阅读说明：本技术 一种生物催化法生产2,5-二甲基吡嗪的方法 (Method for producing 2, 5-dimethylpyrazine by biocatalysis method ) 是由 郭宏明 于 2020-04-27 设计创作，主要内容包括：本发明公开了一种生物催化法生产2,5-二甲基吡嗪的方法,涉及生物催化技术领域。本发明于LB培养基中添加氨苄青霉素作为液体培养基,培养2,5-二甲基吡嗪合成酶菌株,得种子培养物,对所得种子培养物进行发酵、补糖处理,得发酵处理物,再对发酵处理物进行诱导处理,得2,5-二甲基吡嗪合成酶,接着以L-苏氨酸为底物,采用所得酶进行生物催化,得到2,5-二甲基吡嗪。本发明公布了一种全新的合成路线,反应条件温和,原料价格低廉,并可避免化学合成造成的环境污染。本发明解决了目前制备2,5-二甲基吡嗪常用采用化学合成,合成过程的条件苛刻,并有毒气散发和爆炸的危险,往往会造成严重污染,所用试剂成本高的问题。(The invention discloses a method for producing 2, 5-dimethylpyrazine by a biocatalysis method, and relates to the technical field of biocatalysis. Ampicillin is added into an LB culture medium to be used as a liquid culture medium, a 2, 5-dimethylpyrazine synthetase strain is cultured to obtain a seed culture, the obtained seed culture is fermented and subjected to sugar supplement treatment to obtain a fermentation treatment product, the fermentation treatment product is subjected to induction treatment to obtain 2, 5-dimethylpyrazine synthetase, and then L-threonine is used as a substrate and the obtained enzyme is adopted for biological catalysis to obtain the 2, 5-dimethylpyrazine. The invention discloses a brand new synthetic route, which has mild reaction conditions and low raw material price and can avoid environmental pollution caused by chemical synthesis. The invention solves the problems that the existing method for preparing 2, 5-dimethyl pyrazine usually adopts chemical synthesis, the synthesis process has harsh conditions, toxic gas emission and explosion risks, serious pollution is often caused, and the cost of used reagents is high.)

1. A method for producing 2, 5-dimethylpyrazine by a biocatalysis method is characterized by comprising the following steps:

(1) adding antibiotics into an LB culture medium to serve as a liquid culture medium;

(2) culturing the 2, 5-dimethyl pyrazine synthetase strain by using the liquid culture medium obtained in the step (1) to obtain a seed culture;

(3) fermenting the seed culture obtained in the step (2);

(4) performing sugar supplement treatment on the fermentation process in the step (3), and continuing fermentation to obtain a fermentation treatment product;

(5) performing induction treatment on the fermentation treatment product obtained in the step (4) to obtain 2, 5-dimethyl pyrazine synthetase;

(6) and (3) performing biocatalysis by using the 2, 5-dimethyl pyrazine synthetase obtained in the step (5) by using L-threonine as a substrate.

2. The method for producing 2, 5-dimethylpyrazine by the biocatalytic method according to claim 1, wherein the LB culture medium: taking 11-14 parts by weight of KH₂PO₄3 to 5 parts of (NH)₄)₂SO₄1.2-2.5 parts of citric acid monohydrate, 0.4-0.8 part of MgSO₄·7H₂Mixing O, 8-12 parts of dextrose monohydrate and 1000 parts of water, and sterilizing to obtain an LB culture medium;

the steps (1) to (5) constitute a process of high-density fermentation expression of the 2, 5-dimethylpyrazine synthetase strain.

3. The biocatalytic process for producing 2, 5-dimethylpyrazine according to claim 1, comprising the steps of:

(1) preparation of liquid medium: taking 11-14 parts by weight of KH₂PO₄3 to 5 parts of (NH)₄)₂SO₄1.2-2.5 parts of citric acid monohydrate, 0.4-0.8 part of MgSO₄·7H₂Mixing O, 8-12 parts of monohydrate glucose and 1000 parts of water, sterilizing, cooling to 20-39 ℃, adding 1 per mill of antibiotic by mass of sterile water, and mixing to obtain a liquid culture medium;

(2) preparation of seed culture: measuring a 2, 5-dimethyl pyrazine synthetase strain according to 5% of inoculation amount, culturing in a liquid culture medium, controlling the temperature to be 37 ℃, and culturing for 4-6 h to obtain a seed culture;

(3) controlling the fermentation process: adjusting the pH value of the seed culture obtained in the step (2), keeping the internal dissolved oxygen amount to be 30-60% of the total mass by airtight ventilation, oscillating a shaking table, and performing heat preservation fermentation treatment;

(4) sugar supplement treatment: performing sugar supplement treatment on the fermentation process in the step (3), controlling the dissolved oxygen to be 20-30% of the total mass, and controlling the concentration of residual sugar to be 0-0.1 g/L;

(5) and (3) induction treatment: controlling the OD of the thallus after 12-14 h of fermentation culture process₆₀₀The value is 20-40, the temperature is reduced, and an inducer is added according to the mass volume ratio of 0.01-1% to shake the shaking table to induce enzyme expression for 10-24h, so that 2, 5-dimethyl pyrazine synthetase is obtained;

(6) biocatalysis: taking 78-100 parts of Tris-HCl buffer solution, 5-80 parts of 2, 5-dimethylpyrazine synthetase, 1-100 parts of substrate and 0-6 parts of NAD (nicotinamide adenine dinucleotide)⁺Mixing the solutions, reacting, discharging, and inactivating enzyme to obtain 2, 5-dimethyl pyrazine.

4. The biocatalytic method for producing 2, 5-dimethylpyrazine according to claim 3, which comprises the following steps:

(1) preparation of liquid medium: taking 11-14 parts by weight of KH₂PO₄3 to 5 parts of (NH)₄)₂SO₄1.2-2.5 parts of citric acid monohydrate, 0.4-0.8 part of MgSO₄·7H₂Mixing O, 8-12 parts of monohydrate glucose and 1000 parts of water, preserving heat at 121 ℃, sterilizing for 12-20 min, naturally cooling to 20-39 ℃, adding antibiotic with the mass of sterile water being 1 per mill, and uniformly mixing to obtain a liquid culture medium;

(2) preparation of seed culture: measuring 2, 5-dimethyl pyrazine synthetase strain liquid according to 5% of inoculation amountCulturing in a culture medium at 37 deg.C for 4-6 hr, and controlling OD₆₀₀1.8-2.0 to obtain a seed culture;

(3) controlling the fermentation process: adjusting the pH value of the seed culture obtained in the step (2) to 4-8, moving the seed culture to a test tube capable of ventilating, sealing the test tube, controlling the ventilating rate to be 1-1.5 mL/min, maintaining the dissolved oxygen amount in the test tube to be 30-60% of the total mass, oscillating a shaking table at 20-40 ℃ at 220r/min, performing heat preservation fermentation treatment for 6-8 h, and controlling the OD₆₀₀The value is 9.7 to 10.3;

(4) sugar supplement treatment: supplementing glucose monohydrate in the 8 th-30 th hour period of the fermentation process in the step (3), controlling the dissolved oxygen to be 20-30% of the total mass, and controlling the concentration of residual sugar to be 0-0.1 g/L;

(5) and (3) induction treatment: controlling the OD of the thallus after 12-14 h of fermentation culture process₆₀₀When the value is 20-40, cooling to 20-37 ℃, adding an inducer according to the mass-volume ratio of 0.01-1%, mixing, and inducing enzyme expression for 10-24 hours by a shaking table at 220r/min to obtain 2, 5-dimethyl pyrazine synthetase;

(6) biocatalysis: taking 78-100 parts by weight of Tris-HCl buffer solution with the concentration of 0.5M and the pH value of 6.0-8.5, 5-80 parts by weight of 2, 5-dimethylpyrazine synthetase, 1-100 parts by weight of substrate and 0-6 parts by weight of NAD with the concentration of 0-1 mM⁺Mixing the solutions, reacting for 25-30 h at 20-40 ℃, discharging, and inactivating enzyme to obtain the 2, 5-dimethyl pyrazine.

5. The biocatalytic process for producing 2, 5-dimethylpyrazine in accordance with claim 4, wherein the antibiotic used in step (1) is ampicillin at a concentration of 50 μ g/mL.

6. The method for producing 2, 5-dimethylpyrazine according to claim 4, wherein the enzyme inducer in step (5) is any one of lactose solution with concentration of 5g/L and isopropyl thiogalactoside with concentration of 30-50 ppm.

7. The method for producing 2, 5-dimethylpyrazine according to claim 4, wherein the enzyme in step (5) or step (6) is 2, 5-dimethylpyrazine synthase.

8. The method for producing 2, 5-dimethylpyrazine according to claim 7, wherein the enzyme form of the 2, 5-dimethylpyrazine synthase is any one of enzyme powder, enzyme solution, immobilized enzyme, 2, 5-dimethylpyrazine synthase-producing bacterial cells, and 2, 5-dimethylpyrazine synthase-producing cells.

9. The method for producing 2, 5-dimethylpyrazine according to claim 8, wherein the 2, 5-dimethylpyrazine synthase is crude enzyme or pure enzyme.

10. The method for producing 2, 5-dimethylpyrazine according to claim 4, wherein the substrate in step (6) is L-threonine with a concentration of 200 mM.

Technical Field

The invention belongs to the technical field of biocatalysis, and particularly relates to a method for producing 2, 5-dimethylpyrazine by a biocatalysis method.

Background

2, 5-dimethylpyrazine is a colorless to yellowish liquid having a boiling point of 155 ℃ and a flash point of 64 ℃ and is miscible in water and organic solvents. The natural product is present in bulb cabbage, pork, rum, cacao, coffee, potato chips, shrimp, etc. The product has nut flavor, peanut flavor, mildew flavor, loamy flavor, potato flavor, fat flavor, and cocoa powder flavor, 7.5mg/kg solution has nut flavor, potato flavor, and fat flavor, is an allowable spice in GB-2760-86 of China, and is mainly used for preparing cocoa, coffee, meat, nut flavor, potato flavor, etc., and can also be used in dye and pharmaceutical industry.

At present, the synthesis methods of 2, 5-dimethyl pyrazine mainly comprise the following methods:

1. synthesized by ketone compounds containing active methylene. Firstly, reducing ketone containing active methylene into alpha-aminoketone by nitrite, then dehydrating and cyclizing to generate dihydropyrazine, and further dehydrogenating to generate pyrazine. The method has many reaction steps and is not suitable for large-scale industrial production.

2. Monoisopropanolamine and ethylenediamine are used as raw materials, a catalyst required by the reaction is prepared by an ammonia water precipitation method, and alpha-diamine and alpha-ethanolamine are used for synthesizing 2, 5-dimethylpyrazine. Although the process route is short, the selectivity is poor, a single product cannot be obtained, and the product separation is difficult.

3. In the presence of ammonium salt, 2, 5-dimethyl pyrazine can be obtained by heating acrolein and ammonia in glycerol, but the acrolein is a highly toxic substance and the reaction process is easy to explode, so the method is not suitable.

4. The alpha-diamine and the propylene oxide are synthesized, the method adopts gas-solid phase contact catalytic reaction, the product is a mixture of 2, 5-dimethyl pyrazine and 2, 6-dimethyl pyrazine, and the product is difficult to separate and purify.

5. The method takes isopropanolamine as a raw material to synthesize the 2, 5-dimethyl pyrazine through gas-solid catalysis, results of different catalysts are greatly different, the yield is low when the aluminosilicate zeolite and the zinc oxide are used as the catalysts, and the industrial preparation cost of the silver-containing catalyst is high.

At present, chemical synthesis is commonly used for preparing 2, 5-dimethyl pyrazine, the conditions of the synthesis process are harsh, and the synthesis process has the risks of toxic gas emission and explosion, so that the problems of serious pollution and high cost of used reagents are often caused. There is therefore an urgent need for a completely new and mild process which reduces the production costs.

Disclosure of Invention

The invention aims to: in order to solve the problems that chemical synthesis is commonly adopted for preparing 2, 5-dimethylpyrazine at present, the conditions of the synthesis process are harsh, toxic gas emission and explosion risks exist, serious pollution is caused frequently, and the cost of used reagents is high, the method for producing the 2, 5-dimethylpyrazine by using the biocatalysis method is provided.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for producing 2, 5-dimethyl pyrazine by a biocatalysis method comprises the following steps:

(1) adding antibiotics into an LB culture medium to serve as a liquid culture medium;

(2) culturing the 2, 5-dimethyl pyrazine synthetase strain by using the liquid culture medium obtained in the step (1) to obtain a seed culture;

(3) fermenting the seed culture obtained in the step (2);

(4) performing sugar supplement treatment on the fermentation process in the step (3), and continuing fermentation to obtain a fermentation treatment product;

(5) performing induction treatment on the fermentation treatment product obtained in the step (4) to obtain 2, 5-dimethyl pyrazine synthetase;

(6) and (3) performing biocatalysis by using the 2, 5-dimethyl pyrazine synthetase obtained in the step (5) by using L-threonine as a substrate.

As a further description of the above technical solution:

the LB culture medium: taking 11-14 parts by weight of KH₂PO₄3 to 5 parts of (NH)₄)₂SO₄1.2-2.5 parts of citric acid monohydrate, 0.4-0.8 part of MgSO₄·7H₂Mixing O, 8-12 parts of monohydrate glucose and 1000 parts of water, and sterilizing to obtain an LB culture medium;

the steps (1) to (5) constitute a process of high-density fermentation expression of the 2, 5-dimethylpyrazine synthetase strain.

As a further description of the above technical solution:

the method for producing the 2, 5-dimethylpyrazine by the biocatalysis method comprises the following steps:

(1) preparation of liquid medium: taking 11-14 parts by weight of KH₂PO₄3 to 5 parts of (NH)₄)₂SO₄1.2-2.5 parts of citric acid monohydrate, 0.4-0.8 part of MgSO₄·7H₂Mixing O, 8-12 parts of monohydrate glucose and 1000 parts of water, sterilizing, cooling to 20-39 ℃, adding 1 per mill of antibiotic by mass of sterile water, and mixing to obtain a liquid culture medium;

(2) preparation of seed culture: measuring a 2, 5-dimethyl pyrazine synthetase strain according to 5% of inoculation amount, culturing in a liquid culture medium, controlling the temperature to be 37 ℃, and culturing for 4-6 h to obtain a seed culture;

(3) controlling the fermentation process: adjusting the pH value of the seed culture obtained in the step (2), keeping the internal dissolved oxygen amount to be 30-60% of the total mass by airtight ventilation, oscillating a shaking table at 37 ℃, and performing heat preservation fermentation treatment;

(4) sugar supplement treatment: carrying out sugar supplement treatment on the fermentation process in the step (3), controlling dissolved oxygen to be 20-30% of the total mass, adjusting sugar supplement rate according to the dissolved oxygen, and controlling residual sugar concentration to be 0-0.1 g/L;

(5) and (3) induction treatment: controlling the OD of the thallus after 12-14 h of fermentation culture process₆₀₀Cooling when the value is 20-40, adding an inducer according to the mass-volume ratio of 0.01-1%, and carrying out shake table induced enzyme expression for 10-24h to obtain 2, 5-dimethyl pyrazine synthetase;

(6) biocatalysis: taking 78-100 parts of Tris-HCl buffer solution, 5-80 parts of 2, 5-dimethylpyrazine synthetase, 1-100 parts of substrate and 0-6 parts of NAD (nicotinamide adenine dinucleotide)⁺And mixing the solutions, reacting at 20-40 ℃, and discharging to obtain the 2, 5-dimethyl pyrazine.

As a further description of the above technical solution:

the method for producing 2, 5-dimethylpyrazine by a biocatalysis method specifically comprises the following steps:

(1) preparation of liquid medium: taking 11-14 parts by weight of KH₂PO₄3 to 5 parts of (NH)₄)₂SO₄1.2-2.5 parts of citric acid monohydrate, 0.4-0.8 part of MgSO₄·7H₂Mixing O, 8-12 parts of monohydrate glucose and 1000 parts of water, preserving heat at 121 ℃, sterilizing for 12-20 min, naturally cooling to 20-39 ℃, adding antibiotic with the mass of 1 per mill of sterile water, and mixingMixing uniformly to obtain a liquid culture medium;

(2) preparation of seed culture: measuring 2, 5-dimethyl pyrazine synthetase strain according to 5% of inoculation amount, culturing in liquid culture medium at 37 deg.C for 4-6 h, and controlling OD₆₀₀1.8-2.0 to obtain a seed culture;

(3) controlling the fermentation process: adjusting the pH value of the seed culture obtained in the step (2) to 4-8, moving the seed culture to a test tube capable of ventilating, sealing the test tube, controlling the ventilating rate to be 1-1.5 mL/min, maintaining the dissolved oxygen amount in the test tube to be 30-60% of the total mass, oscillating a shaking table at 20-40 ℃ at 220r/min, performing heat preservation fermentation treatment for 6-8 h, and controlling the OD₆₀₀The value is 9.7 to 10.3;

(4) sugar supplement treatment: supplementing glucose monohydrate in the 8 th-30 th hour period of the fermentation process in the step (3), controlling the dissolved oxygen to be 20-30% of the total mass, and controlling the concentration of residual sugar to be 0-0.1 g/L;

(5) and (3) induction treatment: controlling the OD of the thallus after 12-14 h of fermentation culture process₆₀₀When the value is 20-40, cooling to 20-37 ℃, adding an inducer according to the mass-volume ratio of 0.01-1%, mixing, and inducing enzyme expression for 10-24 hours by a shaking table at 220r/min to obtain 2, 5-dimethyl pyrazine synthetase;

(6) biocatalysis: taking 78-100 parts by weight of Tris-HCl buffer solution with the concentration of 0.5M and the pH value of 6.0-8.5, 5-80 parts by weight of 2, 5-dimethylpyrazine synthetase, 1-100 parts by weight of substrate and 0-6 parts by weight of NAD with the concentration of 0-1 mM⁺Mixing the solutions, reacting for 25-30 h at 20-40 ℃, discharging, and inactivating enzyme to obtain the 2, 5-dimethyl pyrazine.

As a further description of the above technical solution:

the antibiotic in the step (1) is ampicillin with the concentration of 50 mug/mL.

As a further description of the above technical solution:

the enzyme inducer in the step (5) is any one of lactose solution with the concentration of 5g/L and isopropyl thiogalactoside with the concentration of 30-50 ppm.

As a further description of the above technical solution:

the enzyme in the step (5) or the step (6) is 2, 5-dimethyl pyrazine synthetase.

As a further description of the above technical solution:

the enzyme form of the 2, 5-dimethyl pyrazine synthetase is any one of enzyme powder, enzyme liquid, immobilized enzyme, thallus for producing the 2, 5-dimethyl pyrazine synthetase and cells for producing the 2, 5-dimethyl pyrazine synthetase.

As a further description of the above technical solution:

the 2, 5-dimethyl pyrazine synthetase is crude enzyme or pure enzyme.

As a further description of the above technical solution:

the substrate in the step (6) is L-threonine with a concentration of 200 mM.

As a further description of the above technical solution:

the 2, 5-dimethyl pyrazine synthetase strain is a 2, 5-dimethyl pyrazine production strain (DMP 009).

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

(1) adding ampicillin into an LB culture medium as a liquid culture medium, culturing a 2, 5-dimethylpyrazine synthetase strain to obtain a seed culture, fermenting and sugar supplementing the obtained seed culture to obtain a fermentation treatment product, then performing induction treatment on the fermentation treatment product to obtain 2, 5-dimethylpyrazine synthetase, then performing biocatalysis by using L-threonine as a substrate and using the obtained enzyme to dissociate hydrogen on threonine hydroxyl under the action of the 2, 5-dimethylpyrazine synthetase, automatically separating carboxyl, simultaneously performing condensation rearrangement to form an intermediate, and performing automatic cyclization on the two-molecular intermediate to obtain 2, 5-dimethylpyrazine;

(2) the results obtained from the examples of the invention are plotted to conclude the following: as can be seen from FIG. 2, 5-dimethylpyrazine synthetase with different final bacterial cell concentrations can promote substrate conversion, and the conversion rate is improved along with the extension of the action time, and the action time is gradually gentle after 8 h; as is clear from FIG. 3, the conversion effect was stably exhibited under various pH conditions, and the pH was 6.0 in this pH rangeThe conversion rate is lowest when the pH value is 8.0; as can be seen from FIG. 4, the different NADs⁺Under the action of concentration, the substrate conversion can be completed, wherein the conversion efficiency is highest when the concentration is 1mM, and the conversion efficiency is lowest when the concentration is 0, and NAD can be seen in a certain concentration range⁺The increase in concentration has an effect on the increase in conversion; as can be seen from FIG. 5, the conversion effect of the substrate can be improved by adding the yeast powder, and in a certain concentration range, the conversion effect can be improved along with the increase of the use amount of the yeast powder, while the mass conversion effect is reduced by increasing the content of the peptone, and the conversion effect when the addition amount of the peptone is 1% is close to the effect when no yeast powder or peptone is added; as can be seen from FIG. 6, according to the bioreactor reaction curve, the conversion effect of OD 25 is higher than that of OD 12.5, which indicates that the final concentration of the bacterial cells is positively correlated with the conversion effect within a certain concentration range; in conclusion, the 2, 5-dimethyl pyrazine can be obtained by the conversion of the obtained enzyme under different conversion conditions, and the conditions can be optimized to pH 8.0 and NAD⁺The concentration is 1mM, the yeast powder accounts for 1 percent, and the OD value of the thallus is 25;

(3) the invention discloses a brand new synthetic route, which has mild reaction conditions and low raw material price, can avoid environmental pollution caused by chemical synthesis, and solves the problems that the existing method for preparing 2, 5-dimethyl pyrazine usually adopts chemical synthesis, the synthesis process has harsh conditions, toxic gas emission and explosion risks, serious pollution is caused and the cost of used reagents is high.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a catalytic scheme of the present invention;

FIG. 2 is a graph of the effect of reaction time on catalytic performance;

FIG. 3 is a graph showing the effect of reaction pH on catalytic performance;

FIG. 4 is coenzyme factor NAD⁺Shadow of concentration on catalytic effectSound chart

FIG. 5 is a graph of the effect of yeast or peptone addition on the catalytic reaction;

FIG. 6 is a graph showing the effect of expanding the reaction system of the bioreactor;

FIG. 7 is a graph of high density fermentation.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The antibiotic was ampicillin at a concentration of 50. mu.g/mL.

The enzyme inducer is any one of lactose solution with the concentration of 5g/L and isopropyl thiogalactoside (IPTG) with the concentration of 30-50 ppm.

The substrate was L-threonine at a concentration of 200 mM.

The 2, 5-dimethyl pyrazine synthetase strain is a 2, 5-dimethyl pyrazine production strain (DMP 009).

The enzyme is 2, 5-dimethyl pyrazine synthetase, the enzyme form of the 2, 5-dimethyl pyrazine synthetase is not limited, and the enzyme is any one of enzyme powder, enzyme liquid, immobilized enzyme and thallus or cell for producing the 2, 5-dimethyl pyrazine synthetase, and the 2, 5-dimethyl pyrazine synthetase can be crude enzyme or pure enzyme.

The steps (1) to (5) constitute a process of high-density fermentation expression of the 2, 5-dimethyl pyrazine synthetase strain.

A method for producing 2, 5-dimethylpyrazine by a biocatalytic method comprises the following steps:

(1) preparation of liquid medium: taking 11-14 parts by weight of KH₂PO₄3 to 5 parts of (NH)₄)₂SO₄1.2-2.5 parts of citric acid monohydrate, 0.4-0.8 part of MgSO₄·7H₂Mixing O, 8-12 parts of monohydrate glucose and 1000 parts of water, preserving heat and sterilizing at 121 ℃ for 12-20 min, and naturally cooling toAdding antibiotics with the mass of 1 per mill of the sterile water at the temperature of 20-39 ℃, and uniformly mixing to obtain a liquid culture medium;

(2) preparation of seed culture: measuring a 2, 5-dimethyl pyrazine synthetase strain according to 5% of inoculation amount, culturing in a liquid culture medium at a temperature of 20-37 ℃ for 4-6 h, and controlling OD₆₀₀1.8-2.0 to obtain a seed culture;

(3) controlling the fermentation process: adjusting the pH value of the seed culture obtained in the step (2) to 4-8 by using HCl solution with the concentration of 1M, moving the seed culture to a test tube capable of ventilating, sealing the test tube, controlling the ventilating rate to be 1-1.5 mL/min, maintaining the dissolved oxygen amount in the test tube to be 30-60% of the total mass, oscillating a shaking table at 20-40 ℃ at 220r/min, performing heat preservation fermentation for 6-8 h, and controlling OD₆₀₀The value is 9.7 to 10.3;

(4) sugar supplement treatment: supplementing glucose monohydrate in the 8 th-30 th hour period of the fermentation process in the step (3), controlling the dissolved oxygen to be 20-30% of the total mass, and controlling the concentration of residual sugar to be 0-0.1 g/L;

(5) and (3) induction treatment: controlling the OD of the thallus after 12-14 h of fermentation culture process₆₀₀When the value is 20-40, cooling to 20-37 ℃, adding an inducer according to the mass-volume ratio of 0.01-1%, and inducing enzyme expression for 10-24h by oscillating a shaker at 220r/min to obtain 2, 5-dimethyl pyrazine synthetase;

(6) biocatalysis: taking 78-100 parts by weight of Tris-HCl buffer solution with the concentration of 0.5M and the pH value of 6.0-8.5 and 5-80 parts by weight of OD₆₀₀20-40 parts of 2, 5-dimethylpyrazine synthase, 1-100 parts of substrate, 0-6 parts of NAD with concentration of 0-1 mM⁺Mixing the solution in a ventilating test tube, reacting at 20-40 ℃ for 25-30 h at 200rpm, discharging, and inactivating enzyme to obtain the 2, 5-dimethyl pyrazine.