CN110699398B - Method for preparing A-ring degradation product by resting cell transformation of phytosterol - Google Patents
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Abstract
The invention discloses a method for preparing an A-ring degradation product by transforming plant sterol by resting cells, belonging to the technical field of steroid preparation. The invention comprises the steps of seed culture, resting cell transformation, separation, 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 plant sterol by resting cells has the advantages of high normalized content, short conversion time and small pollution, and can save production finished products and improve economic benefit 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 resting 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.
Mycobacterium fortuitum (Mycobacterium fortuitum) NK-XHX-103 is described in the patent No. CN 103756940A and is generally used only for converting phytosterol into delta-lactone, and the current general research is to improve the yield of delta-lactone by controlling the reaction conditions.
Disclosure of Invention
Therefore, the invention aims to provide a method for preparing an A-ring degradation product by transforming plant sterol by resting cells, which can reduce waste oil generation and transformation time and improve the 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 resting cells, which comprises the following steps:
(1) Resting cell transformation: inoculating Mycobacterium fortuitum (Mycobacterium fortuitum) NK-XHX-103 strain into a transformation culture medium after slant culture and seed culture, filtering and separating, fermenting and transforming the phytosterol, and fermenting and transforming the phytosterol to obtain a fermentation product;
(2) Separating and extracting: filtering after conversion is completed, extracting and layering filtrate by using chloroform after acidification, extracting a water layer by using chloroform for 2-3 times, and concentrating chloroform 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 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 ℃ and 200rpm for 48h 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;
(4) Seed pot culture: inoculating the secondary seed liquid into a seed tank according to the volume ratio of 10%; after inoculation, the culture was carried out at 30℃and 200rpm, at an air flow rate of 0.5VVM and a pot pressure of 0.05MPa for 48h to 72h.
Preferably, the collection number of Mycobacterium fortuitum (Mycobacterium fortuitum) NK-XHX-103 is CCTCC M2013543.
Preferably, after the seed culture in step (1) is completed, the seed bacterial liquid is filtered, and the filter cake is rinsed with 20mM phosphate buffer solution having ph=8.0 and suction-filtered.
Preferably, the formulation of the fermentation base medium described in step (1): 10-100g/L of phytosterol, 10-400g/L of hydroxypropyl cyclodextrin, 20-200g/L of bacteria, 5g/L of PPE and the balance of 20mM PBS (pH=8.0);
preferably, the phytosterols are crushed to 200 mesh.
Preferably, the extraction method in the step (2) specifically includes: 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 broth into the upper water layer, extracting for 2 times repeatedly, mixing the obtained chloroform layers, and concentrating under reduced pressure; the concentrated substance is added with 4V (relative to the substrate amount) toluene, the temperature is raised to 60 ℃, the pressure is reduced, the concentration is carried out to a small volume, the temperature is reduced to 0-4 ℃, the filtration is carried out, and the filter cake is leached by a small amount of toluene.
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 preparing the A-ring degradation product by transforming the plant sterol by resting cells has the advantages of short transformation time, high product yield and normalization content, realization of oil-free transformation, little pollution and good economic benefit, and is more suitable for industrial production.
(2) The invention adopts a resting cell transformation method, has shorter reaction route, omits a plurality of reaction steps and post-treatment steps, is beneficial to improving the reaction yield and reduces the intermediate loss. The preparation method of the invention can also reduce the use of chemical reagents 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.
The materials used in the examples below were all commercially available from conventional sources. 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 Mycobacterium fortuitum seed culture
And (3) strain: mycobacterium fortuitum (Mycobacterium fortuitum) NK-XHX-103
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, and pH=7.5-8.0.
Sterilizing at 121deg.C for 30 min. After solidification, inoculating 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 conversion
(1) First-stage 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, pH=7.5-8.0; sterilizing at 121deg.C for 30 min, and cooling to room temperature.
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.
(2) Two-stage 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.
Inoculation under sterile conditions, inoculum size: 10%. After inoculation, shaking culture was carried out at 30℃and 200rpm for 48 hours.
3. Seed culture in 50 liter jar
The secondary seed solution was inoculated into a 50 liter tank at a volume ratio of 10% and the post-inoculation volume was 30 liters.
Conversion conditions: 30 ℃,200rpm, air flow rate 0.5VVM, tank pressure 0.05MPa, culture time: 48h-72h.
EXAMPLE 2 fermentation conversion
(1) Seed culture was performed as in example 1;
(2) Seed separation
The cultivated seeds were filtered, and the filter cake was rinsed with 20mM phosphate buffer at ph=8.0 and suction filtered.
(3) Fermentation conversion in 10L tank
The conversion was carried out in a 10L tank. Metering volume: 6L. Post inoculation volume: 6L.
The formula of the transformation medium comprises: phytosterol: 10g/L, hydroxypropyl cyclodextrin: 10g/L, mycobacterium fortuitum: 20g/L, PPE 5g/L, balance 20mM PBS (pH=8.0).
Conversion conditions: 30 ℃,200rpm, air flow of 0.5vvm, tank pressure of 0.05MPa, conversion time of 88h, TLC plate monitoring conversion condition, and finishing conversion.
(4) Extraction of
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 1.2L of chloroform, stirring and extracting for 30 minutes, standing for more than 8 hours, and layering; separating the lower chloroform layer, concentrating under reduced pressure to dry; adding chloroform into the upper water layer, extracting for 2 times again, using 1.2L chloroform each time, and concentrating under reduced pressure; and adding 240 ml of toluene into the concentrated substance, heating to 60 ℃, concentrating under reduced pressure to a small volume, cooling to 0-4 ℃, filtering, and leaching a filter cake with a small amount of toluene to obtain a crude product of the A ring degradation product.
(5) 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. 15.2 g of white needle-like crystals are collected, and the normalized content is 99.11 percent.
EXAMPLE 3 fermentation conversion
(1) Seed culture was performed as in example 2;
(2) Seed separation
The cultivated seeds were filtered, and the filter cake was rinsed with 20mM phosphate buffer at ph=8.0 and suction filtered.
(3) Fermentation conversion in 10L tank
The conversion was carried out in a 10L tank. Metering volume: 6L. Post inoculation volume: 6L.
The formula of the transformation medium comprises: phytosterol: 20g/L, hydroxypropyl cyclodextrin: 40g/L, mycobacterium fortuitum: 40g/L, PPE 5g/L, balance 20mM PBS (pH=8.0).
Conversion conditions: 30 ℃,200rpm, air flow of 0.5vvm, tank pressure of 0.05MPa, conversion time of 104h, TLC plate monitoring conversion condition, and finishing conversion.
(4) Extracting, hydrolyzing and refining
The post-treatment was carried out according to the extraction, hydrolysis and purification method of example 2, and the amount of the other reagents was doubled, and the volume of reduced pressure concentration was doubled, except for chloroform used for the aqueous phase extraction. 31.5 g of white needle-like crystals are collected, and the normalized content is 98.99%.
EXAMPLE 4 fermentation conversion
(1) Seed culture was performed as in example 2;
(2) Seed separation
The cultivated seeds were filtered, and the filter cake was rinsed with 20mM phosphate buffer at ph=8.0 and suction filtered.
(3) Fermentation conversion in 10L tank
The conversion was carried out in a 10L tank. Metering volume: 6L. Post inoculation volume: 6L.
The formula of the transformation medium comprises: 40g/L of phytosterol, 160g/L of hydroxypropyl cyclodextrin, 80g/L of mycobacterium fortuitum, 5g/L of PPE and the balance of 20mM PBS; ph=7.5-8.0.
Conversion conditions: 30 ℃,200rpm, air flow of 0.5vvm, tank pressure of 0.05MPa, conversion time 96h, TLC plate monitoring conversion, and finishing conversion.
(4) Extracting, hydrolyzing and refining
The post-treatment was carried out according to the extraction, hydrolysis and purification method of example 2, and the amount of the other reagents was 4 times that of example 2, except for chloroform used for the extraction of the water phase, and the volume of concentration under reduced pressure was 4 times that of example 2. 61.1 g of white needle-like crystals with normalized content of 99.42 percent are collected.
EXAMPLE 5 fermentation conversion
(1) Seed culture was performed as in example 2;
(2) Seed separation
The cultivated seeds were filtered, and the filter cake was rinsed with 20mM phosphate buffer at ph=8.0 and suction filtered.
(3) Fermentation conversion in 10L tank
The conversion was carried out in a 10L tank. Metering volume: 6L. Post inoculation volume: 6L.
The formula of the transformation medium comprises: phytosterol: 80g/L, hydroxypropyl cyclodextrin: 320g/L, mycobacterium fortuitum: 160g/L, PPE 5g/L, balance 20mM PBS (pH=8.0).
Conversion conditions: 30 ℃,200rpm, air flow of 0.5vvm, tank pressure of 0.05MPa, conversion time of 128h, TLC plate monitoring conversion condition, and finishing conversion.
(4) Extracting, hydrolyzing and refining
The post-treatment was carried out according to the extraction, hydrolysis and purification method of example 2, except that chloroform was used for the extraction of the water phase, the amount of the other reagent was 8 times that of example 2, and the volume of the concentrated solution was 8 times that of example 2. 117.4 g of white needle-like crystals with normalized content of 99.12 percent are collected.
EXAMPLE 6 fermentation conversion
(1) Seed culture was performed as in example 2;
(2) Seed separation
The cultivated seeds were filtered, and the filter cake was rinsed with 20mM phosphate buffer at ph=8.0 and suction filtered.
(3) Fermentation conversion in 10L tank
The conversion was carried out in a 10L tank. Metering volume: 6L. Post inoculation volume: 6L.
The formula of the transformation medium comprises: phytosterol: 100g/L, hydroxypropyl cyclodextrin: 400g/L, mycobacterium fortuitum: 200g/L, 5g/L of PPE, the balance 20mM PBS (pH=8.0).
Conversion conditions: 30 ℃,200rpm, air flow of 0.5vvm, tank pressure of 0.05MPa, conversion time 144h, TLC plate monitoring conversion condition, and finishing conversion.
(4) Extracting, hydrolyzing and refining
The post-treatment was carried out according to the extraction, hydrolysis and purification method of example 2, except that chloroform was used for the extraction of the water phase, the amount of the other reagent was 8 times that of example 2, and the volume of the concentrated solution under reduced pressure was 10 times that of example 2. 145.8 g of white needle-like crystals are collected, and the normalized content is 98.99%.
Claims (3)
1. A method for preparing an A-ring degradation product by transforming plant sterol by resting cells, comprising the following steps:
(1) Resting cell transformation: inoculating Mycobacterium fortuitum (Mycobacterium fortuitum) NK-XHX-103 strain into a transformation culture medium after slant culture and liquid seed culture, and fermenting and transforming the phytosterol after filtration and separation to obtain a fermentation product;
(2) Separating and extracting: filtering after conversion is completed, extracting the water layer for 2-3 times by using chloroform after acidification, and concentrating the 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;
(4) Seed pot culture: inoculating the secondary seed liquid into a seed tank according to the volume ratio of 10%; after inoculation, culturing for 48-72 h at 30 ℃,200rpm, air flow of 0.5VVM and tank pressure of 0.05 MPa;
filtering the seed bacterial liquid after the seed culture in the step (1), leaching a filter cake by using 20mM phosphate buffer solution with pH value of 8.0, and filtering the filter cake by suction;
the formulation of the transformation medium of step (1): 10-100g/L of phytosterol, 10-400g/L of hydroxypropyl cyclodextrin, 20-200g/L of mycobacterium fortuitum, 5g/L of PPE and the balance of 20mM PBS; ph=7.5-8.0.
2. The method for preparing an A-ring degradation product by transforming plant sterols with resting cells according to claim 1, wherein the extraction method of step (2) specifically comprises: filtering or centrifuging the converted bacterial liquid, regulating the pH value of the filtrate to 2.0, and stirring for 30 minutes; adding chloroform for extraction, standing for more than 8 hours, and layering; separating the lower chloroform layer, concentrating under reduced pressure to dry; adding chloroform into the upper water layer, extracting for 2 times repeatedly, and concentrating under reduced pressure; adding toluene into the concentrated substance, heating to 60 ℃, pulping, cooling to 0-4 ℃, filtering, and leaching the filter cake with a small amount of toluene.
3. The method for preparing an A ring degradation product by transforming plant sterol with resting cells according to claim 1, wherein the refining method of the step (3) is specifically as follows: adding methanol and active carbon into the crude product obtained by extraction, heating to 60 ℃, stirring and decoloring for 2 hours, filtering, and washing carbon with a small amount of methanol; concentrating under reduced pressure at 60deg.C, adding a small amount of water to dry residual methanol, adding toluene, concentrating under reduced pressure again, removing water, cooling to 0-4deg.C, crystallizing for 2 hr, vacuum filtering, leaching, and oven drying at 70deg.C to obtain solid product.
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