阅读说明：本技术 静息细胞法制备孕甾-5-烯-3β,21-二醇的方法 (Method for preparing pregna-5-ene-3 beta, 21-diol by resting cell method ) 是由 孟浩 刘喜荣 杨芳 曾春玲 赵小娟 于 2020-07-23 设计创作，主要内容包括：本发明涉及甾体类药物中间体的生产方法,具体来说是一种静息细胞法制备孕甾-5-烯-3β,21-二醇的方法。发明内容包括(1)植物甾醇的3位保护、(2)静息细胞转化、(3)提取、(4)水解、(5)精制步骤。本发明以植物甾醇为原料生产孕甾-5-烯-3β,21-二醇,原料易得,降低了生产成本；本发明采用保护剂对植物甾醇3位羟基进行官能团保护,发酵后水解可直接制得孕甾-5-烯-3β,21-二醇,副产物少,收率更高,反应路线更短,省掉了许多传统制备方法所需要的反应步骤和后处理步骤。(The invention relates to a production method of steroid medicine intermediate, in particular to a method for preparing pregna-5-ene-3 beta, 21-diol by resting cell method. The invention comprises the steps of (1) protecting 3-position of phytosterol, (2) transforming resting cells, (3) extracting, (4) hydrolyzing and (5) refining. The invention takes the phytosterol as the raw material to produce pregna-5-ene-3 beta, 21-diol, the raw material is easy to obtain, and the production cost is reduced; the invention adopts the protective agent to protect the functional group of the 3-hydroxyl of the phytosterol, and the pregna-5-ene-3 beta, 21-diol can be directly prepared by hydrolysis after fermentation, thereby having less by-products, higher yield and shorter reaction route, and saving a plurality of reaction steps and post-treatment steps required by the traditional preparation method.)

1. A method for preparing pregn-5-ene-3 beta, 21-diol by a resting cell method is characterized by comprising the following steps:

(1) and 3, protecting: utilizing methylal as a protective agent, protecting 3-position hydroxyl of phytosterol to obtain phytosterol etherate, and adding phosphorus pentoxide as a catalyst and diatomite as a filter aid in the reaction process;

(2) transformation of resting cells: carrying out slant culture and liquid seed culture on a Mycobacterium (Mycobacterium sp.) B-NRRL 3683 mutation strain, filtering and separating seeds, adding the seeds into a PBS buffer system, and carrying out fermentation conversion on the phytosterol etherate to obtain a fermentation product;

(3) extraction: standing and layering the fermentation product obtained in the step (2), and pumping the upper layer bacterial liquid into a beaker for later use; filtering the lower layer solid to obtain a solid product, combining the filtrate with the upper layer bacterial liquid, and applying the filtrate to the conversion of the next batch of resting cells;

(4) performing hydrolysis, namely extracting the solid product obtained in the step (3) by using ethyl acetate, heating and concentrating under reduced pressure, and hydrolyzing by using hydrochloric acid to obtain a hydrolysate;

(5) refining: and (4) refining and purifying the hydrolysate obtained in the step (4) to obtain a pregna-5-ene-3 beta, 21-diol product.

2. The method for preparing pregn-5-ene-3 β, 21-diol by resting cell method according to claim 1, wherein the mass ratio of methylal to phytosterol in step (1) is 3-20: 1.

3. The method for preparing pregn-5-ene-3 β, 21-diol by resting cell method according to claim 1, wherein the method for seed culture of B-NRRL 3683 mutant strain in step (2) comprises the following steps:

(1) slant culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, agar 20g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; after inoculation, culturing at 30 ℃ for 4-5 days;

(2) first-order seed culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; after inoculation, shake culturing is carried out for 48h at 30 ℃ and 200 rpm;

(3) secondary seed culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; inoculating the primary seed liquid to a secondary seed culture medium according to the volume ratio of 10%, and after inoculation, performing shake culture at 30 ℃ and 200rpm for 48 hours;

(4) seed tank culture:

the formula of the culture medium is as follows: 0.1-10g/L of peptone, 0.1-10g/L of yeast extract, 0.1-10g/L of glucose, 0.1-10g/L of disodium hydrogen phosphate, and pH 7.5-8.0, preparing a culture medium according to the formula, and sterilizing at 121 ℃ for 30 min; inoculating the secondary seed liquid to a seed culture medium according to the volume ratio of 10%, wherein the culture parameters are as follows: culturing at 28-32 deg.C, air flow rate of 0.5-1.0vvm, stirring speed of 50-500rpm, and pot pressure of 0.05-0.06MPa for 14-96 h.

4. The method for preparing pregn-5-ene-3 β, 21-diol by resting cell method according to claim 1, wherein the transformation medium formula in step (2) comprises the following components by mass percent: 1-10% of phytosterol etherate, 1-40% of hydroxypropyl-beta-cyclodextrin, 2-20% of thalli and the balance of PBS (20 mM of pH 8.0).

5. The method for preparing pregn-5-ene-3 β, 21-diol according to claim 4, wherein the phytosterol etherate is ground to 200 mesh.

6. The method for preparing pregn-5-ene-3 β, 21-diol according to claim 1, wherein the transformation method of resting cells in step (2) comprises: preparing a conversion system according to the formula, and converting at the conditions of 28-32 ℃, stirring speed of 50-500rpm, air flow of 0.5-1.0vvm and tank pressure of 0.05-0.06 MPa.

7. The method for preparing pregn-5-ene-3 β, 21-diol by resting cell method according to claim 1, wherein the hydrolysis method in step (4) is specifically: dissolving the solid product with ethyl acetate, stirring for 1h, performing suction filtration, taking the filter cake as solid waste after being washed by water, combining the filtrates, concentrating at 45 ℃ under reduced pressure until no fraction is produced, adding 5% hydrochloric acid, heating to 60 ℃, hydrolyzing for 1-2h, performing HPLC tracking until the reaction is complete, performing suction filtration, discarding the filtrate, and collecting the filter cake to obtain the hydrolysate.

8. The method for preparing pregn-5-ene-3 β, 21-diol by resting cell method according to claim 1, wherein the refining method in step (5) comprises: drying the hydrolysate at 70 ℃, adding methanol for dissolving, performing suction filtration, heating and concentrating the filtrate under reduced pressure to a small volume, cooling, performing suction filtration, and drying to obtain a crude product; adding petroleum ether into the crude product, heating, refluxing, pulping, cooling, filtering to obtain white solid, oven drying, adding methanol, heating to dissolve, concentrating under reduced pressure to paste, cooling to 0-4 deg.C, growing crystal for 2 hr, vacuum filtering, and oven drying.

Technical Field

The invention relates to a production method of steroid medicine intermediate, in particular to a method for preparing pregna-5-ene-3 beta, 21-diol by resting cell method.

Background

Pregna-5-ene-3 beta, 21-diol is an important intermediate in steroid synthesis, the traditional process is to utilize a similar structure of a 3-position ketone group, selectively reduce the 3-position ketone group into a product obtained by a beta hydroxyl group by a chemical method, a certain amount of 3-position alpha hydroxyl isomer is often carried, the yield is low, the steps are complicated, and various organic reagents are used to easily cause pollution.

The strain Mycobacterium sp.B-NRRL 3683 is described in U.S. Pat. No. 4755463 and is generally used only for the fermentation of phytosterols to 4-AD and ADD, and it is currently widely studied to improve the yield of 4-AD and ADD by controlling the reaction conditions.

Disclosure of Invention

In view of the above technical problems, the present invention aims to provide a method for preparing pregna-5-ene-3 β, 21-diol by converting phytosterol into resting cells, which has characteristics of high yield, high quality, low pollution and easy operation. In order to achieve the purpose, the invention adopts the technical scheme that:

a method for preparing pregn-5-ene-3 beta, 21-diol by a resting cell method comprises the following steps:

(1) and 3, protecting: utilizing methylal as a protective agent, protecting 3-position hydroxyl of phytosterol to obtain phytosterol etherate, and adding phosphorus pentoxide as a catalyst and diatomite as a filter aid in the reaction process;

(2) transformation of resting cells: carrying out slant culture and liquid seed culture on a Mycobacterium (Mycobacterium sp.) B-NRRL 3683 mutation strain, filtering and separating seeds, adding the seeds into a PBS buffer system, and carrying out fermentation conversion on the phytosterol etherate to obtain a fermentation product;

(3) extraction: standing and layering the fermentation product obtained in the step (2), and pumping the upper layer bacterial liquid into a beaker for later use; filtering the lower layer solid to obtain a solid product, combining the filtrate with the upper layer bacterial liquid, and applying the filtrate for the next batch of conversion;

(4) performing hydrolysis, namely extracting the solid product obtained in the step (3) by using ethyl acetate, heating and concentrating under reduced pressure, and hydrolyzing by using hydrochloric acid to obtain a hydrolysate;

(5) refining: and (4) refining and purifying the hydrolysate obtained in the step (4) to obtain a pregna-5-ene-3 beta, 21-diol product.

Preferably, the mass ratio of the methylal to the phytosterol in the step (1) is 10-40: 1.

Preferably, the method for seed culture of B-NRRL 3683 mutant strain in the step (2) comprises the following steps:

(1) slant culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, agar 20g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; after inoculation, culturing at 30 ℃ for 4-5 days;

(2) first-order seed culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; after inoculation, shake culturing is carried out for 48h at 30 ℃ and 200 rpm;

(3) secondary seed culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; inoculating the primary seed liquid to a secondary seed culture medium according to the volume ratio of 10%, and after inoculation, performing shake culture at 30 ℃ and 200rpm for 48 hours;

(4) seed tank culture:

the formula of the culture medium is as follows: 0.1-10g/L of peptone, 0.1-10g/L of yeast extract, 0.1-10g/L of glucose, 0.1-10g/L of disodium hydrogen phosphate, and pH 7.5-8.0, preparing a culture medium according to the formula, and sterilizing at 121 ℃ for 30 min; inoculating the secondary seed liquid to a seed culture medium according to the volume ratio of 10%, wherein the culture parameters are as follows: culturing at 28-32 deg.C, air flow rate of 0.5-1.0vvm, stirring speed of 50-500rpm, and pot pressure of 0.05-0.06MPa for 14-96 h.

Preferably, the transformation medium formula in the step (2) comprises the following components in percentage by mass: 1-10% of phytosterol etherate, 1-40% of hydroxypropyl-beta-cyclodextrin, 2-20% of thalli and the balance of PBS (20 mM of pH 8.0).

Preferably, the phytosterol etherate is ground to 200 mesh.

Preferably, the resting cell transformation method in the step (2) is specifically as follows: preparing a conversion system according to the formula, and converting at the conditions of 28-32 ℃, stirring speed of 50-500rpm, air flow of 0.5-1.0vvm and tank pressure of 0.05-0.06 MPa.

Preferably, the hydrolysis method in the step (4) is specifically as follows: dissolving the solid product with ethyl acetate, stirring for 1h, performing suction filtration, taking the filter cake as solid waste after being washed by water, combining the filtrates, concentrating at 45 ℃ under reduced pressure until no fraction is produced, adding 5% hydrochloric acid, heating to 60 ℃, hydrolyzing for 1-2h, performing HPLC tracking until the reaction is complete, performing suction filtration, discarding the filtrate, and collecting the filter cake to obtain the hydrolysate.

Preferably, the refining method in the step (5) is specifically as follows: drying the hydrolysate at 70 ℃, adding methanol for dissolving, performing suction filtration, heating and concentrating the filtrate under reduced pressure to a small volume, cooling, performing suction filtration, and drying to obtain a crude product; adding petroleum ether into the crude product, heating, refluxing, pulping, cooling, filtering to obtain white solid, oven drying, adding methanol, heating to dissolve, concentrating under reduced pressure to paste, cooling to 0-4 deg.C, growing crystal for 2 hr, vacuum filtering, and oven drying.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention takes the phytosterol as the raw material to produce pregna-5-ene-3 beta, 21-diol, the raw material is easy to obtain, and the production cost is reduced.

2. The activity of 3-hydroxyl of phytosterol is very high, and the phytosterol is easily oxidized in the conversion process to cause conversion failure.

3. The invention adopts a resting cell conversion method, the conversion system has single nutrition, the risk of contamination is low, the bacterial quantity is adjustable, the feeding concentration of a substrate can be increased, the conversion reaction time is shortened, the pH value of a phosphate buffer system can be adjusted, the enzyme activity in the conversion process is relatively stable, the good conversion capability is ensured, simultaneously the needed cyclodextrin and the bacteria can be repeatedly utilized, and the separation between the cyclodextrin and the products is convenient.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but 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.