, which is substituted into the standard curve. Potency = (0.0079*peak area+0.0065)*dilution factor*standard labeled potency 298.

[0060] The yield of coenzyme A synthesis was 1823.508 units / ml, and the ratio of reduced coenzyme A to oxidized coenzyme A was 99.99:0.01.

[0061] The 30th reaction 8 hours spectrum is as follows Figure 3 As shown, the peak area of ​​coenzyme A is 73.5528 mAU*min, which is substituted into the standard curve. Potency = (0.0079*peak area+0.0065)*dilution factor*standard labeled potency 298.

[0062] The yield of coenzyme A synthesis was 1750.950 units / ml, and the ratio of reduced coenzyme A to oxidized coenzyme A was 99.98:0.02.

[0063] From the above, it can be seen that the immobilized enzyme is easy to separate from the reaction liquid, the immobilized enzyme can be reused, and the activity stability is high. The immobilized enzyme has high catalytic activity, and the coenzyme A yield is more than 1700 units / ml. Compared with the synthesis of bacterial cells, the reaction liquid components reduce the impurities derived from the metabolism of bacterial cells, and the subsequent extraction and purification are easy. Compared with the immobilized ammonia-producing Brevibacterium cells, the immobilized enzyme synthesized coenzyme A has higher enzyme activity in the weak electric field reaction tank, better enzyme activity retention, and 1700 units / ml efficiency is maintained for 30 batches of continuous reaction. In addition, the proportion of oxidized coenzyme A is greatly reduced, and there is no need to add mercaptoethanol for reduction, which reduces the addition of impurities and avoids the unpleasant odor in the finished coenzyme A.

[0064] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by ordinary technicians in this technical field within the essential scope of the present invention also fall within the protection scope of the present invention.

Claims

1. A method for synthesizing coenzyme A by combining magnetic immobilized enzyme with weak electric field, characterized in that: The following steps are involved: S1. Bacteria seed culture: Brevibacterium ammoniagenes was inoculated on liquid culture medium and cultured at 30-40°C and 220 rpm for 24 h; S2. Fermentation culture: The qualified shake flask seeds in step S1 were inoculated on the seed tank medium at an inoculum of 10%, and cultured at 30-40°C and 220 rpm for 24 h; S3. Collecting and preserving bacteria: The liquid obtained by fermentation culture in step S2 is centrifuged to separate the bacterial cells and the supernatant to obtain bacterial cells, and then stored at -20°C; S4. Preparation of magnetic immobilized carrier and immobilization of intracellular enzyme protein of Brevibacterium ammoniagenes: Add polyvinyl alcohol, superparamagnetic Fe3O4 nanoparticles, sodium chloride and deionized water into a container to fully dissolve them, then add mixed hydroxyethyl methacrylate, divinylbenzene and mixed porogen n-heptane and ethyl acetate, stir at 200-300rpm, heat to 75°C for reaction for 2 hours, then heat to 85°C for reaction for 3 hours, and finally heat to 95°C for curing for 6 hours; wash the obtained resin carrier with hot water, extract with acetone for 8 hours, and dry in vacuum at 50-60°C; after drying, use a rubidium magnet to screen the magnetic carrier, retain the adsorbed ones, and exclude the immobilized carrier without magnetism; The bacterial cells of Brevibacterium ammoniagenes are taken out and thawed, dissolved in 500mM imidazole-50mM PBS-200mM NaCl at a concentration of 15%, broken by a high-pressure homogenizer to release intracellular Coenzyme A synthetase, centrifuged at 4000-5000rpm to obtain Coenzyme A synthetase liquid, and the Coenzyme A synthetase liquid is stirred for 4 hours or more on an activated magnetic hydroxy resin carrier at a rate of 30-60mg protein / 1g, so that the iminocarbonic acid groups on the carrier react with the amino groups on the enzyme molecules, and immobilized to obtain magnetic immobilized Coenzyme A synthetase; The activation method of the magnetic hydroxy resin carrier is as follows: adding the carrier to a 1% cyanogen bromide solution and stirring for 4 hours or more to activate the hydroxyl groups on the carrier to produce iminocarbonic acid derivatives; S5. Synthesis of Coenzyme A by Magnetic Immobilized Coenzyme A Synthetase: Add the magnetic immobilized coenzyme A synthetase to the weak electric field reaction tank, wash the magnetic immobilized coenzyme A synthetase with purified water, filter and dry for later use, the mass volume ratio of the magnetic immobilized coenzyme A synthetase to the substrate reaction liquid is 1:10-5:10; pump the reaction liquid to the weak electric field reaction tank, set the voltage of the weak electric field controller to 12V and the current to 2A, and generate coenzyme A by enzyme-catalyzed stirring reaction at a temperature of 32-39°C; The substrate reaction solution is prepared in a preparation tank, and the substrate reaction solution includes the following raw materials: 10-30 mmol / L calcium pantothenate, 50-150 mmol / L adenosine triphosphate, 20-60 mmol / L magnesium chloride, and 15-35 mmol / L cysteine; then the pH of the substrate reaction solution is adjusted to 6.5-8.5, and the temperature is raised to 35-39°C for insulation.

2. The method for synthesizing coenzyme A by combining magnetic immobilized enzyme with weak electric field according to claim 1, characterized in that: The liquid culture medium comprises the following components by weight percentage: 1-3% glucose, 1-3% peptone, 1-3% yeast extract, 0.2-0.5% magnesium sulfate, and 0.15-0.45% sodium chloride.

3. The method for synthesizing coenzyme A by combining magnetic immobilized enzyme with weak electric field according to claim 1, characterized in that: In step S1, the pH of the liquid culture medium is adjusted to 6.0-7.5 before sterilization, and then sterilized at 121° C. for 20 min.

4. The method for synthesizing coenzyme A by combining magnetic immobilized enzyme with weak electric field according to claim 1, characterized in that: The seed tank culture medium comprises the following components by weight percentage: 3-8% sucrose, 1-3% peptone, 0.5-2.0% magnesium sulfate, 1-3% KH2PO4, 1-3% K2HPO4, and 0.5-1.0% urea.

5. The method for synthesizing coenzyme A by combining magnetic immobilized enzyme with weak electric field according to claim 4, characterized in that: In step S2, the sucrose in the seed tank culture medium is 3-8%, the peptone is 1-3%, and the magnesium sulfate is 0.5-2.0%. The pH is adjusted to 7.0-8.0 before sterilization, and then sterilized at 115°C for 20 minutes; the KH2PO4 in the seed tank culture medium is 1-3%, K2HPO4 is 1-3%, and the urea is 0.5-1.0%, and sterilized at 105°C for 15 minutes.

6. The method for synthesizing coenzyme A by combining magnetic immobilized enzyme with weak electric field according to claim 1, characterized in that: The weak electric field reaction tank includes: a jacketed tank body, a stirring paddle, a stirring motor and a weak electric field controller; the jacketed tank body accessories include an enzyme-fixing partition, a product discharge port, an enzyme-fixing discharge port and internally fixed weak electric field positive and negative plates; the working output voltage of the weak electric field controller is 6~24V, and the controllable current range is 0.5~20A.

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