Fermentation process of high-expression nitrile hydratase

Abstract

The invention discloses a high-expression nitrile hydratase fermentation process which comprises the following steps: marking a strain on an LB plate culture medium, and culturing for 16 hours in a constant-temperature incubator at 37 DEG C; selecting monoclonal cells, transferring the monoclonal cells into a 40ml LB culture medium shake flask, and carrying out shake flask culture at 37 DEG C and 220rpm for 8 hours; transferring 40mL of the primary seed solution into a 150ml secondary seed culture medium shake flask, and carrying out shake cultivation for 8 hours at 37 DEG C and 220rpm (revolutions per minute); transferring 150mL of the secondary seed solution into a 10L fermentation tank filled with a fermentation culture medium, carrying out fermentation culture at 37 DEG C until OD60060 or so, cooling to 24 DEG C, and adding an inducer for induction; fermenting and culturing in a tank: detecting the OD value of the fermentation liquor, sampling every 6-8 hours, detecting the OD value and the enzyme activity of the fermentation liquor, and putting in the tank until the enzyme activity does not rise any more. According to the method, fermentation conditions of screened strains are optimized, so that the unit enzyme activity of nitrile hydratase is improved, the use amount of bacterial sludge is reduced, the fermentation period is shortened, and the production cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of microbial enzyme fermentation technology, and in particular to a fermentation process for high expression of nitrile hydratase. Background Technology

[0002] Nicotinamide is the amide form of vitamin B3 (niacin) and a key component of coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) in organisms. It is widely involved in processes such as energy metabolism, DNA repair, and cell signaling.

[0003] Nicotinamide has wide applications in pharmaceuticals, health products, cosmetics, and feed additives. Nicotinamide (the amide form of vitamin B3) is a multifunctional biomolecule with biological functions involving cell metabolism, antioxidation, anti-inflammation, skin health, and immune regulation. Nicotinamide is the amide form of nicotinic acid and one of the precursors of nicotinamide adenine dinucleotide (NAD+). It can be used as a dietary supplement or clinical treatment to supplement NAD+ levels in the body, participating in key functions such as cell metabolism and DNA repair. Nicotinamide is inexpensive, widely available, and has good biocompatibility. It also possesses multiple biological functions, including antibacterial, anti-inflammatory, and cellular immune regulation, and has significant effects on improving skin diseases and neurodegenerative diseases.

[0004] Currently, the main methods for synthesizing nicotinamide are chemical synthesis and biosynthesis. Chemical synthesis suffers from several drawbacks, including harsh reaction conditions, high operational risks, severe environmental pollution and difficulty in treating waste, high toxicity of raw materials and high residue risks, difficulties in product separation, limited purity and yield, complex operation, high overall cost, and significant safety risks. Biosynthesis, on the other hand, offers advantages such as environmental friendliness, superior product characteristics, mild reaction conditions, substrate adaptability and optimizability, and great potential for industrial production.

[0005] The main drawbacks of biosynthesis are the high cost of raw materials, the expensive inducers (such as IPTG), the high price of special nitrogen sources (such as yeast extract), and the low enzyme activity in industrial production. Furthermore, temperature variations during fermentation can inactivate nitrile hydratases. Higher temperatures can alter the enzyme structure, leading to reduced or even lost enzyme activity, while low temperatures may also inhibit enzyme activity. All of these factors limit temperature control and efficiency improvements in fermentation. Summary of the Invention

[0006] The purpose of this invention is to provide a fermentation process that highly expresses nitrile hydratase, thereby increasing the unit enzyme activity of nitrile hydratase, reducing the amount of sludge used, shortening the fermentation cycle, and reducing production costs, thus solving the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A fermentation process for high expression of nitrile hydratase includes the following steps:

[0009] Step 1: Activation of bacterial strain: Streak the bacterial strain on LB agar plates and incubate at 37°C for 16 hours;

[0010] Step 2: Primary seed culture: Pick a single clone and transfer it to a 40ml LB medium shake flask, and culture at 37℃ and 220rpm for 8h.

[0011] Step 3: Secondary seed culture: Transfer 40 mL of primary seed culture to a 150 mL secondary seed culture medium shake flask and culture at 37 °C and 220 rpm for 8 h.

[0012] Step 4: Fermentation in a fermenter: Transfer 150 mL of the secondary seed culture to a 10 L fermenter containing fermentation medium and ferment at 37 °C until OD reaches the target level. 600 At around 60°C, cool down to 18°C ​​and add an inducing agent to induce induction;

[0013] Step 5: Detect the OD value of the fermentation broth: Take samples every 6-8 hours to detect the OD value and enzyme activity of the fermentation broth. Once the enzyme activity stops rising, the broth can be transferred to the tank.

[0014] Furthermore, the fermentation tank culture in step 4 includes the following process:

[0015] Step 401: After the fermenter is cleaned by alkaline boiling and acid washing, it is sterilized in an empty state. pH and dissolved oxygen electrodes are installed, and a pressure leak test is performed. After the condition is normal, the prepared culture medium is fully dissolved and added to the fermenter to a final volume of 3.5L. Defoaming is added, and the fermenter is sterilized at 121℃ for 20 minutes. After the temperature drops to 37℃, the pH is adjusted to 7.0 with ammonia water, and dissolved oxygen is measured.

[0016] Step 402: Inoculation volume: 50 mL seed culture; initial conditions: temperature: 37℃, pH: 7.0, air flow rate: 0.3 m3 / h, tank pressure: 0.05 MPa, rotation speed: 200 rpm.

[0017] Step 403: After 3 hours of fermentation, start increasing the speed, reaching 800 rpm after 6 hours. At this time, dissolved oxygen will rise after 1.5 hours. The pH of the fermentation broth will first rise and then fall. After the dissolved oxygen rises back to above 60, the speed will decrease. At this time, start feeding. During this period, pay attention to the pH value, which will rise to about 7.2. Control the feeding rate to ensure that the pH value does not rise again or has a downward trend.

[0018] Step 404: Measure the OD value about 2 hours after feeding. When the OD reaches 50 or above, start cooling and induction. After induction, take samples every 4 hours to test OD and enzyme activity. Adjust the feeding speed according to the specific growth rate u, and control u≤0.02.

[0019] Further, the standard dissolved oxygen conditions in step 401 are: temperature: 37℃, pH: 7.0, air volume: 1.0m3 / h, tank pressure: 0.05MPa, and rotation speed: 700rpm.

[0020] Furthermore, in step 402, the fermentation process is carried out according to the reaction conditions of the tank. When the rotation speed is increased to 400 rpm, the air volume and rotation speed are adjusted alternately, the dissolved oxygen is controlled at 25-35%, the maximum rotation speed is 800 rpm, and the maximum air volume is 1.0 m3 / L.

[0021] Furthermore, the volume of the fermentation medium in step 4 is 4L.

[0022] Furthermore, the components of the LB medium include: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 15 g / L agar strips.

[0023] Furthermore, the components of the secondary seed culture medium include: 10.5 g / L K2HPO4, 4.5 g / L KH2PO4, 1 g / L (NH4)2SO4, 0.5 g / L sodium citrate dihydrate, 10 g / L FM902 yeast extract, and 5 g / L glycerol.

[0024] Furthermore, the fermentation medium comprises the following components: K2HPO4 12 g / L, KH2PO4 4 g / L, (NH4)2SO4 1.2 g / L, sodium citrate dihydrate 0.3 g / L, FM902 yeast extract 10 g / L, glycerol 20 g / L, and magnesium sulfate heptahydrate 1.2 g / L.

[0025] Furthermore, 50 μg / mL of Kana antibiotic was added to both LB medium and secondary seed medium. The preparation method of Kana antibiotic is as follows: 50 g / L stock solution, filtered through a 0.2 μm filter membrane for sterilization, stored at -20℃, and the working concentration is 1.0 per thousand.

[0026] Furthermore, the feed in step 403 is a fermentation feed medium, which is prepared as follows: 300 g / L glycerol, 55 g / L corn steep liquor powder, sterilized in an autoclave at 121°C for 20 min.

[0027] The inducing agents in step 404 are lactose inducer and cobalt chloride. The working concentration of lactose inducer and cobalt chloride is calculated based on the fermentation volume, which is 15 g / L + 0.4 g / L. They are prepared separately and sterilized in an autoclave at 121°C for 20 min.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] This invention obtains a strain that highly expresses nitrile hydratase and equips it with a complete fermentation process. The fermentation conditions of the screened strain are optimized to determine the optimal values ​​of each factor in the fermentation culture process of nitrile hydratase, achieving an enzyme activity of ≥15000u / mL. This provides an efficient and rapid production method for the synthesis of nitrile hydratase and can form a complete tricyanopyridine to nicotinamide industrial chain. Attached Figure Description

[0030] Figure 1 This is a flowchart of the fermentation process for high expression of nitrile hydratase in this invention;

[0031] Figure 2 This invention relates to the detection of enzyme activity and OD in shake flasks. 600 A bar chart of values;

[0032] Figure 3 This is a bar graph illustrating the effect of induction temperature on enzyme activity in this invention.

[0033] Figure 4 This is a bar graph showing the effect of the amount of inducer used in this invention on enzyme activity.

[0034] Figure 5 This is a bar graph illustrating the effect of induction time on enzyme activity according to the present invention.

[0035] Figure 6 This is a bar graph showing the effect of the specific growth rate on enzyme activity and fermentation according to the present invention. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Strains source: The strains used in the experiments of this invention were self-constructed in the molecular laboratory.

[0038] The experimental instruments used in the experiment are as follows:

[0039] Clean bench, constant temperature incubator, shaker, spectrophotometer, protein electrophoresis apparatus, liquid chromatograph, 10L fermenter, PCR instrument, -80℃ refrigerator.

[0040] like Figure 1 As shown, a fermentation process for high expression of nitrile hydratase includes the following steps:

[0041] Step 1: Activation of bacterial strain: The bacterial strain was streaked on LB agar plates (Kana: 50ug / mL) and incubated at 37℃ for 16 hours;

[0042] Step 2: Primary seed culture: Pick a single clone and transfer it to a 40ml LB medium (Kana: 50ug / mL) shake flask, and culture at 37℃ and 220rpm for 8h.

[0043] Step 3: Secondary seed culture: Transfer 40 mL of primary seed culture to a 150 mL secondary seed culture medium (Kana: 50 ug / mL) shake flask and culture at 37 °C and 220 rpm for 8 h.

[0044] Step 4: Fermentation in a fermentation tank: Transfer 150 mL of the secondary seed culture to a 10 L fermentation tank containing fermentation medium (4 L of substrate), and ferment at 37 °C until OD reaches 1000. 600 At around 60°C, cool down to 18°C, add an inducing agent to induce induction, as detailed below:

[0045] (1) After the fermenter is cleaned by alkaline boiling and acid washing, it is sterilized in the air. pH and dissolved oxygen electrodes are installed, and a pressure leak test is performed. After it is normal, the prepared culture medium is fully dissolved and added to the fermenter to a final volume of 3.5L. Defoaming is added, and the fermenter is sterilized at 121℃ for 20 minutes. After the temperature drops to 37℃, the pH is adjusted to 7.0 with ammonia water, and dissolved oxygen standardization is performed (dissolved oxygen standardization conditions: temperature: 37℃, pH: 7.0, air volume: 1.0m3 / h, tank pressure: 0.05MPa, rotation speed: 700rpm).

[0046] (2) Flame inoculation: Inoculation volume: 50 mL seed liquid; initial conditions: temperature: 37℃, pH: 7.0, air volume: 0.3 m3 / h, tank pressure: 0.05 MPa, rotation speed: 200 rpm. (During fermentation, the air volume and rotation speed are adjusted alternately according to the tank reaction conditions when the rotation speed is increased to 400 rpm, the dissolved oxygen is controlled at 25-35%, the maximum rotation speed is 800 rpm, and the maximum air volume is 1.0 m3 / L);

[0047] (3) After fermentation for about 3 hours, start increasing the speed. It will reach 800 rpm in about 6 hours. At this time, dissolved oxygen will rise after 1.5 hours. During this period, the pH of the fermentation liquid will rise first and then fall. When the dissolved oxygen rises back to above 60, the speed will drop. At this time, start feeding. During this period, pay attention to the pH value, which will rise to about 7.2. Control the feeding rate to ensure that the pH value does not rise or has a downward trend.

[0048] (4) Measure the OD value about 2 hours after feeding. Start cooling and induction when the OD reaches above 50. Take samples every 4 hours after induction to test OD and enzyme activity. Adjust the feeding rate according to the specific growth rate u, and control u ≤ 0.02 for optimal results.

[0049] Step 5: Detect the OD value of the fermentation broth: Take samples every 6-8 hours to detect the OD value and enzyme activity of the fermentation broth. Once the enzyme activity stops rising, the broth can be transferred to the tank.

[0050] The LB medium consists of: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 15 g / L agar strips.

[0051] The components of the secondary seed culture medium include: K2HPO4 10.5 g / L, KH2PO4 4.5 g / L, (NH4)2SO4 1 g / L, sodium citrate dihydrate 0.5 g / L, FM902 yeast extract 10 g / L, and glycerol 5 g / L.

[0052] The fermentation medium consists of: 12 g / L K2HPO4, 4 g / L KH2PO4, 1.2 g / L (NH4)2SO4, 0.3 g / L sodium citrate dihydrate, 10 g / L FM902 yeast extract, 20 g / L glycerol, and 1.2 g / L magnesium sulfate heptahydrate.

[0053] Both LB medium and secondary seed medium were supplemented with 50 μg / mL of Kana antibiotic. The preparation method of Kana antibiotic is as follows: 50 g / L stock solution, filtered through a 0.2 μm filter membrane for sterilization, stored at -20℃, and the working concentration is 1.0 per thousand.

[0054] The feed was a fed-batch fermentation medium, prepared as follows: 300 g / L glycerol, 55 g / L corn steep liquor powder, sterilized in an autoclave at 121°C for 20 minutes.

[0055] The inducing agents used were lactose inducer and cobalt chloride. The working concentration of lactose inducer and cobalt chloride was calculated based on the fermentation volume, and was 15 g / L + 0.4 g / L. They were prepared separately and sterilized in an autoclave at 121℃ for 20 min.

[0056] Based on the bacterial strains obtained above, the following experiments were conducted in this embodiment:

[0057] Step 1: Transform and screen strains to obtain strains with high enzyme activity.

[0058] After the genes obtained from the initial screening were amplified, they were ligated into the pET42 vector backbone, and the plasmid was mutated using a random mutagenesis kit. Protein detection was then performed after the mutation.

[0059] ① Activate the new bacterial strain in glycerol tubes on LB (K+) plates and incubate at 37℃ for 16 hours; pick a single colony from the plate and transfer it to an LB (K+) single tube, and incubate at 37℃ and 220 rpm on a shaker for 16 hours;

[0060] ② Transfer the single-tube bacterial culture to an LB(K+) shake flask at a transfer volume of 3%, and incubate at 37°C and 220 rpm on a shaker until OD reaches 0.5. 600 Value 0.4-0.6, add lactose, and induce culture at 18℃ and 220rpm for 20h on a shaker;

[0061] ③ Take the shake flask and measure enzyme activity and OD. 600 Value, result as Figure 2 As shown, SDS-PAGE detection was performed.

[0062] ④ Select single colonies with normal protein expression and high enzyme activity as detected by SDS-PAGE, mix them, activate them on LB(K+) plates, incubate them in a constant temperature incubator at 37℃ for 16 hours, and then store them in 20% glycerol at -80℃.

[0063] Step 2: Optimization of three key parameters: temperature, time, and inducer concentration.

[0064] (1) Temperature has a significant impact on the stability, inclusion body formation, and soluble expression of exogenous proteins. By setting a series of temperature gradients (15℃, 18℃, 21℃, 24℃, 27℃, 30℃, and 33℃) to induce expression, the protein expression level and activity at each temperature were measured to determine the optimal expression temperature. Figure 3 Data showed that the enzyme activity was highest at an induction temperature of 24℃. As the temperature increased, the OD value of the cells gradually increased, but the enzyme activity did not increase further.

[0065] (2) Effect of inducer dosage on enzyme activity

[0066] Lactose is used to induce the expression of recombinant proteins in *E. coli*. It works by binding to repressor proteins, releasing the repression of the operon, and thus initiating transcription of the target gene. When galactosidase degrades lactose into galactose and glucose, some lactose is rearranged to form isolaxose. Lactose is metabolized, and its concentration may not be constant throughout the induction process. Therefore, the amount of lactose used becomes a critical factor in recombinant protein expression; insufficient lactose results in poor protein expression, while excessive lactose is wasteful. Therefore, if... Figure 4 As shown, fermentation in a 10L tank was conducted using strains with high enzyme activity at unit volume concentrations of 8 g / L, 10 g / L, 12 g / L, 15 g / L, 17 g / L, and 19 g / L, respectively. The enzyme activity reached its maximum at an inducer concentration of 12 g / L; further increasing the inducer concentration did not result in further increases in enzyme activity.

[0067] (3) Effect of induction time on enzyme activity

[0068] The effect of induction time on enzyme activity is mainly reflected in the synthesis and accumulation of enzyme proteins. With prolonged induction time, the expression level of enzyme proteins increases; however, when their accumulation within the cell reaches a certain level, they may become toxic to the recombinant bacteria, leading to a decrease in the synthesis rate or even product degradation. Therefore, it is necessary to balance the relationship between induction time and enzyme activity during the induction process. Figure 5As shown, with increasing induction time, the bacterial cells gradually age and autolyze in the later stages, resulting in a decrease in OD and enzyme activity. Therefore, the appropriate incubation time is between 48 and 51 hours.

[0069] Step 3: Effect of growth rate on enzyme activity

[0070] Under suitable growth conditions, the specific growth rate is high, microbial metabolism is active, and enzyme activity is relatively high. However, under unfavorable conditions, such as excessively high or low dissolved oxygen, excessively high or low temperatures, or unsuitable pH, the specific growth rate decreases, thus affecting enzyme synthesis and activity. The specific growth rate of the induced cells can be controlled by adjusting the feeding rate. Figure 6 As shown, when u is around 0.02, the fermentation cycle is relatively long, and the feeding rate becomes the main factor limiting cell growth. Cell growth is slow, and as lactose, as a carbon source, is metabolized during fermentation, its concentration gradually decreases. Under the influence of limiting substances, the cells enter the stationary phase prematurely. When u is around 0.04, the feeding rate is high, and the dissolved oxygen during fermentation is low, becoming the main factor limiting cell growth. The cells produce a large amount of acetic acid, and excessive acetic acid production affects cell growth and protein expression.

[0071] Step 4: Catalytic verification is shown in the table below:

[0072]

[0073] As shown in the table above, in the experiment of converting nicotinamide with 3-CN using a 1L reaction system via whole-cell catalysis, the highest nicotinamide production reached 450.727 g / L with a 3 g / L cell feed rate and constant-rate addition of 70% 3-CN for 10 hours, with a 3-CN residue of 265 mg / L and a molar conversion rate of 99.93%. Therefore, this indicates that the fermentation process of the present invention can maximize the expression of enzyme activity in the cells, achieve a high catalytic effect with a relatively small amount of cells, further reduce industrial production costs, and improve production efficiency.

[0074] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A fermentation process for high expression of nitrile hydratase, characterized in that, Includes the following steps: Step 1: Activation of bacterial strain: Streak the bacterial strain on LB agar plates and incubate at 37°C for 16 hours; Step 2: Primary seed culture: Pick a single clone and transfer it to a 40ml LB medium shake flask, and culture at 37℃ and 220rpm for 8h. Step 3: Secondary seed culture: Transfer 40 mL of primary seed culture to a 150 mL secondary seed culture medium shake flask and culture at 37 °C and 220 rpm for 8 h. Step 4: Fermentation in a fermenter: Transfer 150 mL of the secondary seed culture to a 10 L fermenter containing fermentation medium and ferment at 37 °C until OD reaches the target level. 600 At around 60°C, cool down to 18°C ​​and add an inducing agent to induce induction; Step 5: Detect the OD value of the fermentation broth: Take samples every 6-8 hours to detect the OD value and enzyme activity of the fermentation broth. Once the enzyme activity stops rising, the broth can be transferred to the tank.

2. The fermentation process for high expression of nitrile hydratase as described in claim 1, characterized in that, Step 4, fermentation and culture in the tank, includes the following process: Step 401: After the fermenter is cleaned by alkaline boiling and acid washing, it is sterilized in an empty state. pH and dissolved oxygen electrodes are installed, and a pressure leak test is performed. After the condition is normal, the prepared culture medium is fully dissolved and added to the fermenter to a final volume of 3.5L. Defoaming is added, and the fermenter is sterilized at 121℃ for 20 minutes. After the temperature drops to 37℃, the pH is adjusted to 7.0 with ammonia water, and dissolved oxygen is measured. Step 402: Inoculation volume: 50 mL seed culture; Initial conditions: Temperature: 37℃, pH: 7.0, Airflow: 0.3 m³ / h, Tank pressure: 0.05MPa, rotational speed: 200rpm; Step 403: After 3 hours of fermentation, start increasing the speed, reaching 800 rpm after 6 hours. At this time, dissolved oxygen will rise after 1.5 hours. The pH of the fermentation broth will first rise and then fall. After the dissolved oxygen rises back to above 60, the speed will decrease. At this time, start feeding. During this period, pay attention to the pH value, which will rise to about 7.

2. Control the feeding rate to ensure that the pH value does not rise again or has a downward trend. Step 404: Measure the OD value 2 hours after feeding. Start cooling and induction when the OD reaches 50 or above. After induction, take samples every 4 hours to test OD and enzyme activity. Adjust the feeding speed according to the specific growth rate u, and control u≤0.

02.

3. The fermentation process for high expression of nitrile hydratase as described in claim 1, characterized in that, Standard dissolved oxygen conditions for step 401: Temperature: 37℃, pH: 7.0, Airflow: 1.0 m³ / h, Tank pressure: 0.05MPa, speed: 700rpm.

4. The fermentation process for high expression of nitrile hydratase as described in claim 1, characterized in that, In step 402, the fermentation process is carried out according to the reaction conditions of the tank. When the rotation speed is increased to 400 rpm, the air volume and rotation speed are adjusted alternately, the dissolved oxygen is controlled at 25-35%, the maximum rotation speed is 800 rpm, and the maximum air volume is 1.0 m3 / L.

5. The fermentation process for high expression of nitrile hydratase as described in claim 1, characterized in that, The volume of the fermentation medium in step 4 is 4L.

6. The fermentation process for high expression of nitrile hydratase as described in claim 1, characterized in that, The components of the LB medium include: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 15 g / L agar strips.

7. The fermentation process for high expression of nitrile hydratase as described in claim 1, characterized in that, The components of the secondary seed culture medium include: 10.5 g / L K2HPO4, 4.5 g / L KH2PO4, 1 g / L (NH4)2SO4, 0.5 g / L sodium citrate dihydrate, 10 g / L FM902 yeast extract, and 5 g / L glycerol.

8. The fermentation process for high expression of nitrile hydratase as described in claim 1, characterized in that, The fermentation medium comprises the following components: K2HPO4 12 g / L, KH2PO4 4 g / L, (NH4)2SO4 1.2 g / L, sodium citrate dihydrate 0.3 g / L, FM902 yeast extract 10 g / L, glycerol 20 g / L, and magnesium sulfate heptahydrate 1.2 g / L.

9. The fermentation process for high expression of nitrile hydratase as described in claim 1, characterized in that, Both LB medium and secondary seed medium were supplemented with 50 μg / mL of Kana antibiotic. The preparation method of Kana antibiotic is as follows: 50 g / L stock solution, filtered through a 0.2 μm filter membrane for sterilization, stored at -20℃, and the working concentration is 1.0 per thousand.

10. The fermentation process for high expression of nitrile hydratase as described in claim 2, characterized in that, The feed in step 403 is a fermentation feed medium, which is prepared as follows: 300 g / L glycerol, 55 g / L corn steep liquor powder, sterilized in an autoclave at 121°C for 20 min. The inducing agents in step 404 are lactose inducer and cobalt chloride. The working concentration of lactose inducer and cobalt chloride is calculated based on the fermentation volume, which is 15 g / L + 0.4 g / L. They are prepared separately and sterilized in an autoclave at 121°C for 20 min.

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