阅读说明：本技术 ***c1,2位的生物脱氢方法 (Biological dehydrogenation method of methyltestosterone C1,2 site ) 是由 唐艳平 刘喜荣 孟浩 曾春玲 赵小娟 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,具体涉及一种甲睾酮C1,2位的生物脱氢方法。本发明以甲睾酮为底物,以简单诺卡氏菌菌液为酶源,同时将大豆油100-200ml/L和吐温-80 0.1-2g/L加入转化体系中,于29-31℃下进行转化反应,反应完全,反应物经分离纯化得到脱氢甲睾酮。本发明在采用普通直接转化法的基础上,通过加入大豆油和吐温-80,可以大幅度提高转化率,转化率可达到99％以上；同时产物的降解率可降低至,反应专一性强,转化时间相对较快,产品质量高；并且无需无菌环境,常规室外操作即可,操作简单,适合工业化大批量生产。(The invention relates to a production method of steroid medicine intermediates, in particular to a biological dehydrogenation method of methyltestosterone C1, 2. The method takes methyltestosterone as a substrate, takes simple Nocardia bacterial liquid as an enzyme source, simultaneously adds 200ml/L soybean oil and 800.1-2 g/L Tween into a conversion system, carries out conversion reaction at 29-31 ℃, and obtains dehydromethyltestosterone after the reaction product is separated and purified. On the basis of adopting a common direct conversion method, the conversion rate can be greatly improved by adding the soybean oil and the Tween-80, and can reach more than 99 percent; meanwhile, the degradation rate of the product can be reduced, the reaction specificity is strong, the conversion time is relatively fast, and the product quality is high; and the method does not need sterile environment, can be operated in a conventional outdoor manner, is simple to operate and is suitable for industrial mass production.)

1. A method for biological dehydrogenation of methyltestosterone at C1,2 position, which comprises the following steps: taking 17 alpha methyl-17 beta hydroxyandrost-4-en-3-one as a substrate, taking simple Nocardia bacterium liquid as an enzyme source, simultaneously adding 1-16% (v/v) of soybean oil and 0.2% (w/v) of tween-800.05 into a conversion system, carrying out conversion reaction at 29-31 ℃, completely reacting, and separating and purifying reactants to obtain 17 alpha methyl-17 beta hydroxyandrost-1, 4-diene-3-one.

2. The method for the biological dehydrogenation of methyltestosterone C1,2 according to claim 1, wherein the substrate is added in an amount of 0.5-10% by mass/volume (w/v).

3. The method for biologically dehydrogenating methyltestosterone C1,2 according to claim 1, wherein the preparation method of the Nocardia simplex bacterial liquid comprises the following steps:

(1) slant culture: the slant culture medium comprises the following substances in percentage by mass volume (w/v): 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 0.01-2% of agar powder; sterilizing at pH7.0-7.2 at 121 deg.C for 30min, and inoculating Nocardia simplex strain;

(2) first-order seed culture: the primary seed culture medium comprises the following substances in percentage by mass volume (w/v): 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of potassium dihydrogen phosphate and 0.01-0.05% of yeast extract; sterilizing at 121 deg.C for 30min at pH7.0-7.2, inoculating Nocardia simplex slant strain, and performing shake culture to obtain first-stage seed solution;

(3) secondary seed culture: the secondary seed culture medium comprises the following substances in percentage by mass volume (w/v): 0.1 to 1 percent of glucose, 0.1 to 1 percent of corn steep liquor, 0.1 to 0.5 percent of peptone, 0.01 to 0.25 percent of monopotassium phosphate, 0.05 percent of 17 alpha methyl-17 beta hydroxyandrost-4-en-3-one and 0.02 percent of natural enemy; sterilizing at 121 ℃ for 30min at the pH of 7.0-7.2, inoculating the primary seed liquid into the secondary seed culture medium, and performing shaking culture to obtain the Nocardia simplex bacterium liquid.

4. The method for biologically dehydrogenating methyltestosterone C1,2 according to claim 3, wherein OD in said Nocardia simplex bacterial liquid₆₀₀The value is 0.5-2.0.

5. The method for the biological dehydrogenation of methyltestosterone C1,2 according to claim 3, wherein the slant culture conditions are as follows: culturing at 25-40 deg.C for 1-5 days.

6. The method for the biological dehydrogenation of methyltestosterone C1,2 according to claim 3, wherein the conditions of the primary seed culture are as follows: shaking at 25-40 deg.C, rotating at 50-200rpm, and culturing for 10-72 h.

7. The method for the biological dehydrogenation of methyltestosterone C1,2 according to claim 3, wherein the conditions of said secondary seed culture are as follows: shaking at 25-40 deg.C, rotating at 50-200rpm, and culturing for 10-72 h.

8. The method for the biological dehydrogenation of methyltestosterone C1,2 according to claim 1, wherein when the conversion reaction is carried out in a 10L fermentor, the conditions are as follows: air flow rate of 0.05-0.6m³The pressure of the tank is 0.01-0.1M Pa.

9. The method for biologically dehydrogenating methyltestosterone C1,2 according to claim 1, wherein the separation and purification method specifically comprises: heating the conversion solution to 80 ℃ and stirring, keeping the temperature, standing and layering, separating an upper oil phase, cooling to normal temperature, adding 3L of methanol, stirring, standing and layering, concentrating an upper methanol layer under reduced pressure, extracting a lower oil layer for 2 times, combining the upper methanol layer and the lower methanol layer, concentrating the mixture at 50-70 ℃ to paste, adding water to dry the methanol in the mixture, cooling to room temperature, carrying out suction filtration to obtain a white-like solid, and drying the solid at 70 ℃; adding petroleum ether after drying, heating to 60 ℃, refluxing and pulping, cooling to below 30 ℃, performing suction filtration, leaching a filter cake with petroleum ether, drying after suction filtration.

10. The biological dehydrogenation method of methyltestosterone C1,2 according to claim 1, wherein the biological dehydrogenation method specifically comprises: the preparation method comprises the following steps of performing slant culture, primary seed culture and secondary seed culture on the Nocardia simplex to prepare a bacterial liquid, and adding 120g of 17 alpha methyl-17 beta hydroxyandrost-4-en-3-one, 960ml of soybean oil and 806 g of tween-806 in a conversion system by taking the volume of the bacterial liquid as 6L; the transformation system carries out transformation reaction at 30 ℃, the reaction is complete, and the reactant is separated and purified to obtain 17 alpha methyl-17 beta hydroxyandrosta-1, 4-diene-3-ketone.

Technical Field

The invention relates to a production method of steroid medicine intermediates, in particular to a biological dehydrogenation method of methyltestosterone C1, 2.

Background

Steroid compound C1, 2-position dehydrogenation have two kinds of chemical methods and biological methods, the traditional chemical method adopts arsenic dioxide dehydrogenation, the process is simple, the yield is considerable, but the product contains a certain amount of arsenic, which can not reach the regulation of related standards, and the biological method has the characteristics of strong specificity, rapid reaction, high yield and the like, so the biological method has gradually replaced the chemical method in recent years.

The reaction process of the dehydrogenation of methyltestosterone C1, 2-position is shown in figure 1. Methyltestosterone undergoes dehydrogenation at the C1 and 2 positions, and there are 4 reaction systems in the current biological method: general direct transformation, two-fluid phase transformation, protoplast transformation, and disrupted cell transformation. The ordinary direct conversion is to directly add the bacterial liquid into a fermentation medium for conversion, and the bacterial liquid does not need any treatment but needs aseptic operation, thereby increasing the difficulty of the operation. The double liquid phase transformation technology is mainly applied to separation and purification of biomolecules and cells in the early stage, and is gradually applied to biotransformation in recent years, the key factor in the technology is selection of a phase forming medium, but selection of an organic solvent with good compatibility to cells is difficult. Protoplast transformation can overcome the difficulty that intracellular enzyme catalyzes microbial transformation due to the influence of cell membrane permeability, but has complex and fussy operation and is not beneficial to mass production. The conversion rate of the steroid compounds by the microorganisms is improved by increasing the dissolubility of the substrate, strengthening mass transfer and increasing the effective contact of enzyme and the substrate through ultrasonic disruption conversion, but the ultrasonic disruption is more difficult for industrial production. The highest conversion in these 4 systems was the normal direct conversion, but it was also only 59.5%, leading to high costs.

Disclosure of Invention

Aiming at the technical problems, the invention aims to provide a method for preparing 17 alpha methyl-17 beta hydroxyandrost-1, 4-diene-3-one by quickly converting 17 alpha methyl-17 beta hydroxyandrost-4-en-3-one with high purity and strong singleness through simple nocardia microorganisms, and can solve the problems of complex operation, low conversion rate and high degradation rate in the conventional methyltestosterone dehydrogenation method.

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

a method for biological dehydrogenation of methyltestosterone at position C1,2, said method comprising: taking 17 alpha methyl-17 beta hydroxyandrost-4-en-3-one as a substrate, taking simple Nocardia bacterium liquid as an enzyme source, simultaneously adding 1-16% (v/v) of soybean oil and 0.2% (w/v) of tween-800.05 into a conversion system, carrying out conversion reaction at 29-31 ℃, completely reacting, and separating and purifying reactants to obtain 17 alpha methyl-17 beta hydroxyandrost-1, 4-diene-3-one.

Further, the addition amount of the substrate is 0.5-10% by mass volume percentage (w/v).

Further, the preparation method of the nocardia simplex bacterial liquid comprises the following steps:

(1) slant culture: the slant culture medium comprises the following substances in percentage by mass volume (w/v): 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 0.01-2% of agar powder; sterilizing at pH7.0-7.2 at 121 deg.C for 30min, and inoculating Nocardia simplex strain;

(2) first-order seed culture: the primary seed culture medium comprises the following substances in percentage by mass volume (w/v): 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of potassium dihydrogen phosphate and 0.01-0.05% of yeast extract; sterilizing at 121 deg.C for 30min at pH7.0-7.2, inoculating Nocardia simplex slant strain, and performing shake culture to obtain first-stage seed solution;

(3) secondary seed culture: the secondary seed culture medium comprises the following substances in percentage by mass volume (w/v): 0.1 to 1 percent of glucose, 0.1 to 1 percent of corn steep liquor, 0.1 to 0.5 percent of peptone, 0.01 to 0.25 percent of monopotassium phosphate, 0.05 percent of 17 alpha methyl-17 beta hydroxyandrost-4-en-3-one and 0.02 percent of natural enemy; sterilizing at 121 ℃ for 30min at the pH of 7.0-7.2, inoculating the primary seed liquid into the secondary seed culture medium, and performing shaking culture to obtain the Nocardia simplex bacterium liquid.

Further, OD in the Nocardia simplex bacterium liquid₆₀₀The value is 0.5-2.0.

Further, the slant culture conditions are as follows: culturing at 25-40 deg.C for 1-5 days.

Further, the conditions of the primary seed culture are as follows: shaking at 25-40 deg.C, rotating at 50-200rpm, and culturing for 10-72 h.

Further, the conditions of the secondary seed culture are as follows: shaking at 25-40 deg.C, rotating at 50-200rpm, and culturing for 10-72 h.

Further, when the conversion reaction is carried out in a 10L fermenter, the conditions are: air flow rate of 0.05-0.6m³The tank pressure is 0.01-0.1 MPa.

Further, the separation and purification method specifically comprises the following steps: heating the conversion solution to 80 ℃ and stirring, keeping the temperature, standing and layering, separating an upper oil phase, cooling to normal temperature, adding 3L of methanol, stirring, standing and layering, concentrating an upper methanol layer under reduced pressure, extracting a lower oil layer for 2 times, combining the upper methanol layer and the lower methanol layer, concentrating the mixture at 50-70 ℃ to paste, adding water to dry the methanol in the mixture, cooling to room temperature, carrying out suction filtration to obtain a white-like solid, and drying the solid at 70 ℃; after drying, adding petroleum ether, heating to 60 ℃, refluxing and pulping, cooling to below 30 ℃, performing suction filtration, leaching a filter cake with petroleum ether, drying after suction filtration.

Specifically, the method comprises the following steps: the preparation method comprises the following steps of performing slant culture, primary seed culture and secondary seed culture on the Nocardia simplex to prepare a bacterial liquid, and adding 120g of 17 alpha methyl-17 beta hydroxyandrost-4-en-3-one, 960ml of soybean oil and 806 g of tween-806 in a conversion system by taking the volume of the bacterial liquid as 6L; the transformation system carries out transformation reaction at 30 ℃, the reaction is complete, and the reactant is separated and purified to obtain 17 alpha methyl-17 beta hydroxyandrosta-1, 4-diene-3-ketone.

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

on the basis of adopting a common direct conversion method, the conversion rate can be greatly improved by adding the soybean oil and the Tween-80, and can reach more than 99 percent; meanwhile, the degradation rate of the product is low, the reaction specificity is strong, the conversion time is relatively fast, and the product quality is high; and the method does not need sterile environment, can be operated in a conventional outdoor manner, is simple to operate and is suitable for industrial mass production.

Drawings

FIG. 1 shows the reaction sequence of dehydrogenation of methyltestosterone C1 at position 2.

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. 17 alpha methyl-17 beta hydroxyandrost-4-en-3-one is hereinafter abbreviated methyltestosterone and 17 alpha methyl-17 beta hydroxyandrost-1, 4-diene-3-one is hereinafter abbreviated dehydromethyltestosterone.