阅读说明：本技术 一种鸟嘌呤的合成方法 (Synthetic method of guanine ) 是由 朱文佳 王海波 李嫦 张小兰 于 2020-12-28 设计创作，主要内容包括：本发明属于食品微生物、发酵技术领域,具体涉及一种鸟嘌呤的合成方法。其合成步骤主要为：以微生物发酵制备鸟苷发酵液,以鸟苷发酵液合成反应得到鸟嘌呤,通过提取方法制得鸟嘌呤成品。本发明提供了一种简单、高效的合成鸟嘌呤的方案。通过鸟苷为原料,水为溶剂,酸水解鸟苷制备鸟嘌呤。特别是通过酸直接水解微生物发酵制备的鸟苷发酵液,直接得到鸟嘌呤,生产工序大幅减少,生产成本低。由于在生产过程中,水为溶剂,生产环境干净,环保性好。所得的鸟嘌呤成品收率高,纯度高。(The invention belongs to the technical field of food microorganisms and fermentation, and particularly relates to a synthetic method of guanine. The synthesis steps mainly comprise: preparing guanosine fermentation liquor by microbial fermentation, obtaining guanine by guanosine fermentation liquor synthesis reaction, and preparing a guanine finished product by an extraction method. The invention provides a simple and efficient scheme for synthesizing guanine. The preparation of guanine is carried out by taking guanosine as raw material and water as solvent and hydrolyzing guanosine. Particularly, the guanine fermentation liquid prepared by the microbial fermentation is directly hydrolyzed by acid to directly obtain the guanine, the production procedures are greatly reduced, and the production cost is low. In the production process, water is used as a solvent, so that the production environment is clean and the environmental protection property is good. The obtained guanine finished product has high yield and high purity.)

1. A synthetic method of guanine comprises the following steps:

A. preparing a guanosine fermentation liquor by microbial fermentation;

B. and B, taking the guanosine fermentation liquor prepared in the step A as a raw material, and dispersing the guanosine fermentation liquor by using water, wherein the weight ratio of guanosine to water is 1: (5-20), adjusting pH to 0.6-1.2 with acid to dissolve and clear guanosine fermentation liquor, heating to 85-95 ℃, and reacting for 2-5 hours to convert the guanosine fermentation liquor into guanine generating liquor;

C. c, cooling and crystallizing the guanine generating liquid in the step B to obtain a solid phase which is a guanine reactant; adding water into the purine reactant, wherein the weight ratio of the guanine reactant to the water is 1 (3-10), heating to 35-99 ℃, adjusting the pH range to 0.1-1.0 by using acid to obtain guanine solution clear liquid, and cooling and crystallizing the guanine solution clear liquid to obtain a solid phase which is a guanine hydrochloride wet refined product;

D. and C, adding water into the guanine hydrochloride wet refined product in the step C, wherein the weight ratio of the guanine hydrochloride wet refined product to the water is as follows: and (10-20), adjusting the pH value to ensure that the pH value of a solid-liquid phase is 7.0-9.0, cooling and crystallizing to obtain a solid phase which is a guanine wet refined product, and drying the guanine wet refined product to obtain a guanine finished product.

2. The method for synthesizing guanine according to claim 1, wherein: the microorganism of step A is Bacillus subtilis.

3. The method for synthesizing guanine according to claim 1, wherein: the fermentation process of the step A comprises the following steps:

(1) slant culture: culturing the bacillus subtilis strain on a slant at the temperature of 25-36 ℃ for 16-32h and the pH value of 6.5-7.5, wherein the components of a culture medium are calculated by g/L: 1-3 parts of glucose, 3-10 parts of peptone, 5-15 parts of beef extract, 2-10 parts of yeast powder, 1.0-2.5 parts of sodium chloride and 15-30 parts of agar;

(2) seed culture: transferring the slant culture strain to a seed culture medium, wherein the culture medium conditions are as follows: the pH value is 6.5-7.5, the temperature is 25-36 ℃, the rotating speed of a shaking table is 80-220rpm, the culture is carried out for 8-24h, and the components of a culture medium are calculated by g/L: 10-30 parts of glucose, 3-10 parts of peptone, 8-10 parts of beef extract, 2-10 parts of yeast powder, 15-25ml/L of corn steep liquor, 3-7 parts of urea, 5-10ml/L of soybean meal hydrolysate, 3-8 parts of monosodium glutamate, 1.5-2.5 parts of NaCl1 and KH₂PO₄ 0.5-1.5，MgSO₄·7H₂O 0.2-0.8；

(3) Fermentation culture: inoculating the seed culture solution into a fermentation tank containing a culture medium for fermentation at 32-38 ℃ for 40-96 h; the components of the culture medium are calculated in g/L: 10-30 parts of glucose, 3-10 parts of peptone, 8-10 parts of beef extract, 2-10 parts of yeast powder, 15-25ml/L of corn steep liquor, 3-7 parts of urea, 5-10ml/L of soybean meal hydrolysate, 3-8 parts of monosodium glutamate, 1.5-2.5 parts of NaCl1 and KH₂PO₄ 0.5-1.5，MgSO₄·7H₂0.2-0.8 of O; the content of guanosine fermentation liquor reaches 66.3 g/L.

4. The method for synthesizing guanine according to claim 1, wherein: the acid used to adjust the pH range of step B, C is hydrochloric acid; and D, adjusting the pH range by using ammonia water.

5. The method for synthesizing guanine according to claim 1, wherein: and the step C also comprises the process of adding activated carbon into the guanine clear solution for decoloring, filtering to remove the activated carbon, and then cooling and crystallizing.

6. The method according to claim 5, wherein: the dosage of the activated carbon is 0.5-5.0% of the pure weight of guanine.

7. The method for synthesizing guanine according to claim 1, wherein: and C, heating the guanine hydrochloride salt solution clear solution in the step C within the range of 80-99 ℃.

8. The method for synthesizing guanine according to claim 1, wherein: and the drying operation is to dry the wet guanine fine product in a heating mode to obtain a guanine finished product.

9. The method for synthesizing guanine according to claim 1, wherein: the step C, D of cooling crystallization is that the temperature is reduced to-2 ℃ to 10 ℃, the heat preservation time is 0-3 hours, and then the solid phase and the liquid phase are separated.

Technical Field

The invention belongs to the technical field of food microorganisms and fermentation, and particularly relates to a synthetic method of guanine.

Background

Guanine is mainly used as an intermediate of acyclovir, an antiviral drug. Thioguanine, and open-ring guanine. The synthesis method of guanine comprises the following steps: patent document 1: 201610436294.1, patent document 2: 201810878118.2, patent document 3: 201910746229.2, patent document 4: 99118145.X, patent document 5: 99121054.9, patent document 6: 201310022550.9, patent document 7: 201810651148.X, patent documents 1, 3, 4, 5, and using different starting compounds as raw materials to prepare guanine. Patent 2 is the recovery of guanine. Patent 6, 7 are the production of guanosine by fermentation. The prior technical scheme can be divided into two types, one type is chemical synthesis, the quality is poor, the environmental protection pressure is large, the other type is that guanosine is adopted as a raw material, guanine is prepared by the reaction of acetic anhydride and guanosine, acetic acid is used as a solvent in the reaction, an anhydrous environment is required, and the production cost is high.

Disclosure of Invention

Aiming at the technical defects, the invention provides a synthetic method of guanine with low production cost.

In order to solve the technical problems, the technical scheme of the invention is as follows: a synthetic method of guanine comprises the following steps:

A. preparing a guanosine fermentation liquor by microbial fermentation;

B. and B, taking the guanosine fermentation liquor prepared in the step A as a raw material, and dispersing the guanosine fermentation liquor by using water, wherein the weight ratio of guanosine to water is 1: (5-20), adjusting pH to 0.6-1.2 with acid to dissolve and clear guanosine fermentation liquor, heating to 85-95 ℃, and reacting for 2-5 hours to convert the guanosine fermentation liquor into guanine generating liquor; in this step, the pH is adjusted with particular attention, and when the pH is too low, the by-products are increased and when the reaction is too high, the reaction is incomplete. The reaction time is long due to low temperature, which is not suitable for the production period, and the by-products are more due to high temperature.

C. C, cooling and crystallizing the guanine generating liquid in the step B to obtain a solid phase which is a guanine reactant; adding water into the purine reactant, wherein the weight ratio of the guanine reactant to the water is 1 (3-10), heating to 35-99 ℃, adjusting the pH range to 0.1-1.0 by using acid to obtain guanine solution clear liquid, and cooling and crystallizing the guanine solution clear liquid to obtain a solid phase which is a guanine hydrochloride wet refined product;

D. and C, adding water into the guanine hydrochloride wet refined product in the step C, wherein the weight ratio of the guanine hydrochloride wet refined product to the water is as follows: and (10-20), adjusting the pH value to ensure that the pH value of a solid-liquid phase is 7.0-9.0, cooling and crystallizing to obtain a solid phase which is a guanine wet refined product, and drying the guanine wet refined product to obtain a guanine finished product.

Further: in the above method for synthesizing guanine, the microorganism of step A is Bacillus subtilis.

The fermentation process of the step A comprises the following steps:

(1) slant culture: culturing the bacillus subtilis strain on a slant at the temperature of 25-36 ℃ for 16-32h and the pH value of 6.5-7.5, wherein the components of a culture medium are calculated by g/L: 1-3 parts of glucose, 3-10 parts of peptone, 5-15 parts of beef extract, 2-10 parts of yeast powder, 1.0-2.5 parts of sodium chloride and 15-30 parts of agar;

(2) seed culture: transferring the slant culture strain to a seed culture medium, wherein the culture medium conditions are as follows: the pH value is 6.5-7.5, the temperature is 25-36 ℃, the rotating speed of a shaking table is 80-220rpm, the culture is carried out for 8-24h, and the components of a culture medium are calculated by g/L: 10-30 parts of glucose, 3-10 parts of peptone, 8-10 parts of beef extract, 2-10 parts of yeast powder, 15-25ml/L of corn steep liquor, 3-7 parts of urea, 5-10ml/L of soybean meal hydrolysate, 3-8 parts of monosodium glutamate, 1.5-2.5 parts of NaCl1 and KH₂PO₄ 0.5-1.5，MgSO₄·7H₂O 0.2-0.8；

(3) Fermentation culture: inoculating the seed culture solution into a fermentation tank containing a culture medium for fermentation at 32-38 ℃ for 40-96 h; the components of the culture medium are calculated in g/L: 10-30 parts of glucose, 3-10 parts of peptone, 8-10 parts of beef extract, 2-10 parts of yeast powder, 15-25ml/L of corn steep liquor, 3-7 parts of urea, 5-10ml/L of soybean meal hydrolysate, 3-8 parts of monosodium glutamate, 1.5-2.5 parts of NaCl1 and KH₂PO₄ 0.5-1.5，MgSO₄·7H₂0.2-0.8 of O; the content of guanosine fermentation liquor reaches 66.3 g/L.

The acid used to adjust the pH range of step B, C is hydrochloric acid; and D, adjusting the pH range by using ammonia water.

And the step C also comprises the process of adding activated carbon into the guanine clear solution for decoloring, filtering to remove the activated carbon, and then cooling and crystallizing.

The dosage of the activated carbon is 0.5-5.0% of the pure weight of guanine.

And C, heating the guanine hydrochloride clear solution in the step C to 80-99 ℃, and adding a large amount of hydrochloric acid if the temperature is low.

And the drying operation is to dry the wet guanine fine product in a heating mode to obtain a guanine finished product.

The step C, D of cooling crystallization is that the temperature is reduced to-2 ℃ to 10 ℃, the heat preservation time is 0-3 hours, and then the solid phase and the liquid phase are separated.

Compared with the prior art, the invention has the innovation points that: the invention provides a simple and efficient scheme for synthesizing guanine. The preparation of guanine is carried out by taking guanosine as raw material and water as solvent and hydrolyzing guanosine. Particularly, the guanine fermentation liquid prepared by the microbial fermentation is directly hydrolyzed by acid to directly obtain the guanine, the production procedures are greatly reduced, and the production cost is low. In the production process, water is used as a solvent, so that the production environment is clean and the environmental protection property is good. The obtained guanine finished product has high yield and high purity.

Detailed Description

The present invention is further illustrated by the following specific examples. The following procedures, which are not described in detail, can be performed according to the molecular biology laboratory manual.

Example 1

Preparing guanosine by adopting microbial fermentation, removing thalli after the fermentation is finished to obtain 1000L of guanosine fermentation liquor, detecting the guanosine content (the guanosine content of the guanosine fermentation liquor is detected to be 7.8 percent by a chromatographic column), and adjusting the pH value to be 1.0 by using hydrochloric acid. Heating to 90 deg.C, reacting for 3 hr, cooling to 5 deg.C, and holding for 2 hr. Separating solid phase from liquid phase to obtain guanine reactant as solid phase. Dissolving guanine reactant in water, and adding proper water to ensure that the weight ratio of guanine to water is as follows: 1:7, heating to 95 ℃, and adjusting the pH range to 0.5 to dissolve and clear the guanine. Adding proper amount of active carbon for decolorization, wherein the use amount of the active carbon is 2 percent of the weight (kg) of guanine. Filtering to remove active carbon, cooling the filtrate for crystallization at 5 deg.C for 3 hr. Separating solid phase from liquid phase to obtain 'solid phase' which is guanine hydrochloride wet refined product. Adding water properly to ensure that the weight ratio of the guanine to the water is as follows: 1:10, and adjusting the pH value with ammonia water to ensure that the pH value of a solid-liquid phase is 7.0-8.0. Cooling and crystallizing, and separating solid and liquid phases when the temperature is 5 ℃ to obtain a solid phase which is a guanine wet refined product. Drying the guanine wet refined product by a heating mode to obtain 33.5kg of guanine finished products. The molar yield of the guanine finished product is 80.5 percent, and the purity of the finished product is 99.95 percent through high performance liquid chromatography detection.

The process for preparing guanosine by microbial fermentation comprises the following steps:

(1) slant culture: culturing the bacillus subtilis strain on a slant at the temperature of 25-36 ℃ for 16-32h and the pH value of 6.5-7.5, wherein the components of a culture medium are calculated by g/L: 1-3 parts of glucose, 3-10 parts of peptone, 5-15 parts of beef extract, 2-10 parts of yeast powder, 1.0-2.5 parts of sodium chloride and 15-30 parts of agar;

(2) seed culture: transferring the slant culture strain to a seed culture medium, wherein the culture medium conditions are as follows: the pH value is 6.5-7.5, the temperature is 25-36 ℃, the rotating speed of a shaking table is 80-220rpm, the culture is carried out for 8-24h, and the components of a culture medium are calculated by g/L: 10-30 parts of glucose, 3-10 parts of peptone, 8-10 parts of beef extract, 2-10 parts of yeast powder, 15-25ml/L of corn steep liquor, 3-7 parts of urea, 5-10ml/L of soybean meal hydrolysate, 3-8 parts of monosodium glutamate, 1.5-2.5 parts of NaCl1, KH2PO40.5-1.5 parts of NaCl2, and 0.8 part of MgSO 4.7 7 H2O0.2;

(3) fermentation culture: inoculating the seed culture solution into a fermentation tank containing a culture medium for fermentation at 32-38 ℃ for 40-96 h; the components of the culture medium are calculated in g/L: 10-30 parts of glucose, 3-10 parts of peptone, 8-10 parts of beef extract, 2-10 parts of yeast powder, 15-25ml/L of corn steep liquor, 3-7 parts of urea, 5-10ml/L of soybean meal hydrolysate, 3-8 parts of monosodium glutamate, 1.5-2.5 parts of NaCl1, KH2PO40.5-1.5 parts of NaCl2, and 0.2-0.8 part of MgSO4 & 7H2O 0.2; the content of guanosine fermentation liquor reaches 66.3 g/L.

Example 2

Preparing guanosine by adopting microbial fermentation, removing thalli after the fermentation is finished to obtain 1000L of guanosine fermentation liquor, detecting the guanosine content (the guanosine content of the guanosine fermentation liquor is detected to be 7.8 percent by a chromatographic column), adding water to adjust the specific gravity of the guanosine and the water to be 1: 20, pH0.8 adjusted with hydrochloric acid. Heating to 95 ℃, keeping the temperature for reaction for 2 hours, then cooling to 0 ℃, and keeping the temperature for 2 hours. Separating solid phase from liquid phase to obtain guanine reactant as solid phase. Dissolving guanine reactant in water, and adding proper water to ensure that the weight ratio of guanine to water is as follows: 1:7, heating to 95 ℃, and adjusting the pH range to 0.5 to dissolve and clear the guanine. Adding proper amount of active carbon for decolorization, wherein the use amount of the active carbon is 2 percent of the weight (kg) of guanine. Filtering to remove active carbon, cooling the filtrate for crystallization at 0 deg.C for 3 hr. Separating solid phase from liquid phase to obtain 'solid phase' which is guanine hydrochloride wet refined product. Adding water properly to ensure that the weight ratio of the guanine to the water is as follows: 1:10, and adjusting the pH value with ammonia water to ensure that the pH value of a solid-liquid phase is 7.0-8.0. Cooling and crystallizing, and separating solid and liquid phases when the temperature is 5 ℃ to obtain a solid phase which is a guanine wet refined product. Drying the guanine wet refined product by a heating mode to obtain 34.0kg of guanine finished products. The molar yield of the guanine finished product is 81.69%, and the purity of the finished product is 99.94% by high performance liquid chromatography detection.

The microbial fermentation process for preparing guanosine is the same as in example 1.

Example 3

Preparing guanosine by adopting microbial fermentation, removing thalli after the fermentation is finished to obtain 1000L of guanosine fermentation liquor, detecting the guanosine content (the guanosine content of the guanosine fermentation liquor is detected to be 6.5 percent by a chromatographic column), and concentrating the fermentation liquor to ensure that the proportion of guanosine to water is 1: 5, adjusting pH to 1.2 with hydrochloric acid. Heating to 85 deg.C, reacting for 4 hr, cooling to 0 deg.C, and maintaining for 2 hr. Separating solid phase from liquid phase to obtain guanine reactant as solid phase. Dissolving guanine reactant in water, and adding proper water to ensure that the weight ratio of guanine to water is as follows: 1:7, heating to 95 ℃, and adjusting the pH range to 0.5 to dissolve and clear the guanine. Adding proper amount of active carbon for decolorization, wherein the use amount of the active carbon is 2 percent of the weight (kg) of guanine. Filtering to remove active carbon, cooling the filtrate for crystallization at 0 deg.C for 3 hr. Separating solid phase from liquid phase to obtain 'solid phase' which is guanine hydrochloride wet refined product. Adding water properly to ensure that the weight ratio of the guanine to the water is as follows: 1:10, and adjusting the pH value with ammonia water to ensure that the pH value of a solid-liquid phase is 7.0-8.0. Cooling and crystallizing, and separating solid and liquid phases when the temperature is 5 ℃ to obtain a solid phase which is a guanine wet refined product. And drying the wet guanine fine product in a heating mode to obtain 28.06kg of guanine finished product. The molar yield of the guanine finished product is 81.0 percent, and the purity of the finished product is 99.90 percent by high performance liquid chromatography detection.

The microbial fermentation process for preparing guanosine is the same as in example 1.

Comparative example 1

70kg of finished product guanosine is adopted, and water is added to ensure that the proportion of guanosine to water is 1: 5, adjusting pH to 1.2 with hydrochloric acid.

Heating to 85 deg.C, reacting for 4 hr, cooling to 0 deg.C, and maintaining for 2 hr. Separating solid phase from liquid phase to obtain guanine reactant as solid phase. Dissolving guanine reactant in water, and adding proper water to ensure that the weight ratio of guanine to water is as follows: 1:7, heating to 95 ℃, and adjusting the pH range to 0.5 to dissolve and clear the guanine. Adding proper amount of active carbon for decolorization, wherein the use amount of the active carbon is 2 percent of the weight (kg) of guanine. Filtering to remove active carbon, cooling the filtrate for crystallization at 0 deg.C for 3 hr. Separating solid phase from liquid phase to obtain 'solid phase' which is guanine hydrochloride wet refined product. Adding water properly to ensure that the weight ratio of the guanine to the water is as follows: 1:10, and adjusting the pH value with ammonia water to ensure that the pH value of a solid-liquid phase is 7.0-8.0. Cooling and crystallizing, and separating solid and liquid phases when the temperature is 5 ℃ to obtain a solid phase which is a guanine wet refined product. And drying the wet guanine fine product in a heating mode to obtain 31.0kg of guanine finished products. The molar yield of the guanine finished product is 83.0 percent, the purity of the finished product is 99.90 percent through high performance liquid chromatography detection, but the extraction yield from the guanosine fermentation liquid to guanosine is about 90 percent, so the total molar yield is about 74.7 percent. Indicating that the overall yield of guanine produced directly from guanosine is lower than the yield of guanine produced from a guanosine fermentation broth.

It should be understood that the examples are merely for illustrative purposes and are not intended to limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.