CN102477404B - Method for producing ADD from phytosterols by microbial transformation and culture medium thereof - Google Patents
Method for producing ADD from phytosterols by microbial transformation and culture medium thereof Download PDFInfo
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- CN102477404B CN102477404B CN201010557123.7A CN201010557123A CN102477404B CN 102477404 B CN102477404 B CN 102477404B CN 201010557123 A CN201010557123 A CN 201010557123A CN 102477404 B CN102477404 B CN 102477404B
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Abstract
The invention relates to a method for producing ADD (androstadienedione) from phytosterols by microbial transformation and a culture medium thereof. The fermentation medium contains the following substances which can dissolve or disperse in water: 0.1 g/L to 100 g/L of glucose, 10 g/L to 100 g/L of soybean powders, 0.5 g/L to 10 g/L of potassium dihydrogen phosphate, 0.1 g/L to 10 g/L of calcium carbonate, 0.001 g/L to 0.01 g/L of ferrous sulfate, 0.0001 g/L to 0.001 g/L of manganese chloride, and 10 g/L to 100 g/L of phytosterols. The pH of the fermentation medium is 5.0 to 8.0; and the fermentation medium is free of phytosterol cosolvents or dispersants. By the method and culture medium provided by the invention, the rate of transformation from phytosterols to ADD is increased, the concentration of substrates is increased to increase the transformation efficiency, and the specificity of transformation is improved and the separation and purification process is simplified.
Description
Technical field
The present invention relates to pharmacy field, particularly, method and used medium that to the present invention relates to a plant sterols be ADD through microbial transformation.
Background technology
Rostadienedione (being called for short ADD) is a kind of important intermediate of synthesizing steroid hormone medicine, by it, can be synthesized and be comprised more than 10 kinds such as dexamethasone, prednisolone, Betamethasone Valerate widely used hormonal medicaments clinically.The chemical structural formula of ADD is as shown in formula I below.
Formula I
Synthetic rostadienedione has two kinds of methods at present: the one, and with dioscin, be starting raw material, through 11 step chemosynthesis reactions, obtain.The 2nd, by plant sterol, through microbial transformation, directly obtain ADD.Wherein microbial transformation has reaction conditions gentleness, the aspect advantages such as energy-conserving and environment-protective, determine whether it has economy and comprise reaction substrate concentration, transformation efficiency and conversion specificity, transform specificity and refer to ADD and by product AD (androstane-4-alkene-3 in conversion process, 17-diketone) ratio, its ratio is higher, illustrates that it transforms specificity better.US Patent No. P4345029 has described employing mycobacterium mutation (Mycobacterium phlei) conversion and has obtained AD and ADD; US Patent No. P7259005 has described and has adopted another kind of mycobacterium (Mycobacterium fortuitum) transformation phytosterin to obtain AD and ADD, but its transformation efficiency and specificity are all not high, transformation efficiency is the highest only 64%, and in conversion secondary fermentation liquid, the maximum concentration of AD and ADD only has 2177mg/L.From economic angle, calculate, above-mentioned technical indicator shows that it is without industrial value.
How to improve the economy that directly obtains ADD through microbial transformation by plant sterol and become its key of instead of chemical synthesis method completely.
Summary of the invention
In order to solve above-mentioned problems of the prior art, the invention provides the fermention medium using in a kind of method that is ADD through microbial transformation at plant sterol.The present invention also provides the method that a plant sterols is ADD through microbial transformation.
Particularly, the invention provides:
(1) fermention medium using in the method that is ADD at plant sterol through microbial transformation, it includes the following material that dissolves or be dispersed in water:
Glucose: 0.1-100g/L,
Analysis for soybean powder: 10-100g/L,
Potassium primary phosphate: 0.5-10g/L,
Calcium carbonate: 0.1-10g/L,
Ferrous sulfate: 0.001-0.01g/L,
Manganous chloride tetrahydrate: 0.0001-0.001g/L, and
Plant sterol: 10-100g/L;
The pH of described fermention medium is 5.0-8.0; And, in described fermention medium, do not contain plant sterol solubility promoter or dispersion agent.
(2), according to the fermention medium (1) described, it includes the following material that dissolves or be dispersed in water:
Glucose: 25g/L,
Analysis for soybean powder: 40g/L,
Potassium primary phosphate: 2g/L,
Calcium carbonate: 1g/L,
Ferrous sulfate: 0.0051g/L,
Manganous chloride tetrahydrate: 0.0005g/L, and
Plant sterol: 50g/L;
The pH of described fermention medium is 7.2; And, in described fermention medium, do not contain plant sterol solubility promoter or dispersion agent.
(3) according to the fermention medium (1) or (2) described, wherein said plant sterol solubility promoter or dispersion agent are polyoxyethylene glycol, Trisun Oil R 80 or ethylene glycol.
The method that (4) one plant sterols are ADD through microbial transformation, the method comprises, uses according to the fermention medium described in any one in (1) to (3) when fermentation culture mycobacterium (Mycobacterium).
(5) according to the method described in (4), in the method, when reaching 30-50 hour age, seeding tank kind inoculates, and inoculum size is 5-10% (v/v); Then, at the temperature of 28-32 ℃, ferment, and dissolved oxygen is controlled as 35-80%.
(6) according to the method described in (5), in the method, when reaching 42 hours age, described seeding tank kind inoculates, and inoculum size is 5% (v/v); Then, at the temperature of 30 ℃, ferment, and dissolved oxygen is controlled as 35-45%.
The present invention is devoted to improve plant sterol and by bio-transformation, synthesizes the method for rostadienedione transformation efficiency.Through the inventor, study discovery, the key link that improves transformation efficiency relates to the design of zymotechnique.The inventor finds fermention medium and fermenting process to comprise that the processing parameters such as pH, mixing speed, ventilation, feed supplement, temperature are optimized design, can significantly improve transformation efficiency.Zymotechnique after optimization, fermentation substrate concentration can reach 50g/L, and transformation efficiency can reach 70%, and after fermentation ends, in fermented liquid, the weight ratio of ADD and AD is 98: 2, is significantly higher than the technical indicator of open report.
Embodiment
Below the invention will be further described for the description by embodiment, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modifications or improvement, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
Fermention medium of the present invention includes the following material that dissolves or be dispersed in water: glucose: 0.1-100g/L, is preferably 25g/L; Analysis for soybean powder: 10-100g/L, is preferably 40g/L; Potassium primary phosphate: 0.5-10g/L, is preferably 2g/L; Calcium carbonate: 0.1-10g/L, is preferably 1g/L; Ferrous sulfate: 0.001-0.01g/L, is preferably 0.0051g/L; Manganous chloride tetrahydrate: 0.0001-0.001g/L, is preferably 0.0005g/L; And plant sterol: 10-100g/L, is preferably 50g/L; The pH of described fermention medium is 5.0-8.0, is preferably 7.2; And, in described fermention medium, do not contain plant sterol solubility promoter or dispersion agent, particularly polyoxyethylene glycol, Trisun Oil R 80, ethylene glycol, more particularly Trisun Oil R 80.
In this article, analysis for soybean powder, also referred to as soyflour, is that soya bean is obtained through pulverizing preparation.In the present invention, analysis for soybean powder is preferably by the fine powder of 80 mesh sieves.
In this article, plant sterol is for mainly comprising the mixture of β-sitosterol, Stigmasterol and campesterol.Wherein, the parent nucleus of β-sitosterol, Stigmasterol and campesterol is identical, and its structural formula is respectively:
The structural formula of the structural formula Stigmasterol of β-sitosterol
The structural formula of campesterol
Plant sterol can be purchased from blue sky, Xi'an company.
In this article, plant sterol solubility promoter or dispersion agent refer to the auxiliary agent in order to increase the solubleness of plant sterol in fermention medium or degree of scatter and to add, and comprise polyoxyethylene glycol, Trisun Oil R 80, ethylene glycol etc.
The method that plant sterol of the present invention is ADD through microbial transformation comprises, uses fermention medium of the present invention when fermentation culture mycobacterium (Mycobacterium).
Preferably, in the method for the invention, when seeding tank kind reaches 30-50 hour age, inoculate, inoculum size is 5-10% (v/v); Then, at the temperature of 28-32 ℃, ferment, and dissolved oxygen is controlled as 35-80%.More preferably, when described seeding tank kind reaches 42 hours age, inoculate, inoculum size is 5% (v/v); Then, at the temperature of 30 ℃, ferment, and dissolved oxygen is controlled as 35-45%.
In this article, dissolved oxygen refers to the percent value (DO value) of the oxygen concn in fermented liquid when oxygen concn in the fermented liquid of fermenting process is initial with fermentation.
The zymotechnique technical characterictic the present invention relates to is described below:
(1) the present invention has improved bio-transformation system.According to existing bibliographical information, plant sterol is slightly soluble in water, must increase its solubleness in water, could improve transformation efficiency.Traditional reaction system is that although transformation efficiency has reached more than 80%, substrate plant sterol add-on is 0.1-0.8% only by technology such as aqueous two-phase system and cyclodextrin embeddings, and in fermented liquid, seldom, fermentation unit is very low for ADD output, still cannot suitability for industrialized production.In prior art, some employing vegetables oil, as Trisun Oil R 80 disperses, increase substrate plant sterol add-on.In fact, later stage purifying is very difficult, because product A DD dissolves readily in vegetables oil, need to solve with column chromatography.The present invention develops a kind of bio-transformation system, in substratum, do not add any plant sterol solubility promoter or dispersion agent, allow plant sterol be suspended in substratum and directly transform, guaranteeing under transformation efficiency prerequisite, again for separation and purification of products after fermentation ends offers convenience.
(2) the present invention has optimized fermention medium.To comprise that the nutritive elements such as carbon source, nitrogenous source, metal ion carry out excellent sieve, determined take glucose as carbon source, using ammonium nitrate as nitrogenous source, add calcium salt, magnesium chloride etc. as metal ion reagent, can promote that plant sterol is converted into ADD.
(3) the present invention has optimized the critical process control parameter in fermenting process.To comprising the fermentation key control parameter optimizations such as kind of an age, kind amount, leavening temperature, pH, dissolved oxygen, foam control method, improved the transformation efficiency of ADD.
In the fermented liquid being obtained by the present invention, the measuring method of ADD is as follows:
Condition determination:
Chromatographic column: Kromasil C18 5 μ m 200 * 4.6mm (can purchased from Japanese Shimadzu company);
Moving phase: methyl alcohol: water=80: 20 (V/V);
Flow velocity: 1.0ml/min;
Detect wavelength: 242nm.
The mensuration of typical curve: the ADD standardized solution (50-500 μ g/ml) of preparing a series of different concns, the filtered liquid 20 μ l that get respectively each concentration standard solution inject high performance liquid chromatograph, measure its absorption peak area, then concentration (Y) and absorption peak area (X) are carried out to one-variable linear regression, obtain one-variable linear regression equation.
ADD cubage method: the methanol solution of testing sample being made to suitable concn (50-500 μ g/ml), filtering with microporous membrane with 0.45 μ m, accurately draw filtrate 20 μ l and inject HPLC, record color atlas, by in the above-mentioned regression equation of ADD peak area substitution, calculate ADD amount in sample.
The mycobacteria strain using in following embodiment 1 derives from American Type Culture Collecti (ATCC), and this bacterial classification is submitted permanent preservation in nineteen fifty-nine, and preserving number is NCTC1542, and current granting without any restrictions is to the public.
Embodiment 1: the fermentation that plant sterol bio-transformation is ADD, extraction process
ADD comes from mycobacterium nutrient solution, and its cultivation, extraction and purification step are as follows:
(1) seed culture
The proportioning of the substratum of first order seed bottle, secondary seed tank is identical with compound method, and liquid nutrient medium forms as shown in table 1.
Table 1 seed culture medium composition (gram/every liter)
During preparation, according to proportioning, various starting material are fully dissolved, stir, natural pH value is 6.7 ± 0.2, uses 40% NaOH solution adjusting pH value to 7.0, is then warming up to 118-121 ℃, and steam sterilizing 30 minutes, is cooled to culture temperature and waits to inoculate.
Use inclined-plane directly to inoculate, whole lawns on 1 inclined-plane of scraping, are inoculated in first order seed bottle (250ml/750ml triangular flask), 30 ± 0.5 ℃ of the temperature that controls environment after inoculation, and 220rpm shaking culture, conventionally cultivates 48h and gets final product transferred species.
After first order seed maturation, be transferred in secondary seed tank, inoculum size 5% (V/V), after inoculation, tank temperature control is 30 ± 1 ℃, and tank pressure is controlled 0.04-0.05MPa, regulate the omnidistance DO value of seeding tank rotating speed and air flow control more than 60%, conventionally cultivate 42 ± 6h and can reach transferred species index.
(2) fermentation culture
In fermention medium, contain the organism such as glucose, soybean cake powder, plant sterol, its content is between 0.1-10% (W/V), add in right amount the inorganic salt such as potassium primary phosphate, calcium carbonate, iron vitriol, Manganous chloride tetrahydrate, regulate pH between 6-8 simultaneously.
Fermention medium forms as shown in table 2.
Table 2 fermention medium composition (gram/every liter)
During preparation, according to proportioning, various starting material are fully dissolved, stir, natural pH value is 6.7 ± 0.2, uses 40% NaOH solution adjusting pH value to 7.0, is then warming up to 118-121 ℃, and steam sterilizing 30 minutes, is cooled to culture temperature and treats transferred species.
Inoculum size according to 5%, cultivates mature seed by secondary seed tank and proceeds in fermentor tank, and controlled fermentation process temperature is 30 ℃ ± 1 ℃, and dissolved oxygen is controlled at more than 35%, cultivates 120 hours, and in fermented liquid, ADD content is 30g/L.The weight ratio of ADD: AD is 98: 2.
(3) extract
Get 100ml fermented liquid in 100ml centrifuge tube, centrifugal 10 minutes of 3000rpm.Supernatant liquor is with 50ml ethyl acetate extraction three times; Thalline extracts three times with 50ml ethyl acetate backflow, after filtration, and merging filtrate and extraction supernatant liquor, 60 ℃ ,-0.09MPa is evaporated to dry, obtains the about 2.5g of ADD crude product.
ADD crude product 2.5g 20ml acetic acid ethyl dissolution, adds 0.1g gac, reflux 30 minutes, and filtered while hot, filtrate is concentrated into 10ml, cools to 4 ℃, is incubated 10 hours, filters, dryly obtains ADD product, and product HPLC purity is greater than 98.0%.
Claims (4)
1. the fermention medium using in the method that is ADD at plant sterol through microbial transformation, it is by being comprised of the following material dissolving or be dispersed in water:
Glucose: 25g/L,
Analysis for soybean powder: 40g/L,
Potassium primary phosphate: 2g/L,
Calcium carbonate: 1g/L,
Ferrous sulfate: 0.0051g/L,
Manganous chloride tetrahydrate: 0.0005g/L, and
Plant sterol: 50g/L;
The pH of described fermention medium is 7.2.
2. the method that a plant sterols is ADD through microbial transformation, the method comprises, when fermentation culture mycobacterium (Mycobacterium), use fermention medium according to claim 1, the mycobacterium (Mycobacterium) that wherein said mycobacterium (Mycobacterium) is NCTC1542 for preserving number.
3. according to the method described in claim 2, in the method, when reaching 30-50 hour age, seeding tank kind inoculates, and inoculum size is 5-10% (v/v); Then, at the temperature of 28-32 ℃, ferment, and dissolved oxygen is controlled as 35-80%.
4. according to the method described in claim 3, in the method, when reaching 42 hours age, described seeding tank kind inoculates, and inoculum size is 5% (v/v); Then, at the temperature of 30 ℃, ferment, and dissolved oxygen is controlled as 35-45%.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103789385A (en) * | 2012-10-31 | 2014-05-14 | 天津金耀集团有限公司 | Method for preparing androstane 4-alkene-3,17-diketone by sodium dodecyl sulfate through auxiliary of phytosterol composition |
| CN104561213A (en) * | 2013-10-26 | 2015-04-29 | 山东方明药业集团股份有限公司 | Method for preparing 4-androstenedione by virtue of microbial conversion |
| CN103923966A (en) * | 2014-03-25 | 2014-07-16 | 河北众盛生物科技有限公司 | Method of preparing 4-androstenedione by fermentatively degrading phytosterol by virtue of single water phase system |
| CN110656147B (en) * | 2019-10-16 | 2023-05-05 | 湖南新合新生物医药有限公司 | Biological dehydrogenation method of androstenedione C1,2 position |
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Address after: 100871 Beijing, Haidian District Road, building No. 298, founder of the building, Zhongguancun, layer 5 Patentee after: Peking Founder Group Co., Ltd. Patentee after: CHONGQING DAXIN PHARMACEUTICAL CO., LTD. Patentee after: PKU HEALTHCARE INDUSTRY GROUP Address before: 100871 Beijing, Haidian District Road, building No. 298, founder of the building, Zhongguancun, layer 5 Patentee before: Peking Founder Group Co., Ltd. Patentee before: Chongqing Daxin Pharmaceuticals Co., Ltd. of PKU International HealthCare Group Patentee before: PKU International Healthcare Group Co., Ltd. |
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