CN110656147B - Biological dehydrogenation method of androstenedione C1,2 position - Google Patents
Biological dehydrogenation method of androstenedione C1,2 position Download PDFInfo
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Abstract
The invention relates to a production method of a steroid drug intermediate, in particular to a biological dehydrogenation method of androstenedione C1 and C2. The invention takes 4-androstene-3, 17-dione as a substrate, takes simple nocardia bacteria liquid as an enzyme source, simultaneously adds 100-200ml/L of soybean oil and 0.1-2g/L of tween-80 into a conversion system, carries out conversion reaction at 29-31 ℃, completely reacts, and obtains 1, 4-androstadiene-3, 17-dione by separating and purifying reactants. 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 the conversion rate can reach more than 96 percent; meanwhile, the degradation rate of the product is low, the reaction specificity is strong, the conversion time is relatively quick, and the product quality is high; and the method does not need a sterile environment, is convenient for outdoor operation, is simple to operate, and is suitable for industrialized mass production.
Description
Technical Field
The invention relates to a production method of a steroid drug intermediate, in particular to a biological dehydrogenation method of androstenedione C1 and C2.
Background
The dehydrogenation of steroid C1 and 2 sites is carried out by two 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 and can not meet 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 androstenedione C1,2 dehydrogenation is shown in figure 1. Androstenedione is dehydrogenated at the C1 and 2 positions, and 4 reaction systems exist in the biological method at present: ordinary direct transformation, double liquid phase transformation, protoplast transformation, and disrupted cell transformation. The common direct conversion is to directly add bacterial liquid into a fermentation medium for conversion, and the bacterial liquid does not need to be subjected to any treatment, but needs aseptic operation, thereby increasing the operation difficulty. The two-liquid phase transformation technology is mainly applied to separation and purification of biomolecules and cells in early stage, and is gradually applied to biological transformation in recent years, wherein a key factor in the technology is selection of a phase medium, but selection of an organic solvent with better compatibility to cells is difficult. Protoplast transformation can overcome the difficulty that cell membrane permeability affects intracellular enzyme to catalyze microbial transformation, but has complex and tedious operation, and is not beneficial to mass production. Ultrasonic disruption transformation increases the conversion rate of microorganisms to steroid compounds by increasing substrate solubility, enhancing mass transfer, increasing the effective contact of enzymes with substrates, but ultrasonic disruption is more difficult for industrial production. Of these 4 systems, the highest conversion is common direct conversion, but only about 70% is required, resulting in high costs.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide a method for preparing 1, 4-androstene-3, 17-dione by using simple nocardia microorganism with high purity and strong singleness to convert 4-androstene-3, 17-dione, which can solve the problems of complex operation, low conversion rate and high degradation rate in the existing androstene dione dehydrogenation method.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for biological dehydrogenation of androstenedione at the C1,2 position, characterized in that the method comprises the steps of: the preparation method comprises the steps of taking 4-androstene-3, 17-dione as a substrate, taking simple nocardia liquid as an enzyme source, simultaneously adding 1% -16% of soybean oil and 800.05% -0.2% of tween-into a conversion system, carrying out conversion reaction at 29-31 ℃, and separating and purifying reactants to obtain 1, 4-androstene-3, 17-dione.
Further, the addition amount of the substrate is 0.5% -10%.
Further, the preparation method of the nocardia simply bacterial liquid comprises the following steps:
(1) Slant culture: the slant culture medium comprises the following substances in mass and volume percent (w/v): glucose 0.1-1%, corn steep liquor 0.1-1%, peptone 0.1-0.5%, potassium dihydrogen phosphate 0.01-0.15%, yeast extract 0.01-0.05% and agar powder 0.01-2%; sterilizing at 121deg.C for 30min at pH7.0-7.2, and inoculating simple Nocardia strain;
(2) Primary seed culture: the primary seed culture medium comprises the following substances in mass and volume percent (w/v): glucose 0.1-1%, corn steep liquor 0.1-1%, peptone 0.1-0.5%, potassium dihydrogen phosphate 0.01-0.15% and yeast extract 0.01-0.05%; sterilizing at 121 deg.C at pH7.0-7.2 for 30min, inoculating simple nocardia inclined plane strain, and shake culturing to obtain first-stage seed solution;
(3) Secondary seed culture: the secondary seed culture medium comprises the following substances in mass and volume percent (w/v): glucose 0.1-1%, corn steep liquor 0.1-1%, peptone 0.1-0.5%, potassium dihydrogen phosphate 0.01-0.25%, 4-androstene-3, 17-dione 0.05% and dichlormid 0.02%; sterilizing at 121 deg.C at pH7.0-7.2 for 30min, inoculating the primary seed solution into the secondary seed culture medium, and shake culturing to obtain simple nocardia bacteria solution.
Further, OD in the Nocardia simply bacteria liquid 600 The value is 0.5-2.0.
Further, the conditions of the slant culture are as follows: culturing at 25-40deg.C for 1-5d.
Further, the conditions of the primary seed culture are as follows: shaking culture at 25-40deg.C at 50-200rpm for 10-72 hr.
Further, the conditions of the secondary seed culture are as follows: shaking culture at 25-40deg.C at 50-200rpm for 10-72 hr.
Further, when the conversion reaction is performed in a 10L fermenter, the conditions are as follows: air flow rate of 0.05-0.6m 3 And/h, the tank pressure is 0.01-0.1MPa.
Further, the separation and purification method specifically comprises the following steps: heating the conversion solution to 80 ℃, stirring, preserving heat, standing for layering, separating an upper oil phase, cooling to normal temperature, adding 3L of methanol, stirring, standing for layering, concentrating the upper methanol under reduced pressure, extracting the lower oil layer for 2 times, mixing the upper methanol layer with the lower oil layer, concentrating under reduced pressure, concentrating to paste at 50-70 ℃, adding water to dry the methanol therein, cooling to room temperature, carrying out suction filtration to obtain white-like solid, and drying the solid at 70 ℃; and (3) adding petroleum ether after drying, heating to 60 ℃, refluxing and pulping, cooling to below 30 ℃, carrying out suction filtration, leaching a filter cake by using petroleum ether, and drying after suction drying.
Further, the biological dehydrogenation method specifically comprises the following steps: preparing bacterial liquid from nocardia simply by slant culture, primary seed culture and secondary seed culture, and adding 120g of 4-androstene-3, 17-dione, 960g of soybean oil and 6g of tween-80 into a conversion system according to the volume of the bacterial liquid being 6L; the conversion system is subjected to conversion reaction for 72 hours at 30 ℃, the reaction is complete, and the reactants are separated and purified to obtain the 1, 4-androstadiene-3, 17-dione.
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 the conversion rate can reach more than 96 percent; meanwhile, the degradation rate of the product is low, the reaction specificity is strong, the conversion time is relatively quick, and the product quality is high; and the method does not need a sterile environment, is convenient for outdoor operation, is simple to operate, and is suitable for industrialized mass production.
Drawings
FIG. 1 shows the dehydrogenation reaction of androstenedione at the C1,2 position.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The materials used in the examples below were all commercially available from conventional sources. 4-androstene-3, 17-dione is hereinafter referred to as 4-AD, and 1, 4-androstene-3, 17-dione is hereinafter referred to as ADD.
EXAMPLE 1 simple Nocardia seed culture
And (3) strain: nocardia simply
Slant culture medium: glucose 0.1-1%, corn steep liquor 0.1-1%, peptone 0.1-0.5%, potassium dihydrogen phosphate 0.01-0.15%, yeast extract 0.01-0.05%, and agar powder 0.01-2%; the pH is 7.0-7.2. Culture conditions: 25-40 ℃ and 1-5d.
Primary seed medium: glucose 0.1-1%, corn steep liquor 0.1-1%, peptone 0.1-0.5%, potassium dihydrogen phosphate 0.01-0.15%, and yeast extract 0.01-0.05%; the pH is 7.0-7.2. Culture conditions: 500ml shaking bottle is filled with 100ml culture medium, shaking culture is carried out, the rotation speed is 50-200rpm, the temperature is 25-40 ℃, and the culture time is 10-72h.
Secondary seed medium: glucose 0.1-1%, corn steep liquor 0.1-1%, peptone 0.1-0.5%, potassium dihydrogen phosphate 0.01-0.25%, 4-androstene-3, 17-dione 0.05% and dichlormid 0.02%; the pH is 7.0-7.2. Culture conditions: 500ml shaking bottle is filled with 100ml culture medium, shaking culture is carried out, the rotation speed is 50-200rpm, the temperature is 25-40 ℃, and the culture time is 10-72h.
OD in Nocardia simply bacterial liquid obtained by culture 600 The value is 0.5-2.0.
Example 2 shake flask transformation
Seed culture was performed as in example 1, the primary seed solution was inoculated into a secondary seed medium and cultured for 10 hours, 2g of 4-AD was added to 100ml of the secondary seed after the culture, and the transformation was performed for 72 hours under the conditions: 200rpm, 30.+ -. 1 ℃. At the end of the conversion, the sample was fed into the liquid phase, the normalized ADD content was 82.19%, and the normalized 4-AD content was 17.18%.
Example 3 shake flask transformation
Seed culture was performed as in example 1, the primary seed solution was inoculated into a secondary seed medium and cultured for 16 hours, 2g of 4-AD crushed to 200 mesh was added to 100ml of the secondary seed after culture, and the conversion conditions were 72 hours: 200rpm, 30.+ -. 1 ℃. At the end of the conversion, the sample was fed into the liquid phase, the ADD normalized content was 85.16%, and the 4-AD normalized content was 14.49%.
Example 4 shake flask transformation
Seed culture was performed as in example 1, the primary seed liquid was inoculated into a secondary seed medium and cultured for 24 hours, 2g of 4-AD,16ml of soybean oil was added to 100ml of the secondary seed thus cultured, and the transformation was performed for 72 hours under the conditions: 200rpm, 30.+ -. 1 ℃. At the end of the conversion, the sample was fed into the liquid phase, the normalized ADD content was 91.51%, and the normalized 4-AD content was 8.17%.
Example 5 shake flask transformation
Seed culture was performed as in example 1, the primary seed solution was inoculated into a secondary seed medium and cultured for 36 hours, 2g of 4-AD,0.1g of Tween 80 was added to 100ml of the secondary seed after the culture, and the conditions for transformation were as follows: 200rpm, 30.+ -. 1 ℃. At the end of the conversion, the sample was fed into the liquid phase with an ADD normalized content of 88.39% and a 4-AD normalized content of 11.15%.
Example 6 shake flask transformation
Seed culture was performed as in example 1, the primary seed solution was inoculated into a secondary seed medium and cultured for 48 hours, 2g of 4-AD,16ml of soybean oil, 0.1g of Tween 80 was added to 100ml of the secondary seed after culture, and the transformation conditions were: 200rpm, 30.+ -. 1 ℃. At the end of the conversion, the sample was fed into the liquid phase with an ADD normalized content of 94.48% and a 4-AD normalized content of 5.24%.
Example 7 shake flask transformation
Seed culture was performed as in example 1, the primary seed liquid was inoculated into a secondary seed medium and cultured for 10 hours, 0.5g of 4-AD,10ml of soybean oil, 0.15g of Tween 80 was added to 100ml of the secondary seed thus cultured, and the conditions for transformation were: 200rpm, 30.+ -. 1 ℃. At the end of the conversion, the sample was fed into the liquid phase, the normalized ADD content was 93.65%, and the normalized 4-AD content was 5.81%.
Example 8 shake flask transformation
Seed culture was performed as in example 1, the primary seed solution was inoculated into a secondary seed medium and cultured for 48 hours, 5g of 4-AD,16ml of soybean oil, 0.05g of Tween 80 was added to 100ml of the secondary seed after culture, and the transformation conditions were 80 hours: 200rpm, 30.+ -. 1 ℃. At the end of the conversion, the sample was fed into the liquid phase, the normalized ADD content was 93.44%, and the normalized 4-AD content was 6.07%.
Example 9 shake flask transformation
Seed culture was performed as in example 1, the primary seed solution was inoculated into a secondary seed medium and cultured for 24 hours, 10g of 4-AD,1ml of soybean oil, 0.2g of Tween 80 was added to 100ml of the secondary seed after culture, and the transformation conditions were 80 hours: 200rpm, 30.+ -. 1 ℃. At the end of the conversion, the sample was fed into the liquid phase, the normalized ADD content was 94.10%, and the normalized 4-AD content was 5.58%.
EXAMPLE 10 fermenter conversion and separation purification
600ml of primary seeds were cultivated according to the formulation of example 1.
Preparing a culture medium according to the secondary seed formula of example 1, culturing seeds in a 10L fermentation tank, metering the volume of the seeds to 6L, adding the seeds into the 10L fermentation tank after preparation, sterilizing the seeds for 30min at 121 ℃, cooling the seeds to 30 ℃, and inoculating the seeds to the volume of 6L after inoculation. Seed culture conditions of 30+ -1deg.C, rotation speed of 150-200rpm and air flow rate of 0.2m 3 And/h, pot pressure is 0.05MPa, culture time is 16h, culture is finished, 120g of 4-AD substrate, 6g of tween-80 and 960ml of soybean oil are added, conversion is carried out for 72h, and conversion conditions are as follows: 200rpm, 30+/-1 ℃, the rotating speed of 200-250rpm and the air flow of 0.05-0.6m 3 And/h, the tank pressure is 0.01-0.1MPa.
At the end of the conversion, the liquid phase was sampled, with an ADD of 94.14%,4-AD of 5.09%.
Heating the conversion solution to 80deg.C, stirring for 30min, maintaining the temperature, standing for 4 hr, layering, separating upper layer oil phase, cooling to room temperature, adding 3L methanol, stirring for 30min, standing for 1 hr, layering, and concentrating upper layer methanol under reduced pressure. The lower oil layer was extracted 2 more times, and the upper methanol layer was combined and concentrated under reduced pressure. Concentrating at 50-70deg.C to paste, adding small amount of water, drying with methanol, cooling to room temperature, vacuum filtering to obtain white solid, and oven drying at 70deg.C. After drying, 240ml petroleum ether is added, the mixture is heated to 60 ℃ for refluxing and pulping for 2 hours, cooled to below 30 ℃, filtered, the filter cake is leached by a little petroleum ether, pumped to dryness, and dried in an oven at 70 ℃. 108.5g of white crystals are recovered and the sample is sent to liquid phase for detection, wherein the ADD is 96.68 percent and the 4-AD is 3.17 percent.
Claims (10)
1. A method for biological dehydrogenation of androstenedione at the C1,2 position, characterized in that the method comprises the steps of: 4-androstene-3, 17-dione is used as a substrate, simple nocardia bacteria liquid prepared by slant culture, primary seed culture and secondary seed culture is used as an enzyme source, soybean oil with the volume percentage of 1-16% and tween-80 with the mass and volume percentage of 0.05-0.2% are added into a conversion system, the conversion reaction is carried out at 29-31 ℃, the reaction is complete, and the reactants are separated and purified to obtain the 1, 4-androstadiene-3, 17-dione.
2. The biological dehydrogenation process of androstenedione C1,2 according to claim 1, characterized in that the substrate is added in an amount of 0.5% to 10% by mass volume.
3. The biological dehydrogenation method of androstenedione C1 and C2 according to claim 1, characterized in that the preparation method of the simple nocardia bacterium solution is as follows:
(1) Slant culture: the slant culture medium comprises the following substances in percentage by mass and volume: glucose 0.1-1%, corn steep liquor 0.1-1%, peptone 0.1-0.5%, potassium dihydrogen phosphate 0.01-0.15%, yeast extract 0.01-0.05% and agar powder 0.01-2%; sterilizing at 121deg.C for 30min at pH7.0-7.2, and inoculating simple Nocardia strain;
(2) Primary seed culture: the primary seed culture medium comprises the following substances in percentage by mass and volume: glucose 0.1-1%, corn steep liquor 0.1-1%, peptone 0.1-0.5%, potassium dihydrogen phosphate 0.01-0.15% and yeast extract 0.01-0.05%; sterilizing at 121 deg.C at pH7.0-7.2 for 30min, inoculating simple nocardia inclined plane strain, and shake culturing to obtain first-stage seed solution;
(3) Secondary seed culture: the secondary seed culture medium comprises the following substances in percentage by mass and volume: glucose 0.1-1%, corn steep liquor 0.1-1%, peptone 0.1-0.5%, potassium dihydrogen phosphate 0.01-0.25%, 4-androstene-3, 17-dione 0.05% and dichlormid 0.02%; sterilizing at 121 deg.C at pH7.0-7.2 for 30min, inoculating the primary seed solution into the secondary seed culture medium, and shake culturing to obtain simple nocardia bacteria solution.
4. The method for biological dehydrogenation of androstenedione at C1 and C2 positions according to claim 3, wherein the OD in the nocardia simply bacteria solution 600 The value is 0.5-2.0.
5. A biological dehydrogenation process according to claim 3 wherein the conditions of the slant culture are: culturing at 25-40deg.C for 1-5d.
6. A biological dehydrogenation process according to claim 3 wherein the primary seed culture conditions are: shaking culture at 25-40deg.C at 50-200rpm for 10-72 hr.
7. A biological dehydrogenation process according to claim 3 wherein the secondary seed culture conditions are: shaking culture at 25-40deg.C at 50-200rpm for 10-72 hr.
8. The biological dehydrogenation process of androstenedione C1,2 according to claim 1, characterized in that the conversion reaction is carried out in a 10L fermenter under the following conditions: air flow rate of 0.05-0.6m 3 And/h, the tank pressure is 0.01-0.1MPa.
9. The biological dehydrogenation method for androstenedione C1 and C2 according to claim 1, characterized in that the separation and purification method specifically comprises the following steps: heating the conversion solution to 80 ℃, stirring, preserving heat, standing for layering, separating an upper oil phase, cooling to normal temperature, adding 3L of methanol, stirring, standing for layering, concentrating the upper methanol under reduced pressure, extracting the lower oil layer for 2 times, mixing the upper methanol layer with the lower oil layer, concentrating under reduced pressure, concentrating to paste at 50-70 ℃, adding water to dry the methanol therein, cooling to room temperature, carrying out suction filtration to obtain white-like solid, and drying the solid at 70 ℃; and (3) adding petroleum ether after drying, heating to 60 ℃, refluxing and pulping, cooling to below 30 ℃, carrying out suction filtration, leaching a filter cake by using petroleum ether, and drying after suction drying.
10. The biological dehydrogenation method of androstenedione C1,2 according to claim 1, characterized in that it comprises the following specific steps: preparing bacterial liquid from nocardia simply by slant culture, primary seed culture and secondary seed culture, and adding 120g of 4-androstene-3, 17-dione, 960ml of soybean oil and 6g of tween-80 into a conversion system according to the volume of the bacterial liquid being 6L; the conversion system is subjected to conversion reaction for 72 hours at 30 ℃, the reaction is complete, and the reactants are separated and purified to obtain the 1, 4-androstadiene-3, 17-dione.
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| GB765867A (en) * | 1952-04-10 | 1957-01-16 | Olin Mathieson | Microbiological oxidation of steroids |
| EP1507867B1 (en) * | 2002-02-01 | 2011-08-03 | N.V. Organon | Process for fermentation of phytosterols to androstadienedione |
| CN101525651A (en) * | 2008-03-07 | 2009-09-09 | 安徽建筑工业学院 | Method for preparing androstenedione by biodegradation of phytosterol in two-liquid-phase system |
| CN101402928B (en) * | 2008-10-23 | 2010-07-28 | 江南大学 | Bacterial strain for microbial transformation phytosterin as yield per unit androstane diene diketone |
| CN102477404B (en) * | 2010-11-24 | 2014-04-02 | 北大方正集团有限公司 | Method for producing ADD from phytosterols by microbial transformation and culture medium thereof |
| CN102586139B (en) * | 2012-01-20 | 2013-03-20 | 广东本科生物工程股份有限公司 | High-yield AD/ADD strain and method for high-efficient production of AD/ADD |
| CN103266161B (en) * | 2013-05-24 | 2014-10-15 | 江南大学 | Fermentation strategy for producing androstenedione (ADD) by using recombinant bacillus subtilis |
| CN106282289B (en) * | 2015-05-15 | 2021-03-02 | 上海医药工业研究院 | Preparation method of androstane-1, 4-diene-3, 17-diketone and fermentation medium used by same |
| CN106636160A (en) * | 2016-11-17 | 2017-05-10 | 江南大学 | Method for generating ADD by converting AD through recombined Escherichia coli whole cells |
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