CN110951815A

CN110951815A - Method for preparing intermediate by biologically fermenting ergosterol etherate by using resting cells

Info

Publication number: CN110951815A
Application number: CN201911175986.5A
Authority: CN
Inventors: 赵小娟; 刘喜荣; 孟浩; 曾春玲; 杨芳
Original assignee: Hunan Xinhexin Biological Medicine Co ltd
Current assignee: Hunan Xinhexin Biological Medicine Co ltd
Priority date: 2019-11-26
Filing date: 2019-11-26
Publication date: 2020-04-03

Abstract

The invention belongs to a production method of steroid medicine intermediates, and particularly relates to a method for preparing an intermediate by biologically fermenting ergosterol etherate by resting cells. The invention comprises the following steps: (1) 3-bit protection; (2) transformation of resting cells; (3) extracting; (4) hydrolyzing; (5) and (5) refining. The preparation method has the advantages of low cost, short route, and capability of saving a plurality of reaction steps and post-treatment steps, and the shortening of the reaction route is also beneficial to the improvement of the reaction yield and the reduction of the intermediate loss. The invention adopts the steps of protecting the 3-position hydroxyl group, directly increases the solubility of the fermentation substrate in the fermentation liquor after the fermentation reaction of the growing cell organism, is beneficial to the fermentation, can improve the yield of the product and reduce the generation of impurities. The preparation method of the invention can also reduce the use of chemical reagents, and is beneficial to environmental protection.

Description

Method for preparing intermediate by biologically fermenting ergosterol etherate by using resting cells

Technical Field

The invention belongs to a production method of steroid medicine intermediates, and particularly relates to a method for preparing an intermediate by biologically fermenting ergosterol etherate by resting cells.

Background

The structural formula of an intermediate pregna-5, 7-diene-3 β, 21-diol is shown as follows, the product is an important intermediate in steroid synthesis, a similar structure of a 3-keto group is used in a traditional preparation method, and the 3-keto group is selectively reduced to β hydroxyl group by using a chemical method, but the product obtained by using the chemical method often has a certain amount of 3-position α hydroxyl isomer and needs to be completed by multi-step chemical reactions, wherein a plurality of expensive and environment-friendly reagents are needed, the cost is high, the yield is low, and the product quality is poor.

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

Aiming at the technical problems, the invention aims to provide a method for preparing an intermediate pregna-5, 7-diene-3 β, 21-diol by using resting cell biological fermentation ergosterol etherate, which can solve the problems of complex steps, easy generation of 3-bit α hydroxyl isomer impurities, high cost and low yield in the synthesis process of the intermediate.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing an intermediate pregna-5, 7-diene-3 β, 21-diol by biologically fermenting ergosterol etherate by using resting cells comprises the following steps:

(1) and 3, protecting: using methylal as a protective agent, protecting 3-hydroxy of ergosterol to obtain ergosterol etherate, and adding phosphorus pentoxide as a catalyst and diatomite as a filter aid in the reaction process;

(2) B-NRRL 3683 mutation strain is prepared by taking mycobacteria B-NRRL 3683 as a starting strain, performing slant culture and secondary seed culture, collecting strain suspension prepared by the strains, adding nitrosoguanidine for mutation treatment, diluting the strain suspension after the mutation, coating the diluted strain suspension on a solid plate culture medium, selecting a single colony for ergosterol fermentation, and detecting the yield of pregnane-5, 7 diene-3 β and 21-diol to obtain a target mutation strain capable of producing the intermediate;

(3) extraction: extracting the fermentation product obtained in the step (2) after the conversion is finished to obtain a solid product;

(4) hydrolysis: dissolving the solid product obtained in the step (3) by using ethyl acetate, performing suction filtration after stirring, combining filtrates, performing reduced pressure concentration, and adding hydrochloric acid for hydrolysis;

(5) refining: refining and purifying the hydrolysate obtained in the step (4).

Preferably, the mass ratio of methylal to ergosterol in the step (1) is 3-20: 1.

In order to drive the reaction to proceed forward, methanol generated by the reaction and a small amount of water in the system need to be removed, so that a catalyst of phosphorus pentoxide needs to be added, and the phosphorus pentoxide absorbs the methanol or the water to become phosphate ester or phosphoric acid, has low solubility in methylal, is oily, and can be removed by filtration after being adsorbed by adding a filter aid, and the filter aid is preferably diatomite. The addition amounts of the filter aid and the catalyst are determined according to the purpose and can be obtained through routine experiments of researchers, the preferable addition amount of the filter aid is 1 time of the weight of ergosterol, and the addition amount of the catalyst is 0.5 time of the weight of the ergosterol.

Further, the preparation method of the B-NRRL 3683 mutation strain in the step (2) specifically comprises the following steps:

a. taking mycobacteria B-NRRL 3683 as a starting strain, performing slant culture in an M1 solid culture medium added with agar of 2 mass percent, inoculating slant seeds into an M1 liquid seed culture medium, activating, and performing shaking culture on a shaking table at 30 ℃ and 200rpm for 48 hours; inoculating the activated primary seeds into a new M1 liquid seed culture medium according to the volume ratio of 10% to perform secondary seed culture, and performing shaking culture on a shaker at 30 ℃ and 200rpm for 48 hours;

b. centrifuging the grown secondary seeds at 10000rpm for 5min, collecting thallus, centrifuging and washing twice with pH6.0 potassium phosphate buffer solution, preparing into bacterial suspension with pH6.0 sterile potassium phosphate buffer solution, and diluting to 10%⁸-10⁹Per ml;

c. taking 2ml of the bacterial suspension, 1ml of 0.1mol/L nitrosoguanidine solution and 2ml of 0.2mol/L potassium phosphate buffer solution with the pH value of 6.0, adding the mixture into a centrifuge tube, fully mixing the mixture, placing the centrifuge tube in a water bath at 30 ℃ for dark oscillation treatment for 25 to 35min, centrifugally collecting thalli, washing the thalli for 3 times by PBS, adding 5ml of sterile physiological saline into the centrifuge tube, shaking the mixture evenly, taking out a certain bacterial suspension, diluting the bacterial suspension by the physiological saline, taking 100ul of the diluted bacterial suspension, coating the diluted bacterial suspension on a solid plate culture medium, culturing the bacterial suspension at 30 ℃ in the dark place, selecting a single bacterial colony of a mutant strain with good growth to perform ergosterol fermentation, and detecting the yield of pregna-5, 7 diene-3 β and 21-diol to obtain the target mutant strain capable.

Further, 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 h.

(4) 50L tank 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; the charging volume is 30 liters, the inoculation amount is 10 percent, and after inoculation, the culture is carried out for 48 to 72 hours under the conditions of 30 ℃, 150rpm, 0.5vvm and 0.05 MPa.

Further, after the B-NRRL 3683 mutant strain is seed-cultured, the cultured cell is filtered or centrifuged, and the filter cake is rinsed with 20mM PBS having a pH of 8.0 and stored in PBS for later use.

Further, the conversion system for fermentation conversion in the step (2) comprises the following components, by mass, 1-10% of ergosterol etherate, 1-40% of hydroxypropyl- β -cyclodextrin, 2-20% of thalli, and the balance being made up with 20mM PBS (pH 8.0).

Further, the resting cell transformation in the step (2) is specifically as follows: adding reactants according to the conversion system, and converting at 28-32 ℃, air flow of 0.1-1.0vvm and tank pressure of 0.01-0.1 MPa.

Further, the extraction method in the step (3) comprises the following steps: and stopping stirring after the conversion is finished, standing and layering the fermentation product for 2 hours, filtering and drying the lower-layer solid to obtain a solid product, and combining the filtrate with the bacterial liquid separated from the upper layer for later use.

Further, the hydrolysis method of the step (4) is a conventional method, and specifically, the method may be: 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 hydrochloric acid with volume fraction of 5%, heating to 60 ℃, and hydrolyzing for 1-2 h.

Further, the refining method of the step (5) is a conventional method, and specifically may be: drying the filter cake obtained by hydrolysis, adding methanol for dissolving, performing suction filtration, 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 a white solid, drying, adding methanol, heating to dissolve, concentrating under reduced pressure to obtain a paste, cooling to 0-4 deg.C, growing crystal for 2 hr, filtering, and drying to obtain a refined product.

Compared with the prior art, the invention has the following beneficial effects:

(1) the 3-hydroxyl is protected, the strain B-NRRL 3683 is subjected to specific mutagenesis to obtain a mutagenized strain, and the ergosterol etherate is fermented, so that the 17-side chain can be specifically cut, and the 3-hydroxyl is not cut. Meanwhile, after the 3-position hydroxyl group is protected, the polarity of the product is reduced, the solubility of the product in the vegetable oil is increased, the fermentation reaction is facilitated, and the efficiency and the product yield of the fermentation reaction are increased. The conversion of the substrate ergosterol etherate after comminution is likewise advantageous for increasing the fermentation rate.

(2) The invention provides a new path for preparing an intermediate pregna-5, 7 diene-3 β, 21-diol from ergosterol, the cost of the preparation method of the steroid drug intermediate is lower, compared with the conventional chemical preparation method which needs to be subjected to multi-step synthesis steps, the method has shorter route, a plurality of reaction steps and post-treatment steps are saved, the shortening of the reaction route is also beneficial to the improvement of the reaction yield and the reduction of intermediate loss.

Detailed Description

In order to make the objects, technical solutions and advantages 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. 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.

The materials used in the following examples are all commercially available from conventional sources.

EXAMPLE 1 mutagenesis of species

Starting strains: mycobacterium sp.B-NRRL 3683

(1) And (3) strain culture:

solid slant culture medium: m1+ 2% agar.

Liquid seed culture medium: m1.

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 average fatality rates in 25-35min are 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.

And (3) 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 a single colony with good growth from 1200 single colonies, carrying out ergosterol fermentation by using the selected mutant strain and the starting strain, and detecting the yield of pregnane-5, 7 diene-3 β, 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: 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 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)50 liter jar seed culture

The charge volume is 30 liters, and the inoculation amount is: 10 percent, and culturing for 48 to 72 hours under the conditions of 30 ℃, 150rpm, 0.5vvm and 0.05MPa after inoculation.

(4) Strain isolation

The cultured cells were filtered or centrifuged, and the filter cake was rinsed with 20mM PBS (pH 8.0) and stored in PBS.

Example 3 Ergotosterol 3-position etherification protection

Material proportioning: 1500g of methylal, 100g of ergosterol, 100g of kieselguhr, 50g of phosphorus pentoxide, 4g of sodium carbonate (prepared into a 1% aqueous solution) and 200g of water.

Adding ergosterol and methylal into a reaction bottle in proportion, heating to 25 ℃, stirring until the ergosterol 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 a light yellow solid, drying the light yellow solid in an oven at the temperature of between 40 and 50 ℃ until the weight is constant, and obtaining the ergosterol etherate crude product by the weight of 118.4 g.

Adding acetone with the volume 2 times of that of the crude product of the ergosterol etherate, heating to 50-60 ℃, stirring and refluxing for 30min, cooling to-10 ℃, performing suction filtration, leaching a filter cake by using acetone with the temperature of-10 ℃, drying the filter cake to constant weight at the temperature of 40-50 ℃, and collecting 101.8g of white powder to obtain the ergosterol etherate.

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;

(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 comprises 1 percent of ergosterol etherate, 1 percent of hydroxypropyl- β -cyclodextrin, 5 percent of thalli and the balance of PBS with the pH value of 20 mM8.0.

Transformation conditions are as follows: 30 ℃, 200rpm, air flow of 0.5vvm, tank pressure of 0.05MPa, conversion time of 64h, TLC spot plate monitoring conversion condition, and waiting for conversion completion.

The reaction formula for the biotransformation is shown below:

(4) extraction of

And stopping stirring after the conversion is finished, and standing and layering for 2 hours.

And pumping the upper layer bacterial liquid into a beaker for later use.

And (3) draining the lower-layer solid, merging filtrate with the bacterial liquid separated from the upper layer, applying the filtrate for conversion of the next batch, adding 0.5% of new bacteria and the bacterial liquid of the upper batch into each next batch, and applying the bacterial liquid circularly for 10 times.

(5) Hydrolysis

Material proportioning: ethyl acetate 1L, 5% hydrochloric acid 1.5L.

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 at 45 ℃ under reduced pressure 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

Material proportioning: 3L of methanol and 0.5L of petroleum ether.

And drying a filter cake obtained by hydrolysis at 70 ℃, dissolving the filter cake in 1L 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, refluxing and 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 be pasty, slowly cooling to 0-4 ℃, growing crystals for 2h, carrying out suction filtration and drying to obtain 19.8g of a refined 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 ergosterol, 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 formula of the transformation system comprises 2% of ergosterol etherate, 1% of hydroxypropyl- β -cyclodextrin and 0.5% of new thalli, the bacterial solution in the example 4 is used, and the balance is supplemented by PBS with the pH value of 20mM being 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 42.1g of product, with a normalized content of liquid phase: 98.65 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 ergosterol, 400g of kieselguhr, 200g of phosphorus pentoxide, 16g of sodium carbonate (prepared into a 1% aqueous solution) and 800g of water.

(3) Fermentation transformation

The formula of the transformation system comprises 4% of ergosterol etherate, 10% of hydroxypropyl- β -cyclodextrin, 15% of thalli and the balance of PBS with the pH value of 20mM 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 80.3g of product, with a normalized content of the liquid phase: 98.14 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 ergosterol, 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 formula of the transformation system comprises 8 percent of ergosterol etherate, 20 percent of hydroxypropyl- β -cyclodextrin, 10 percent of thalli and the balance of PBS with the pH value of 20mM 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 the relevant reagents being 8 times the amount used in example 4, to obtain 168.6g of product, with a normalized content of the liquid phase: 98.52 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 ergosterol, 200g of kieselguhr, 100g of phosphorus pentoxide, 10g of sodium carbonate (prepared into a 1% aqueous solution) and 400g of water.

(3) Fermentation transformation

The formula of the transformation system comprises 2% of ergosterol etherate, 5% of hydroxypropyl- β -cyclodextrin, 2% of thalli and the balance of PBS with the pH value of 20 mM8.0.

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 43.5g of product, with a normalized content of the liquid phase: 98.45 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 ergosterol, 1000g of kieselguhr, 500g of phosphorus pentoxide, 40g of sodium carbonate (prepared into a 1% aqueous solution) and 2000g of water.

(3) Fermentation transformation

The formula of the transformation system comprises 10 percent of ergosterol etherate, 40 percent of hydroxypropyl- β -cyclodextrin, 20 percent of thalli and the balance of PBS with the pH value of 20mM 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 203.2g of product, with a normalized content of the liquid phase: 98.43 percent.

Claims

1. A method for preparing an intermediate by biologically fermenting ergosterol etherate by using resting cells, wherein the intermediate is pregna-5, 7 diene-3 β, 21-diol, and the preparation method comprises the following steps:

(5) refining: refining and purifying the hydrolysate obtained in the step (4).

2. The process for preparing an intermediate by biofermentation of ergosterol etherate with resting cells according to claim 1, wherein the mass ratio of methylal to ergosterol in the step (1) is 3-20: 1.

3. The method for preparing the intermediate by biofermentation of ergosterol etherate with resting cells as claimed in claim 1, wherein the preparation method of B-NRRL 3683 mutant strain in the step (2) is specifically as follows:

b. centrifuging the grown second-stage seeds at 10000rpm for 5min, and collectingThe bacterial cells were washed twice with pH6.0 potassium phosphate buffer by centrifugation, suspended in pH6.0 sterile potassium phosphate buffer, and diluted to 10⁸-10⁹Per ml;

4. The process for the biofermentation of ergosterol etherates with resting cells for the preparation of intermediates according to claim 1 or 3, wherein the process for seed culture of B-NRRL 3683 mutagenic species in step (2) comprises the following steps:

5. The method for preparing an intermediate through biofermentation of ergosterol etherate using resting cells as claimed in claim 4, wherein the B-NRRL 3683 mutagenized strain is seed-cultured, the cultured strain is filtered or centrifuged, the filter cake is rinsed with PBS (20 mM pH 8.0) and stored in PBS for further use.

6. The process for preparing an intermediate by biofermentation of ergosterol etherate using resting cells as claimed in claim 1, wherein the transformation system of the fermentation transformation in step (2) comprises the following components, by mass, 1-10% of ergosterol etherate, 1-40% of hydroxypropyl- β -cyclodextrin, 2-20% of thallus, and the balance being made up with 20mM PBS (pH 8.0).

7. The process for the biofermentation of ergosterol etherates with resting cells for the preparation of intermediates according to claim 1, wherein the resting cell transformation in step (2) is in particular: adding reactants according to the conversion system, and converting at 28-32 ℃, air flow of 0.1-1.0vvm and tank pressure of 0.01-0.1 MPa.

8. The method for preparing the intermediate by biofermentation of ergosterol etherate with resting cells as claimed in claim 1, wherein the extraction method of the step (3) is: and stopping stirring after the conversion is finished, standing and layering the fermentation product for 2 hours, filtering and drying the lower-layer solid to obtain a solid product, and combining the filtrate with the bacterial liquid separated from the upper layer for later use.

9. The process for the biofermentation of ergosterol etherates with resting cells for the preparation of intermediates according to claim 1, wherein the hydrolysis process of step (4) is: 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 hydrochloric acid with volume fraction of 5%, heating to 60 ℃, and hydrolyzing for 1-2 h.

10. The process for the biofermentation of ergosterol etherate to intermediates according to claim 1, wherein the refining process of step (5) is: drying the filter cake obtained by hydrolysis, adding methanol for dissolving, performing suction filtration, 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 a white solid, drying, adding methanol, heating to dissolve, concentrating under reduced pressure to obtain a paste, cooling to 0-4 deg.C, growing crystal for 2 hr, filtering, and drying to obtain a refined product.