CN110643649B - Method for preparing A ring degradation product by transforming plant sterol by growing cells - Google Patents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
Abstract
The invention discloses a method for preparing an A-ring degradation product by transforming plant sterol by growing cells, belonging to the technical field of steroid preparation. The invention comprises the steps of slant culture, liquid level seed culture, growing cell transformation, separation and extraction and refining. The invention takes the phytosterol as the raw material to produce the A ring degradation product, the raw material is easy to obtain, and the production cost is reduced; compared with the conventional method for converting the A ring degradation product, the method for preparing the A ring degradation product by converting the phytosterol by the growing cells has the advantages of high product yield, high HPLC purity, short conversion time, small pollution, and production cost saving and economic benefit improvement in actual production.
Description
Technical Field
The invention belongs to the technical field of steroid preparation, and particularly relates to a method for preparing an A-ring degradation product by transforming plant sterols by growing cells.
Background
Recent studies on the preparation of steroid intermediates using microbial transformation of phytosterols have been widely reported and studied, and the related reports were first traced back to 1937 in two patents ber.70470 and ber.702079 issued by Mamoli and vercelone, which disclose the reduction of 17-keto-steroids to 17- β -hydroxysteroids by yeast fermentation. Thereafter, peterson and Murray disclose a method of 11-a-hydroxylation using rhizopus fungi to produce progesterone in U.S. Pat. No. N0.2602769. In 1972, kraych et al disclosed in U.S. Pat. No. 5, 0.3684657 a method for preparing 4-AD, ADD by degrading the fatty chain at position 17 with Mycobacterium sp.NRRL B-3683. In 1973, marsheck et al disclosed in U.S. Pat. No. 5, 0.3759791 that 4-AD was prepared from a steroid starting material having 8 or more carbon atoms at the 17-position of cholestane or the like using Mycobacterium sp.NRRL B-3805.
The degradation product of the ring A is an important intermediate for synthesizing estrone, estradiol and derivatives thereof, but the ring A and the ring B of the steroid nucleus are open in the conversion process, so that the degradation is very easy, and the product is difficult to accumulate, so that the industrialization standard is difficult to reach. At present, the A ring degradation product is mainly produced by converting the plant sterol in an oil phase by utilizing microorganisms, for example, the patent with the patent number of CN104404099A discloses a method for producing the A ring degradation product by fermenting the plant sterol, soybean oil is used in fermentation conversion, extraction is difficult, waste oil is difficult to treat and the environmental impact is large.
Disclosure of Invention
Therefore, the invention aims to provide a method for preparing an A-ring degradation product by transforming plant sterol by growing cells, which can reduce waste oil generation, shorten transformation time and improve yield and purity of the A-ring degradation product.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention provides a method for preparing an A-ring degradation product by transforming plant sterol by growing cells, which comprises the following steps:
(1) Transformation of growing cells: inoculating Mycobacterium fortuitum (Mycobacterium fortuitum) NK-XHX-103 strain into a transformation culture medium after slant culture and liquid seed culture to obtain a fermentation product;
(2) Separating and extracting: filtering after conversion is completed, regulating the pH value of the filtrate to be 2.0, extracting and layering by using chloroform, extracting a water layer by using chloroform for 2 times, layering, merging chloroform layers and concentrating under reduced pressure to obtain a crude product of the A ring degradation product;
(3) Refining: and refining and purifying the crude product of the A ring degradation product to obtain the A ring degradation product.
Preferably, the collection number of Mycobacterium fortuitum (Mycobacterium fortuitum) NK-XHX-103 is CCTCC M2013543.
Preferably, the method for culturing NK-XHX-103 strain seeds in the step (1) comprises the following steps:
(1) Slant culture: the formula of the culture medium comprises: 0.1-10g/L peptone, 0.1-10g/L yeast extract, 0.1-10g/L glucose, 0.1-10g/L disodium hydrogen phosphate and 20g/L agar; ph=7.5-8.0, sterilization at 121 ℃ for 30 minutes;
(2) Primary seed culture: the formula of the culture medium comprises: 0.1-10g/L peptone, 0.1-10g/L yeast extract, 0.1-10g/L glucose and 0.1-10g/L disodium hydrogen phosphate, and shake culturing at 30 deg.C and 200rpm for 48 hr after inoculation;
(3) Secondary seed culture: the formula of the culture medium comprises: 0.1-10g/L peptone, 0.1-10g/L yeast extract, 0.1-10g/L glucose, 0.1-10g/L disodium hydrogen phosphate, pH=7.5-8.0, and sterilizing at 121deg.C for 30 min; inoculating the primary seed solution into a secondary seed culture medium according to the volume ratio of 10%, and performing shake culture at 30 ℃ and 200rpm for 48 hours after inoculation;
preferably, the formulation of the fermentation base medium described in step (1): corn steep liquor 1-6%, sodium nitrate 0.1-0.6%, diammonium phosphate 0.01-0.08%, PPE0.1-0.2%, phytosterol 1-10%, hydroxypropyl cyclodextrin 0.1-40%; sterilizing at 121deg.C for 30 min.
Preferably, the phytosterols are crushed to 200 mesh.
Preferably, the separation and extraction method in the step (4) specifically comprises the following steps: filtering or centrifuging the converted bacterial liquid, removing bacterial cells in the bacterial liquid, adding 20% sulfuric acid into the filtrate to adjust the pH to 2.0, and stirring for 30 minutes; adding 20% volume of chloroform, stirring and extracting for 30 min, standing for more than 8h, and layering; separating the lower chloroform layer, concentrating under reduced pressure to dry; adding chloroform with volume of 20% of fermentation liquor into the upper layer water layer, extracting for 2 times again, mixing the obtained chloroform layers, concentrating under reduced pressure, adding water 0.05V (relative to fermentation liquor), adding dry chloroform, and concentrating into paste; adding 4V (relative to the substrate amount) toluene into the concentrated substance, heating to 60 ℃, concentrating under reduced pressure to a small volume, cooling to 0-4 ℃, filtering, leaching a filter cake with a small amount of toluene, suction-filtering, digging out the filter cake, and drying at 70 ℃.
Preferably, the refining method in the step (3) specifically comprises the following steps: adding methanol 2V (relative to the substrate amount) into the crude product obtained by extraction, heating to 60 ℃ and stirring for decoloring for 2 hours, filtering, and washing carbon with a small amount of methanol; concentrating at 60deg.C under reduced pressure to dry, adding a small amount of water to dry residual methanol, adding 4V toluene, concentrating under reduced pressure, taking out water, concentrating to about 1V, cooling to 0-4deg.C, crystallizing for 2 hr, vacuum filtering, leaching, and oven drying at 70deg.C.
The invention has the beneficial effects that:
(1) The method for converting the A ring degradation product of the phytosterol without oil has the advantages of short conversion time, high product yield and HPLC purity and good economic benefit, and is more suitable for industrial production.
(2) The preparation method of the invention can also reduce the use of chemical reagents, reduce the generation of pollutants and is beneficial to environmental protection.
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.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The phytosterol used in the examples below was beta-sitosterol, available from Vickers biotechnology Co., ltd. Mycobacterium fortuitum (Mycobacterium fortuitum) NK-XHX-103 with accession number CCTCC M2013543 is deposited with China center for type culture Collection.
The invention is further illustrated by the following examples, which are not intended to be limiting.
Example 1 seed culture
(1) Slant culture
The formula comprises the following components: 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 121deg.C for 30 min. After solidification, the mycobacterium is inoculated under aseptic condition.
After inoculation, culturing for 4 days at 30 ℃, and preserving in a refrigerator at 4 ℃ for not more than 1 month.
(2) Shake flask seed culture
The formula comprises the following components: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, and pH=7.5-8.0. Sterilizing at 121deg.C for 30 min. Cooled to room temperature.
Primary culture: inoculation under sterile conditions, inoculum size: 1 ring of bevel thallus is scraped every 100 ml. After inoculation, shaking culture was carried out at 30℃and 200rpm for 48 hours.
Secondary culture: inoculation under sterile conditions, inoculum size: 10%. After inoculation, shaking culture was carried out at 30℃and 200rpm for 48 hours.
EXAMPLE 2 fermentation conversion
(1) Seed culture
Seed culture was performed as in example 1;
(2) Transformation
The formula of the transformation medium comprises: corn steep liquor 5.4%, sodium nitrate 0.54%, diammonium phosphate 0.06%, PPE 0.1%,200 mesh phytosterol 1%, hydroxypropyl cyclodextrin 0.1%, pH=7.5-8.0. 10 liter after inoculation 6 liter of the canned sample.
Sterilizing at 121 ℃ for 30 minutes, cooling to room temperature, inoculating under aseptic condition, and inoculating amount: 20%.
Shake flask transformation conditions: the conversion was monitored by TLC plates at 30℃and 200rpm for 160h, and after conversion was completed, samples were taken and fed to liquid phase detection with a product yield of 33.1%.
(3) Extraction of
After conversion, inactivating for 30 minutes at 80 ℃, cooling to 30 ℃, filtering, and leaching the filter cake with a small amount of clean water. Adding 20% sulfuric acid into the filtrate to adjust pH to about 2.0, stirring for 30 min, adding 1.2L chloroform, stirring and extracting for 30 min, standing for 2h, and layering. The upper aqueous phase was extracted 2 more times with chloroform, each time with 1.2 liters of chloroform; separating out the lower chloroform layer, concentrating under reduced pressure to small volume, adding 300 ml of water, carrying out dry chloroform, concentrating to paste, adding 240 ml of toluene, concentrating under reduced pressure at 60-80 ℃, carrying out water removal from the system, cooling to 0-4 ℃, slowly stirring, growing crystals for 2h, and centrifuging. The filter cake is rinsed with a small amount of cold toluene at 4 ℃, centrifugally dried, discharged and dried at 70 ℃ to obtain white crystals, and the normalized content of the liquid phase is 98.54%.
(4) Refining
120 ml of methanol and 3 g of active carbon are added into the crude product of the A ring degradation product obtained by extraction, the temperature is raised to 60 ℃, the mixture is stirred and decolored for 2 hours, filtered, and the carbon is washed by a small amount of methanol; concentrating at 60deg.C under reduced pressure to dry, adding a small amount of water to dry residual methanol, adding 240 ml of toluene, concentrating under reduced pressure, taking out water, concentrating to about 60 ml, cooling to 0-4deg.C, crystallizing for 2 hr, vacuum filtering, leaching, and oven drying at 70deg.C. White needle-like crystals were collected, with a normalized content of 99.11%.
EXAMPLE 3 fermentation conversion
(1) Seed culture
Seed culture was performed as in example 1;
(2) Transformation
The formula of the transformation medium comprises: corn steep liquor 1%, sodium nitrate 0.1%, diammonium phosphate 0.01%, PPE 0.1%,200 mesh phytosterol 2%, hydroxypropyl cyclodextrin 1%, pH=7.5-8.0. 10 liter after inoculation 6 liter of the canned sample. Inoculating the cultured secondary seeds into sterilized 10-liter tank transformation culture medium, wherein the inoculation amount is 20%, and starting transformation.
Conversion conditions: 29-30 ℃,200rpm, 96h of conversion time, TLC (thin layer chromatography) plate monitoring conversion condition, sampling and sending liquid phase detection after conversion is finished, and the product yield is 32.5%.
(3) Extraction and refining
The post-treatment was carried out according to the extraction and purification method of example 2, except that the chloroform extraction amount was kept the same as that of example 2, and the other reagents were used in an amount 2 times that of example 2 to obtain white needle-like crystals, the normalized content of the liquid phase was 99.32%.
EXAMPLE 4 fermentation conversion
(1) Seed culture
Seed culture was performed as in example 1;
(2) Transformation
The formula of the transformation medium comprises: corn steep liquor 5.4%, sodium nitrate 0.54%, diammonium phosphate 0.06%, PPE 0.1%,200 mesh phytosterol 4%, hydroxypropyl cyclodextrin 2%, pH=7.5-8.0. 10 liter after inoculation 6 liter of the canned sample. Inoculating the cultured secondary seeds into sterilized 10-liter tank transformation culture medium, wherein the inoculation amount is 20%, and starting transformation.
Shake flask transformation conditions: the conversion time is 120h at 30 ℃ and 200rpm, the TLC plate monitors the conversion condition, and after the conversion is finished, the sample is taken and sent to liquid phase detection, and the product yield is 33.1%.
(3) Extraction and refining
The post-treatment was carried out according to the extraction and purification method of example 2, except that the chloroform extraction amount was kept the same as that of example 2, and the other reagents were used in an amount 4 times that of example 2 to obtain white needle-like crystals, the normalized content of the liquid phase was 99.11%.
EXAMPLE 5 fermentation conversion
(1) Seed culture
Seed culture was performed as in example 1;
(2) Transformation
The formula of the transformation medium comprises: corn steep liquor 6%, sodium nitrate 0.6%, diammonium phosphate 0.08%, PPE 0.2%,200 mesh phytosterol 10%, hydroxypropyl cyclodextrin 5%, pH=7.5-8.0. 10 liter after inoculation 6 liter of the canned sample. Inoculating the cultured secondary seeds into sterilized 10-liter tank transformation culture medium, wherein the inoculation amount is 20%, and starting transformation.
Conversion conditions: 29-30 ℃,200rpm, conversion time 186h, TLC plate monitoring conversion, sampling and liquid phase detection after conversion is finished, and product yield is 31.3%.
(3) Extraction and refining
The post-treatment was carried out according to the extraction method of example 2, except that the chloroform extraction amount was kept the same as that of example 2, and the other reagents were 10 times as much as that of example 2 to obtain white crystals, with a normalized liquid phase content of 98.35%.
EXAMPLE 6 fermentation conversion
(1) Seed culture
Seed culture was performed as in example 1;
(2) Transformation
The formula of the transformation medium comprises: corn steep liquor 1%, sodium nitrate 0.1%, diammonium phosphate 0.01%, PPE 0.1%,200 mesh phytosterol 1%, hydroxypropyl cyclodextrin 0.25%, pH=7.5-8.0. 10 liter after inoculation 6 liter of the canned sample. Inoculating the cultured secondary seeds into sterilized 10-liter tank transformation culture medium, wherein the inoculation amount is 20%, and starting transformation.
Conversion conditions: 29-30 ℃,200rpm, 144h of conversion time, TLC plate monitoring conversion condition, sampling and liquid phase detection after conversion is finished, and the product yield is 32.5%.
(3) Extraction of
Post-treatment was performed according to the extraction method of example 2 to obtain white crystals with a normalized liquid phase content of 99.31%.
EXAMPLE 7 fermentation conversion
(1) Seed culture
Seed culture was performed as in example 1;
(2) Transformation
The formula of the transformation medium comprises: corn steep liquor 1%, sodium nitrate 0.1%, diammonium phosphate 0.01%, PPE 0.1%,200 mesh phytosterol 2%, hydroxypropyl cyclodextrin 0.5%, pH=7.5-8.0. 10 liter after inoculation 6 liter of the canned sample. Inoculating the cultured secondary seeds into sterilized 10-liter tank transformation culture medium, wherein the inoculation amount is 20%, and starting transformation.
Conversion conditions: 29-30 ℃,200rpm, conversion time 160h, TLC plate monitoring conversion condition, sampling and liquid phase detection after conversion is finished, and product yield is 32.5%.
(3) Extraction and refining
Post-treatment was performed according to the extraction and purification method of example 3 to obtain white needle-like crystals with a normalized liquid phase content of 99.14%.
Example 8 fermentation conversion
(1) Seed culture
Seed culture was performed as in example 1;
(2) Transformation
The formula of the transformation medium comprises: corn steep liquor 5.4%, sodium nitrate 0.54%, diammonium phosphate 0.06%, PPE 0.1%,200 mesh phytosterol 4%, hydroxypropyl cyclodextrin 1%, pH=7.5-8.0. 10 liter after inoculation 6 liter of the canned sample. Inoculating the cultured secondary seeds into sterilized 10-liter tank transformation culture medium, wherein the inoculation amount is 20%, and starting transformation.
Sterilizing at 121 ℃ for 30 minutes, cooling to room temperature, inoculating under aseptic condition, and inoculating amount: 20%.
Shake flask transformation conditions: conversion was monitored by TLC plates at 30℃at 200rpm for 144h, and after conversion was completed, samples were taken and fed to liquid phase detection with a product yield of 33.1%.
(3) Extraction and refining
Post-treatment was carried out according to the extraction and purification method of example 4 to obtain white needle-like crystals having a normalized liquid phase content of 98.73%.
EXAMPLE 9 fermentation conversion
(1) Seed culture
Seed culture was performed as in example 1;
(3) Transformation
The formula of the transformation medium comprises: corn steep liquor 6%, sodium nitrate 0.6%, diammonium phosphate 0.08%, PPE 0.2%,200 mesh phytosterol 10%, hydroxypropyl cyclodextrin 2.5%, pH=7.5-8.0. 10 liter after inoculation 6 liter of the canned sample. Inoculating the cultured secondary seeds into sterilized 10-liter tank transformation culture medium, wherein the inoculation amount is 20%, and starting transformation.
Conversion conditions: 29-30 ℃,200rpm, 240h of conversion time, monitoring conversion condition by TLC (thin layer chromatography) plate, sampling and sending to liquid phase detection after conversion is finished, and the product yield is 31.3%.
(3) Extraction and refining
The post-treatment was carried out according to the extraction method of example 5 to obtain white crystals with a normalized content of 98.41% in the liquid phase.
EXAMPLE 10 fermentation conversion
(1) Seed culture
Seed culture was performed as in example 1;
(4) Transformation
The formula of the transformation medium comprises: corn steep liquor 6%, sodium nitrate 0.6%, diammonium phosphate 0.08%, PPE 0.2%,200 mesh phytosterol 10%, hydroxypropyl cyclodextrin 40%, pH=7.5-8.0. 10 liter after inoculation 6 liter of the canned sample. Inoculating the cultured secondary seeds into sterilized 10-liter tank transformation culture medium, wherein the inoculation amount is 20%, and starting transformation.
Conversion conditions: 29-30 ℃,200rpm, 16 h conversion time, TLC plate monitoring conversion condition, sampling and liquid phase detection after conversion is finished, and product yield is 32.4%.
(3) Extraction and refining
Post-treatment was performed according to the extraction method of example 5 to obtain white crystals with a normalized liquid phase content of 98.23%.
Claims (3)
1. A method for preparing an A-ring degradation product by transforming plant sterol by growing cells, comprising the following steps:
(1) Transformation of growing cells: inoculating Mycobacterium fortuitum (Mycobacterium fortuitum) NK-XHX-103 strain into a transformation culture medium after slant culture and liquid seed culture to obtain a fermentation product;
(2) Separating and extracting: filtering after conversion is completed, extracting a water layer with chloroform for 2-3 times, and concentrating chloroform under reduced pressure to obtain a crude product of the A ring degradation product;
(3) Refining: refining and purifying the crude product of the A ring degradation product to obtain an A ring degradation product;
the preservation number of the mycobacterium fortuitum (Mycobacterium fortuitum) NK-XHX-103 is CCTCC M2013543;
the method for culturing NK-XHX-103 strain seeds in the step (1) comprises the following steps:
(1) Slant culture: the formula of the culture medium comprises: 0.1-10g/L peptone, 0.1-10g/L yeast extract, 0.1-10g/L glucose, 0.1-10g/L disodium hydrogen phosphate and 20g/L agar; ph=7.5-8.0, sterilization at 121 ℃ for 30 minutes;
(2) Primary seed culture: the formula of the culture medium comprises: 0.1-10g/L peptone, 0.1-10g/L yeast extract, 0.1-10g/L glucose and 0.1-10g/L disodium hydrogen phosphate, and shake culturing at 30 deg.C and 200rpm for 48 hr after inoculation;
(3) Secondary seed culture: the formula of the culture medium comprises: 0.1-10g/L peptone, 0.1-10g/L yeast extract, 0.1-10g/L glucose, 0.1-10g/L disodium hydrogen phosphate, pH=7.5-8.0, and sterilizing at 121deg.C for 30 min; inoculating the primary seed solution into a secondary seed culture medium according to the volume ratio of 10%, and performing shake culture at 30 ℃ and 200rpm for 48 hours after inoculation;
the formula of the transformation medium comprises: corn steep liquor 1-6%, sodium nitrate 0.1-0.6%, diammonium phosphate 0.01-0.08%, PPE0.1-0.2%, phytosterol 1-10%, hydroxypropyl cyclodextrin 0.1-40%; sterilizing at 121deg.C for 30 min.
2. The method for producing a degradation product of a ring by transforming plant sterol with growing cells according to claim 1, wherein the plant sterol is pulverized to 200 mesh.
3. The method for preparing an A-ring degradation product by transforming plant sterols with growing cells according to claim 1, wherein the separation and extraction method in the step (2) is specifically as follows: inactivating, cooling and filtering the converted bacterial liquid, leaching a filter cake by using clear water, adjusting the pH value of the filtrate to 2.0, adding 0.2V chloroform for extraction, standing for 2h and layering; separating out the lower chloroform layer, concentrating under reduced pressure, adding 0.5V toluene, heating to 60-80deg.C, concentrating under reduced pressure, cooling to 0-4deg.C, stirring for 2 hr, centrifuging, eluting the filter cake with 4deg.C cold toluene, centrifuging again, drying, discharging, and oven drying to obtain solid product.
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| CN104388515A (en) * | 2014-11-28 | 2015-03-04 | 江西赣亮医药原料有限公司 | Method for producing 11alpha-OH-ADD from phytosterol by mixed fermentation |
| CN105886418A (en) * | 2014-12-01 | 2016-08-24 | 陈美华 | Application of mycobacterium fortuitum to fermentation production of Biaobochun |
| CN109652338A (en) * | 2019-01-24 | 2019-04-19 | 天津科技大学 | The mycobacterium fortutitum of 9 α-OH-AD of high yield and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103756940A (en) * | 2014-01-22 | 2014-04-30 | 湖南新合新生物医药有限公司 | Mycobacterium fortuitum and application thereof in fermentation production of delta-lactone |
| CN104388515A (en) * | 2014-11-28 | 2015-03-04 | 江西赣亮医药原料有限公司 | Method for producing 11alpha-OH-ADD from phytosterol by mixed fermentation |
| CN105886418A (en) * | 2014-12-01 | 2016-08-24 | 陈美华 | Application of mycobacterium fortuitum to fermentation production of Biaobochun |
| CN109652338A (en) * | 2019-01-24 | 2019-04-19 | 天津科技大学 | The mycobacterium fortutitum of 9 α-OH-AD of high yield and its application |
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