阅读说明：本技术 植物甾醇微生物转化制备add的方法 (Method for preparing ADD by phytosterol microbial transformation ) 是由 杨芳 刘喜荣 孟浩 曾春玲 王敬华 于 2019-10-16 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,具体涉及一种植物甾醇微生物转化制备ADD的方法。本发明包括以下步骤：(1)用分枝杆菌B-NRRL 3683对植物甾醇进行发酵转化,发酵液静置分层得到上层油层；(2)以所述油层为底物,以简单诺卡氏菌的菌液为酶源进行转化反应,反应物经提取精制得到ADD。本发明采用两步转化法,先将植物甾醇转化为4-AD和ADD的混合物,再将富集在油层中的4-AD转化为ADD,两步中采用不同的菌种,整个反应中省略了将4-AD单独提取出来的步骤,转化率高,反应专一性强,转化时间相对较快,同时产物的降解率低,产品质量高,得到的终产物ADD纯度可达99.5％以上,具有极大的应用前景。(The invention relates to a production method of a steroid drug intermediate, in particular to a method for preparing ADD by phytosterol microbial transformation. The invention comprises the following steps: (1) fermenting and converting phytosterol by using mycobacterium B-NRRL 3683, and standing and layering fermentation liquor to obtain an upper oil layer; (2) and (3) carrying out conversion reaction by taking the oil layer as a substrate and a bacterial solution of the nocardia simplicifolia as an enzyme source, and extracting and refining reactants to obtain the ADD. The invention adopts a two-step conversion method, firstly converts the phytosterol into a mixture of 4-AD and ADD, then converts the 4-AD enriched in an oil layer into ADD, adopts different strains in the two steps, omits the step of independently extracting the 4-AD in the whole reaction, has high conversion rate, strong reaction specificity, relatively quick conversion time, low degradation rate of products, high product quality, and high application prospect, and the purity of the obtained final product ADD can reach more than 99.5 percent.)

1. The method for preparing ADD by phytosterol microbial transformation is characterized by comprising the following steps:

(1) carrying out slant culture and shake flask seed culture on mycobacteria (Mycobacterium sp.) B-NRRL 3683, then carrying out fermentation conversion on phytosterol, heating and cooling fermentation liquor after the conversion is finished, standing for layering, and discarding a lower water phase to obtain an upper oil layer;

(2) and (2) taking the oil layer obtained in the step (1) as a substrate, taking a bacterial liquid obtained by performing slant culture and shake flask seed culture on the Nocardia simplex as an enzyme source, adding the bacterial liquid into a conversion system, performing conversion reaction at 28-32 ℃, completely reacting, and extracting and refining reactants to obtain the ADD.

2. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the formula of the slant culture medium of Mycobacterium (Mycobacterium sp.) B-NRRL 3683 in the step (1) is as follows: peptone 0.1-1.0g/L, yeast extract 0.1-1.0g/L, glucose 0.1-1.0g/L, disodium hydrogen phosphate 0.1-1.0g/L, agar 20g/L, pH 7.5-8.0;

the formula of the shake flask seed culture medium in the step (1) is as follows: peptone 0.1-1.0g/L, yeast extract 0.1-1.0g/L, glucose 0.1-1.0g/L, disodium hydrogen phosphate 0.1-1.0g/L, and pH 7.5-8.0.

3. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the formula (w/v) of the transformation medium in the step (1) is as follows: 1-10% of corn steep liquor, 0.1-1% of sodium nitrate, 0.01-0.1% of diammonium phosphate, 0.1-2% of PPE, 1-10% of phytosterol and 16% of soybean oil, wherein the pH value is 7.5-8.0.

4. The method for preparing ADD by microbial transformation of phytosterol according to claim 1 or 3, wherein the transformation conditions in the step (1) are as follows: at 28-32 deg.C, 200rpm, air flow rate of 0.1-1.0vvm, and tank pressure of 0.01-0.1 MPa.

5. The method for preparing ADD through phytosterol microbial conversion as claimed in claim 1, wherein the fermentation broth in step (1) is heated to 80 ℃, kept at the temperature for standing for 2 hours, layered, and the lower aqueous phase is treated with wastewater.

6. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the formula (w/v) of the slant culture medium of Nocardia simplex in the step (2) is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of monopotassium phosphate, 0.01-0.05% of yeast extract, 2% of agar powder and 7.0-7.2% of PH;

the formula (w/v) of the primary seed culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of monopotassium phosphate, 0.01-0.05% of yeast extract and pH value of 7.0-7.2;

the formula (w/v) of the secondary seed culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2.

7. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the formula (w/v) of the transformation medium in the step (2) is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2.

8. The method for preparing ADD by microbial transformation of phytosterol according to claim 1 or 7, wherein the transformation conditions in the step (2) are as follows: at 28-32 deg.C, 200rpm, air flow rate of 0.1-1.0vvm, and tank pressure of 0.01-0.1 MPa.

9. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the extraction method in the step (2) is as follows: heating and standing the conversion product, and removing a water layer and leaving an oil layer; adding methanol into the oil layer, stirring and standing at normal temperature, separating out the upper layer of methanol, concentrating under reduced pressure to dry, adding a small amount of water to carry out suction filtration on residual methanol in the dry oil, cooling and discharging; the separated oil layer is extracted twice with methanol to obtain crude ADD product.

10. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the refining method in the step (2) is as follows: adding a sodium hydroxide solution with the mass concentration of 2% into the ADD crude product, heating for saponification, cooling, performing suction filtration, and leaching to be neutral to obtain a white-like solid; and drying and weighing the white-like solid, adding methanol, heating, stirring, dissolving, adding activated carbon, stirring, decoloring, performing suction filtration and leaching while the white-like solid is hot, concentrating the filtrate under reduced pressure, cooling, growing crystals, performing suction filtration, and drying a filter cake to obtain an ADD fine product.

Technical Field

The invention relates to a production method of a steroid drug intermediate, in particular to a method for preparing ADD by phytosterol microbial transformation.

Background

Androst-4-ene-3,17-dione (androst-4-ene-3,17-dione, abbreviated as androstenedione, AD or 4-AD) and androst-1, 4-diene-3,17-dione (androsta-1,4-diene-3,17-dione, abbreviated as androstenedione, ADD) are important steroid intermediate compounds, androstenedione (4-AD) can be used as precursor of hormone substances such as male hormone, anabolin, etc., and can also be used for synthesizing drugs such as spironolactone, hydrocortisone, prednisone oxide, dexamethasone, etc.; the Androstenedione (ADD) can be used for synthesizing female hormone, oral contraceptive and other medicines.

Currently, there are two main methods for synthesizing androstenedione: firstly, dioscin is used as a starting material and is obtained through 11 steps of chemical synthesis reaction. And secondly, directly obtaining ADD from phytosterol through microbial transformation. The microbial conversion has the advantages of mild reaction conditions, energy conservation, environmental protection and the like, and whether the microbial conversion has economy including reaction substrate concentration, conversion rate and conversion specificity is determined, wherein the conversion specificity refers to the ratio of ADD to byproduct 4-AD in the conversion process, and the higher the ratio is, the better the conversion specificity is. Us patent USP4345029 describes the transformation of Mycobacterium species (Mycobacterium phlei) to AD and ADD; U.S. Pat. No. 3,7259005 describes the transformation of phytosterol into AD and ADD using another Mycobacterium (Mycobacterium fortuitum), but the transformation efficiency and specificity are not high, the transformation rate is only 64% at the maximum, and the industrial value is low.

Disclosure of Invention

In view of the above technical problems, an object of the present invention is to provide a method for preparing ADD by microbial transformation of phytosterol, which can solve the problems of low conversion rate and high degradation rate in the current methods for preparing androstenedione, and can rapidly and highly purify phytosterol to be converted into ADD.

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

a method for preparing ADD by phytosterol microbial transformation comprises the following steps:

(1) carrying out slant culture and shake flask seed culture on mycobacteria (Mycobacterium sp.) B-NRRL 3683, then carrying out fermentation conversion on phytosterol, heating and cooling fermentation liquor after the conversion is finished, standing for layering, and discarding a lower water phase to obtain an upper oil layer;

(2) and (2) taking the oil layer obtained in the step (1) as a substrate, taking a bacterial liquid obtained by performing slant culture and shake flask seed culture on the Nocardia simplex as an enzyme source, adding the bacterial liquid into a conversion system, performing conversion reaction at 28-32 ℃, completely reacting, and extracting and refining reactants to obtain the ADD.

Further, the formula of the slant culture medium of the Mycobacterium (Mycobacterium sp.) B-NRRL 3683 in the step (1) is as follows: peptone 0.1-1.0g/L, yeast extract 0.1-1.0g/L, glucose 0.1-1.0g/L, disodium hydrogen phosphate 0.1-1.0g/L, agar 20g/L, pH 7.5-8.0;

the formula of the shake flask seed culture medium in the step (1) is as follows: peptone 0.1-1.0g/L, yeast extract 0.1-1.0g/L, glucose 0.1-1.0g/L, disodium hydrogen phosphate 0.1-1.0g/L, and pH 7.5-8.0.

Further, the formula (w/v) of the transformation medium in the step (1) is as follows: 1-10% of corn steep liquor, 0.1-1% of sodium nitrate, 0.01-0.1% of diammonium phosphate, 0.1-2% of PPE, 1-10% of phytosterol and 16% of soybean oil, wherein the pH value is 7.5-8.0.

Preferably, the conversion conditions in step (1) are: at 28-32 deg.C, 200rpm, air flow rate of 0.1-1.0vvm, and tank pressure of 0.01-0.1 MPa.

Further, heating the fermentation liquor in the step (1) to 80 ℃, preserving heat, standing for 2 hours, layering, and treating the lower-layer water phase as wastewater.

Further, the formula (w/v) of the slant culture medium of the nocardia simplex in the step (2) is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of monopotassium phosphate, 0.01-0.05% of yeast extract, 2% of agar powder and 7.0-7.2% of PH;

the formula (w/v) of the primary seed culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of monopotassium phosphate, 0.01-0.05% of yeast extract and pH value of 7.0-7.2;

the formula (w/v) of the secondary seed culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2.

Further, the formula (w/v) of the transformation medium in the step (2) is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2.

Preferably, the conversion conditions in step (2) are: at 28-32 deg.C, 200rpm, air flow rate of 0.1-1.0vvm, and tank pressure of 0.01-0.1 MPa.

Further, the extraction method in the step (2) is as follows: heating and standing the conversion product, and removing a water layer and leaving an oil layer; adding methanol into the oil layer, stirring and standing at normal temperature, separating out the upper layer of methanol, concentrating under reduced pressure to dry, adding a small amount of water to carry out suction filtration on residual methanol in the dry oil, cooling and discharging; the separated oil layer is extracted twice with methanol to obtain crude ADD product.

Further, the refining method in the step (2) is as follows: adding a sodium hydroxide solution with the mass concentration of 2% into the ADD crude product, heating for saponification, cooling, performing suction filtration, and leaching to be neutral to obtain a white-like solid; and drying and weighing the white-like solid, adding methanol, heating, stirring, dissolving, adding activated carbon, stirring, decoloring, performing suction filtration and leaching while the white-like solid is hot, concentrating the filtrate under reduced pressure, cooling, growing crystals, performing suction filtration, and drying a filter cake to obtain an ADD fine product.

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

the invention adopts a two-step conversion method, firstly converts the phytosterol into a mixture of 4-AD and ADD, then converts the 4-AD enriched in an oil layer into ADD, adopts different strains in the two steps, omits the step of independently extracting the 4-AD in the whole reaction, has high conversion rate, strong reaction specificity, relatively quick conversion time, low degradation rate of products and high product quality, and the purity of the obtained final product ADD can reach more than 99.5 percent, thereby having great application prospect.

Detailed Description

In order to make the objects, technical solutions and advantages 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. 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 materials used in the following examples are all commercially available from conventional sources.