CN111778307A - Method for preparing pregna-5-ene-3 beta, 21-diol by resting cell method - Google Patents
Method for preparing pregna-5-ene-3 beta, 21-diol by resting cell method Download PDFInfo
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Abstract
The invention relates to a production method of steroid medicine intermediate, in particular to a method for preparing pregna-5-ene-3 beta, 21-diol by resting cell method. The invention comprises the steps of (1) protecting 3-position of phytosterol, (2) transforming resting cells, (3) extracting, (4) hydrolyzing and (5) refining. The invention takes the phytosterol as the raw material to produce pregna-5-ene-3 beta, 21-diol, the raw material is easy to obtain, and the production cost is reduced; the invention adopts the protective agent to protect the functional group of the 3-hydroxyl of the phytosterol, and the pregna-5-ene-3 beta, 21-diol can be directly prepared by hydrolysis after fermentation, thereby having less by-products, higher yield and shorter reaction route, and saving a plurality of reaction steps and post-treatment steps required by the traditional preparation method.
Description
Technical Field
The invention relates to a production method of steroid medicine intermediate, in particular to a method for preparing pregna-5-ene-3 beta, 21-diol by resting cell method.
Background
Pregna-5-ene-3 beta, 21-diol is an important intermediate in steroid synthesis, the traditional process is to utilize a similar structure of a 3-position ketone group, selectively reduce the 3-position ketone group into a product obtained by a beta hydroxyl group by a chemical method, a certain amount of 3-position alpha hydroxyl isomer is often carried, the yield is low, the steps are complicated, and various organic reagents are used to easily cause pollution.
The strain Mycobacterium sp.B-NRRL 3683 is described in U.S. Pat. No. 4755463 and is generally used only for the fermentation of phytosterols to 4-AD and ADD, and it is currently widely studied to improve the yield of 4-AD and ADD by controlling the reaction conditions.
Disclosure of Invention
In view of the above technical problems, the present invention aims to provide a method for preparing pregna-5-ene-3 β, 21-diol by converting phytosterol into resting cells, which has characteristics of high yield, high quality, low pollution and easy operation. In order to achieve the purpose, the invention adopts the technical scheme that:
a method for preparing pregn-5-ene-3 beta, 21-diol by a resting cell method comprises the following steps:
(1) and 3, protecting: utilizing methylal as a protective agent, protecting 3-position hydroxyl of phytosterol to obtain phytosterol etherate, and adding phosphorus pentoxide as a catalyst and diatomite as a filter aid in the reaction process;
(2) transformation of resting cells: carrying out slant culture and liquid seed culture on a Mycobacterium (Mycobacterium sp.) B-NRRL 3683 mutation strain, filtering and separating seeds, adding the seeds into a PBS buffer system, and carrying out fermentation conversion on the phytosterol etherate to obtain a fermentation product;
(3) extraction: standing and layering the fermentation product obtained in the step (2), and pumping the upper layer bacterial liquid into a beaker for later use; filtering the lower layer solid to obtain a solid product, combining the filtrate with the upper layer bacterial liquid, and applying the filtrate for the next batch of conversion;
(4) performing hydrolysis, namely extracting the solid product obtained in the step (3) by using ethyl acetate, heating and concentrating under reduced pressure, and hydrolyzing by using hydrochloric acid to obtain a hydrolysate;
(5) refining: and (4) refining and purifying the hydrolysate obtained in the step (4) to obtain a pregna-5-ene-3 beta, 21-diol product.
Preferably, the mass ratio of the methylal to the phytosterol in the step (1) is 10-40: 1.
Preferably, the method for seed culture of B-NRRL 3683 mutant strain in the step (2) comprises the following steps:
(1) slant culture: the formula of the culture medium is as follows: 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 121 deg.C for 30 min; after inoculation, culturing at 30 ℃ for 4-5 days;
(2) first-order seed culture: the formula of the culture medium is as follows: 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 121 deg.C for 30 min; after inoculation, shake culturing is carried out for 48h at 30 ℃ and 200 rpm;
(3) secondary seed culture: the formula of the culture medium is as follows: 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 121 deg.C for 30 min; inoculating the primary seed liquid to a secondary seed culture medium according to the volume ratio of 10%, and after inoculation, performing shake culture at 30 ℃ and 200rpm for 48 hours;
(4) seed tank culture:
the formula of the culture medium is as follows: 0.1-10g/L of peptone, 0.1-10g/L of yeast extract, 0.1-10g/L of glucose, 0.1-10g/L of disodium hydrogen phosphate, and pH 7.5-8.0, preparing a culture medium according to the formula, and sterilizing at 121 ℃ for 30 min; inoculating the secondary seed liquid to a seed culture medium according to the volume ratio of 10%, wherein the culture parameters are as follows: culturing at 28-32 deg.C, air flow rate of 0.5-1.0vvm, stirring speed of 50-500rpm, and pot pressure of 0.05-0.06MPa for 14-96 h.
Preferably, the transformation medium formula in the step (2) comprises the following components in percentage by mass: 1-10% of phytosterol etherate, 1-40% of hydroxypropyl-beta-cyclodextrin, 2-20% of thalli and the balance of PBS (20 mM of pH 8.0).
Preferably, the phytosterol etherate is ground to 200 mesh.
Preferably, the resting cell transformation method in the step (2) is specifically as follows: preparing a conversion system according to the formula, and converting at the conditions of 28-32 ℃, stirring speed of 50-500rpm, air flow of 0.5-1.0vvm and tank pressure of 0.05-0.06 MPa.
Preferably, the hydrolysis method in the step (4) is specifically as follows: dissolving the solid product with ethyl acetate, stirring for 1h, performing suction filtration, taking the filter cake as solid waste after being washed by water, combining the filtrates, concentrating at 45 ℃ under reduced pressure until no fraction is produced, adding 5% hydrochloric acid, heating to 60 ℃, hydrolyzing for 1-2h, performing HPLC tracking until the reaction is complete, performing suction filtration, discarding the filtrate, and collecting the filter cake to obtain the hydrolysate.
Preferably, the refining method in the step (5) is specifically as follows: drying the hydrolysate at 70 ℃, adding methanol for dissolving, performing suction filtration, heating and concentrating the filtrate under reduced pressure to a small volume, cooling, performing suction filtration, and drying to obtain a crude product; adding petroleum ether into the crude product, heating, refluxing, pulping, cooling, filtering to obtain white solid, oven drying, adding methanol, heating to dissolve, concentrating under reduced pressure to paste, cooling to 0-4 deg.C, growing crystal for 2 hr, vacuum filtering, and oven drying.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention takes the phytosterol as the raw material to produce pregna-5-ene-3 beta, 21-diol, the raw material is easy to obtain, and the production cost is reduced.
2. The activity of 3-hydroxyl of phytosterol is very high, and the phytosterol is easily oxidized in the conversion process to cause conversion failure.
3. The invention adopts a resting cell conversion method, the conversion system has single nutrition, the risk of contamination is low, the bacterial quantity is adjustable, the feeding concentration of a substrate can be increased, the conversion reaction time is shortened, the pH value of a phosphate buffer system can be adjusted, the enzyme activity in the conversion process is relatively stable, the good conversion capability is ensured, simultaneously the needed cyclodextrin and the bacteria can be repeatedly utilized, and the separation between the cyclodextrin and the products is convenient.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments 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, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. 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.
EXAMPLE 1 mutagenesis of species
Starting strains: mycobacterium sp.B-NRRL 3683
(1) And (3) strain culture:
solid slant culture medium: m1+ 2% agar (M1+ 2% agar indicates not clear, but 0.1-10g/L peptone, 0.1-10g/L yeast extract, 0.1-10g/L glucose, 0.1-10g/L disodium hydrogen phosphate, 20g/L agar, pH 7.5-8.0).
Liquid seed culture medium: m1. (M1 is not specific, but is modified into 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).
Culturing strain B-NRRL 3683 in solid slant culture medium, inoculating one ring of the well-grown slant seeds, activating in 500ml triangular flask containing 100ml liquid seed culture medium, shaking and culturing at 30 deg.C and 200rpm for 48 h; taking 10ml of activated primary liquid seeds, inoculating the seeds into a 500ml triangular flask filled with 100ml of liquid seed culture medium for secondary seed culture, and carrying out shake culture on a shaking table at the temperature of 30 ℃ and the rpm of 200 for 48 hours.
(2) Preparation of bacterial suspension
Centrifuging the grown secondary seeds in 10ml centrifuge tube at 10000rpm for 5min, discarding supernatant, collecting thallus, centrifuging and washing twice with pH6.0 potassium phosphate buffer solution, making into bacterial suspension with sterile potassium phosphate buffer solution (pH6.0), and diluting to 108-109One per ml.
(3) Mutagenic treatment with Nitrosoguanidine (NTG)
Taking 2ml of the bacterial suspension, 1ml of 0.1mol/L nitrosoguanidine solution and 2ml of 0.2mol/Lph6.0 potassium phosphate buffer solution, adding the mixture into a centrifuge tube, fully and uniformly mixing, immediately placing the centrifuge tube in a water bath at 30 ℃ for respectively oscillating (in a dark condition) for 25-35 minutes, centrifugally collecting thalli, washing the thalli for 3 times by PBS (phosphate buffer solution) to remove NTG (nitrilotriacetic acid) residues, finally adding 5ml of sterile physiological saline into the centrifuge tube, shaking up the mixture, taking out a certain bacterial suspension, and diluting the bacterial suspension to a certain concentration by the physiological saline for later use. 100ul of the above bacterial suspension was spread on a solid plate medium, and cultured at 30 ℃ in the dark, and the lethality rate was calculated.
Lethality rate (number of colonies at 0 s-number of colonies at different mutagenesis time)/number of colonies at 0s 100%
The calculation shows that the average of 25-35 minutes is 65.2%, 82.1% and 91.7% respectively. When the lethality rate is 80%, the bacterial mutagenesis effect is best, and 30min is the optimal treatment time.
Taking 30min as the optimal mutagenesis time in the test, carrying out mutagenesis treatment on the bacterial suspension of the starting strain according to the method, selecting 8 well-grown single colonies from 1200 single colonies, carrying out phytosterol fermentation on the selected 8 mutant strains and the starting strain, and detecting the yield of pregna-5-ene-3 beta, 21-diol to obtain the target mutagenic strain.
Example 2 seed culture
The strain name is as follows: mycobacterium sp.B-NRRL 3683 mutant strain
(1) Slant culture
The formula is as follows: 0.1-10g/L of peptone, 0.1-10g/L of yeast extract, 0.1-10g/L of glucose, 0.1-10g/L of disodium hydrogen phosphate, 20g/L of agar and 7.5-8.0 of pH;
sterilizing at 121 deg.C for 30 min. After coagulation and forming, inoculation is carried out under aseptic conditions.
After inoculation, the culture is carried out for 4 days at 30 ℃, and the culture is stored in a refrigerator at 4 ℃ for no more than 1 month.
(2) Seed culture in shake flasks
The formula is as follows: 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 121 deg.C for 30 min. And cooling to room temperature.
1. First order seed culture
Inoculation under sterile conditions, inoculum size: scrape 1 ring per 100 ml. After inoculation, the cells were incubated with shaking at 200rpm for 48h at 30 ℃.
2. Second stage seed culture
Inoculation under sterile conditions, inoculum size: 10 percent. After inoculation, the cells were incubated with shaking at 200rpm for 48h at 30 ℃.
3. Seed tank culture:
the formula of the culture medium is as follows: peptone 0.1-1.0g/L, yeast extract 0.1-1.0g/L, glucose 0.1-1.0g/L, disodium hydrogen phosphate 0.1-1.0g/L, and pH 7.5-8.0.
Sterilizing at 121 deg.C for 30 min; inoculating the secondary seed liquid to a seed culture medium according to the volume ratio of 10%, wherein the culture parameters are as follows: culturing at 28-32 deg.C, air flow rate of 0.5-1.0vvm, stirring speed of 50-500rpm, and pot pressure of 0.05-0.06MPa for 14-96 hr.
4. Strain isolation
The fermented seed was isolated by filtration, the filter cake was rinsed with 20mM PBS (pH 8.0) until the filtrate was colorless, and the filter cake was suspended in 20mM PBS (pH 8.0) until needed.
EXAMPLE 3 phytosterol etherification protection at the 3-position
Material proportioning: 1500g of methylal, 100g of phytosterol, 100g of diatomite, 50g of phosphorus pentoxide, 4g of sodium carbonate (prepared into a 1% aqueous solution) and 200g of water.
Adding phytosterol and methylal into a reaction bottle in proportion, heating to 25 ℃, stirring until the phytosterol and methylal are completely dissolved, adding diatomite, slowly adding phosphorus pentoxide, controlling the temperature to be not more than 30 ℃ in the adding process, stirring for 1-1.5 hours at about 25 ℃, detecting that the reaction is complete by thin layer chromatography, heating to above 30 ℃, filtering while hot, washing a filter cake and the reaction bottle by a small amount of water, and drying at 50 ℃. Obtaining light yellow solid, drying in an oven at 40-50 ℃ to constant weight, and obtaining crude phytosterol etherate with weight of 117.1 g.
Heating the obtained crude etherified product and 2 times of acetone by volume to 50-60 ℃, stirring and refluxing for 30min, cooling to-10 ℃, carrying out suction filtration, leaching a filter cake by using-10 ℃ acetone, drying the filter cake to constant weight at 40-50 ℃, and collecting 100.3g of white powder to obtain the phytosterol etherified product.
The reaction formula of 3-position etherification protection is shown as follows:
example 4 resting cell transformation
(1) Seed culture was performed as in example 2;
(2) substrate preparation was performed as in example 3;
material proportioning: 1500g of methylal, 100g of phytosterol, 100g of diatomite, 50g of phosphorus pentoxide, 4g of sodium carbonate (prepared into a 1% aqueous solution) and 200g of water.
(3) Fermentation conversion in 10L tank
The conversion was carried out in a 10L pot. Measuring volume: 6L. Post-inoculation volume: 6L.
The transformation medium used in this example was composed of the following components: 200 mesh phytosterol etherate 1%, hydroxypropyl-beta-cyclodextrin 1%, thallus 5%, and the balance 20mM PBS (pH 8.0).
Transformation conditions are as follows: 30 ℃, 200rpm, air flow of 0.1vvm, tank pressure of 0.05MPa, conversion time of 88h, TLC spot plate monitoring conversion condition, and waiting for conversion completion.
The reaction for resting cell transformation is shown below:
(4) extraction of
After the conversion, the stirring was stopped and the mixture was allowed to stand for 2 hours. Pumping the upper layer bacterial liquid into a beaker for later use; and (3) draining the lower-layer solid, combining the filtrate with the bacterial liquid separated from the upper layer to obtain separated bacterial liquid, and applying the separated bacterial liquid for the next batch of conversion, wherein the bacterial liquid can be recycled for 10 times.
(5) Hydrolysis
Material proportioning: ethyl acetate 1 liter, 5% hydrochloric acid 1.5 liter.
Dissolving the solid product obtained by suction filtration with ethyl acetate, stirring for 1h, suction filtration, leaching filter cakes to obtain solid waste, combining the filtrates, concentrating under reduced pressure at 45 ℃ until no fraction is produced, adding 5% hydrochloric acid, heating to 60 ℃, hydrolyzing for 1-2h, tracking by HPLC (high performance liquid chromatography) until the reaction is complete, suction filtration, discarding the filtrate, and collecting the filter cakes.
The reaction formula of the hydrolysis reaction is shown below:
(6) refining step
Material proportioning: methanol 2l, petroleum ether 0.5 l.
And drying the filter cake obtained by hydrolysis at 70 ℃, dissolving by using 1 liter of methanol, performing suction filtration, concentrating the filtrate under reduced pressure to a small volume, cooling to about 4 ℃, performing suction filtration, and drying. Adding petroleum ether into the obtained product, performing reflux pulping at 70 ℃ for 2-3h, cooling to 25 ℃, filtering to obtain a white-like solid, drying, adding 2L of methanol, heating to 70 ℃ for dissolution, concentrating under reduced pressure to obtain a paste, slowly cooling to 0-4 ℃, growing crystals for 2h, performing suction filtration, and drying to obtain 19.8g of a product, wherein the liquid phase has a normalized content: 98.35 percent.
Example 5 resting cell transformation
(1) Seed culture was performed as in example 2;
(2) substrate preparation was performed as in example 3;
material proportioning: 600g of methylal, 200g of phytosterol, 200g of diatomite, 100g of phosphorus pentoxide, 8g of sodium carbonate (prepared into a 1% aqueous solution) and 400g of water.
(3) Fermentation transformation
The conversion was carried out in a 10L pot. Measuring volume: 6L. Post-inoculation volume: 6L.
The formula of the transformation system is as follows: phytosterol etherate 2%, hydroxypropyl-beta-cyclodextrin 1%, new bacterial cell 0.5%, the bacterial solution of example 4 was used, and the balance was made up with 20mM PBS (pH 8.0).
Transformation conditions are as follows: 30 ℃, 200rpm, air flow of 0.1vvm, tank pressure of 0.01MPa, conversion time of 96h, TLC spot plate monitoring conversion condition, and waiting for conversion completion.
(4) Extracting, hydrolyzing and refining
The post-treatment was carried out according to the extraction, hydrolysis and purification method of example 4, with the amount of the relevant reagents being 2 times the amount of example 4, to obtain 42.3g of product, with a normalized content of the liquid phase: 98.47 percent.
Example 6 resting cell transformation
(1) Seed culture was performed as in example 2;
(2) substrate preparation was performed as in example 3;
material proportioning: 2000g of methylal, 400g of phytosterol, 400g of diatomite, 200g of phosphorus pentoxide, 16g of sodium carbonate (prepared into a 1% aqueous solution) and 800g of water.
(3) Fermentation transformation
The conversion was carried out in a 10L pot. Measuring volume: 6L. Post-inoculation volume: 6L.
The formula of the transformation system is as follows: phytosterol etherate 4%, hydroxypropyl-beta-cyclodextrin 10%, thallus 15%, and the balance of PBS (20 mM, pH 8.0).
Transformation conditions are as follows: at 28 ℃, 200rpm, air flow of 1.0vvm, tank pressure of 0.1MPa, conversion time of 96h, and TLC spot plate monitoring conversion condition until conversion is finished.
(4) Extracting, hydrolyzing and refining
The post-treatment was carried out according to the extraction, hydrolysis and purification method of example 4, with the amount of the relevant reagents being 4 times the amount of example 4, to obtain 80.1g of product, with a normalized content of liquid phase: 98.16 percent.
Example 7 resting cell transformation
(1) Seed culture was performed as in example 2;
(2) substrate preparation was performed as in example 3;
material proportioning: 12000g of methylal, 800g of phytosterol, 800g of diatomite, 400g of phosphorus pentoxide, 30g of sodium carbonate (prepared into a 1% aqueous solution) and 1600g of water.
(3) Fermentation transformation
The conversion was carried out in a 10L pot. Measuring volume: 6L. Post-inoculation volume: 6L.
The formula of the transformation system is as follows: 8% of phytosterol etherate, 20% of hydroxypropyl-beta-cyclodextrin, 10% of thalli and the balance of PBS (20 mM, pH 8.0).
Transformation conditions are as follows: at 28 ℃, 200rpm, air flow of 0.5vvm, tank pressure of 0.1MPa, conversion time of 192h, TLC spot plate monitoring conversion condition, and waiting for conversion completion.
(4) Extracting, hydrolyzing and refining
The post-treatment was carried out according to the extraction, hydrolysis and purification method of example 4, with the amount of relevant reagents being 8 times the amount used in example 4, to obtain 167.2g of product, with a normalized content of liquid phase: 98.36 percent.
Example 8 resting cell transformation
(1) Seed culture was performed as in example 2;
(2) substrate preparation was performed as in example 3;
material proportioning: 4000g of methylal, 200g of phytosterol, 200g of diatomite, 100g of phosphorus pentoxide, 10g of sodium carbonate (prepared into a 1% aqueous solution) and 400g of water.
(3) Fermentation transformation
The conversion was carried out in a 10L pot. Measuring volume: 6L. Post-inoculation volume: 6L.
The formula of the transformation system is as follows: phytosterol etherate 2%, hydroxypropyl-beta-cyclodextrin 5%, thallus 2%, and the balance of PBS with 20mM pH 8.0.
Transformation conditions are as follows: 32 ℃, 200rpm, air flow of 0.1vvm, tank pressure of 0.05MPa, conversion time of 160h, TLC spot plate monitoring conversion condition, and waiting for conversion completion.
(4) Extracting, hydrolyzing and refining
The post-treatment was carried out according to the extraction, hydrolysis and purification method of example 4, with the amount of the relevant reagents being 2 times the amount of example 4, to obtain 41.2g of product, with a normalized content of the liquid phase: 98.65 percent.
Example 9 resting cell transformation
(1) Seed culture was performed as in example 2;
(2) substrate preparation was performed as in example 3;
material proportioning: 10000g of methylal, 1000g of phytosterol, 1100g of diatomite, 500g of phosphorus pentoxide, 60g of sodium carbonate (prepared into a 1% aqueous solution) and 3000g of water.
(3) Fermentation transformation
The conversion was carried out in a 10L pot. Measuring volume: 6L. Post-inoculation volume: 6L.
The formula of the transformation system is as follows: 10% of phytosterol etherate, 40% of hydroxypropyl-beta-cyclodextrin, 20% of thalli and the balance of PBS (20 mM, pH 8.0).
Transformation conditions are as follows: 32 ℃, 200rpm, air flow of 1.0vvm, pot pressure of 0.01MPa, conversion time of 240h, TLC spot plate monitoring conversion condition, and waiting for conversion completion.
(4) Extracting, hydrolyzing and refining
The post-treatment was carried out according to the extraction, hydrolysis and purification method of example 4, with the amount of the relevant reagents being 10 times the amount used in example 4, to obtain 203.4g of product, with a normalized content of the liquid phase: 98.43 percent.
Claims (8)
1. A method for preparing pregn-5-ene-3 beta, 21-diol by a resting cell method is characterized by comprising the following steps:
(1) and 3, protecting: utilizing methylal as a protective agent, protecting 3-position hydroxyl of phytosterol to obtain phytosterol etherate, and adding phosphorus pentoxide as a catalyst and diatomite as a filter aid in the reaction process;
(2) transformation of resting cells: carrying out slant culture and liquid seed culture on a Mycobacterium (Mycobacterium sp.) B-NRRL 3683 mutation strain, filtering and separating seeds, adding the seeds into a PBS buffer system, and carrying out fermentation conversion on the phytosterol etherate to obtain a fermentation product;
(3) extraction: standing and layering the fermentation product obtained in the step (2), and pumping the upper layer bacterial liquid into a beaker for later use; filtering the lower layer solid to obtain a solid product, combining the filtrate with the upper layer bacterial liquid, and applying the filtrate to the conversion of the next batch of resting cells;
(4) performing hydrolysis, namely extracting the solid product obtained in the step (3) by using ethyl acetate, heating and concentrating under reduced pressure, and hydrolyzing by using hydrochloric acid to obtain a hydrolysate;
(5) refining: and (4) refining and purifying the hydrolysate obtained in the step (4) to obtain a pregna-5-ene-3 beta, 21-diol product.
2. The method for preparing pregn-5-ene-3 β, 21-diol by resting cell method according to claim 1, wherein the mass ratio of methylal to phytosterol in step (1) is 3-20: 1.
3. The method for preparing pregn-5-ene-3 β, 21-diol by resting cell method according to claim 1, wherein the method for seed culture of B-NRRL 3683 mutant strain in step (2) comprises the following steps:
(1) slant culture: the formula of the culture medium is as follows: 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 121 deg.C for 30 min; after inoculation, culturing at 30 ℃ for 4-5 days;
(2) first-order seed culture: the formula of the culture medium is as follows: 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 121 deg.C for 30 min; after inoculation, shake culturing is carried out for 48h at 30 ℃ and 200 rpm;
(3) secondary seed culture: the formula of the culture medium is as follows: 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 121 deg.C for 30 min; inoculating the primary seed liquid to a secondary seed culture medium according to the volume ratio of 10%, and after inoculation, performing shake culture at 30 ℃ and 200rpm for 48 hours;
(4) seed tank culture:
the formula of the culture medium is as follows: 0.1-10g/L of peptone, 0.1-10g/L of yeast extract, 0.1-10g/L of glucose, 0.1-10g/L of disodium hydrogen phosphate, and pH 7.5-8.0, preparing a culture medium according to the formula, and sterilizing at 121 ℃ for 30 min; inoculating the secondary seed liquid to a seed culture medium according to the volume ratio of 10%, wherein the culture parameters are as follows: culturing at 28-32 deg.C, air flow rate of 0.5-1.0vvm, stirring speed of 50-500rpm, and pot pressure of 0.05-0.06MPa for 14-96 h.
4. The method for preparing pregn-5-ene-3 β, 21-diol by resting cell method according to claim 1, wherein the transformation medium formula in step (2) comprises the following components by mass percent: 1-10% of phytosterol etherate, 1-40% of hydroxypropyl-beta-cyclodextrin, 2-20% of thalli and the balance of PBS (20 mM of pH 8.0).
5. The method for preparing pregn-5-ene-3 β, 21-diol according to claim 4, wherein the phytosterol etherate is ground to 200 mesh.
6. The method for preparing pregn-5-ene-3 β, 21-diol according to claim 1, wherein the transformation method of resting cells in step (2) comprises: preparing a conversion system according to the formula, and converting at the conditions of 28-32 ℃, stirring speed of 50-500rpm, air flow of 0.5-1.0vvm and tank pressure of 0.05-0.06 MPa.
7. The method for preparing pregn-5-ene-3 β, 21-diol by resting cell method according to claim 1, wherein the hydrolysis method in step (4) is specifically: dissolving the solid product with ethyl acetate, stirring for 1h, performing suction filtration, taking the filter cake as solid waste after being washed by water, combining the filtrates, concentrating at 45 ℃ under reduced pressure until no fraction is produced, adding 5% hydrochloric acid, heating to 60 ℃, hydrolyzing for 1-2h, performing HPLC tracking until the reaction is complete, performing suction filtration, discarding the filtrate, and collecting the filter cake to obtain the hydrolysate.
8. The method for preparing pregn-5-ene-3 β, 21-diol by resting cell method according to claim 1, wherein the refining method in step (5) comprises: drying the hydrolysate at 70 ℃, adding methanol for dissolving, performing suction filtration, heating and concentrating the filtrate under reduced pressure to a small volume, cooling, performing suction filtration, and drying to obtain a crude product; adding petroleum ether into the crude product, heating, refluxing, pulping, cooling, filtering to obtain white solid, oven drying, adding methanol, heating to dissolve, concentrating under reduced pressure to paste, cooling to 0-4 deg.C, growing crystal for 2 hr, vacuum filtering, and oven drying.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110713510A (en) * | 2019-10-16 | 2020-01-21 | 湖南新合新生物医药有限公司 | Method for preparing intermediate by biologically fermenting ergosterol etherate by using resting cells |
| CN110713509A (en) * | 2019-10-16 | 2020-01-21 | 湖南新合新生物医药有限公司 | Method for preparing intermediate by biological fermentation of ergosterol etherate by using growing cells |
| CN112358523A (en) * | 2020-11-14 | 2021-02-12 | 湖南科瑞生物制药股份有限公司 | Method for removing triphenylphosphine oxide |
| CN112375118A (en) * | 2020-11-14 | 2021-02-19 | 湖南科瑞生物制药股份有限公司 | Method for preparing cholesterol and derivatives thereof by taking phytosterol as raw material |
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2020
- 2020-07-23 CN CN202010716189.XA patent/CN111778307A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110713510A (en) * | 2019-10-16 | 2020-01-21 | 湖南新合新生物医药有限公司 | Method for preparing intermediate by biologically fermenting ergosterol etherate by using resting cells |
| CN110713509A (en) * | 2019-10-16 | 2020-01-21 | 湖南新合新生物医药有限公司 | Method for preparing intermediate by biological fermentation of ergosterol etherate by using growing cells |
| CN112358523A (en) * | 2020-11-14 | 2021-02-12 | 湖南科瑞生物制药股份有限公司 | Method for removing triphenylphosphine oxide |
| CN112375118A (en) * | 2020-11-14 | 2021-02-19 | 湖南科瑞生物制药股份有限公司 | Method for preparing cholesterol and derivatives thereof by taking phytosterol as raw material |
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