阅读说明：本技术 一种从植物甾醇制备脱氧胆酸的方法 (Method for preparing deoxycholic acid from phytosterol ) 是由 蒋红平 唐杰 李斌 郭志军 于 2021-02-03 设计创作，主要内容包括：本发明提供了保藏编号为CCTCC NO：M 2020987的诱变菌种在制备脱氧胆酸中的应用。本发明提供了一种新的以植物甾醇为原料,通过生物发酵与化学合成相结合的手段,制备脱氧胆酸的方法,该方法未使用动物源提取,可以以工业废水废渣为原料,绿色环保；通过分支杆菌Mycobacterium sp.NRRL B-3805诱变菌种生物发酵的方法在植物甾醇侧链一步构建胆酸类化合物的羰基侧链,操作简单,收率高,异构体杂质少,有机溶剂使用量少；通过本发明制备脱氧胆酸类化合物,反应试剂简单易得,反应条件温和,反应收率高,适合工业化大生产。(The invention provides a preservation number of CCTCC NO: use of a mutagenized species of M2020987 in the preparation of deoxycholic acid. The invention provides a novel method for preparing deoxycholic acid by taking phytosterol as a raw material through a means of combining biological fermentation and chemical synthesis, and the method does not use animal source extraction, can take industrial wastewater and waste residues as raw materials, and is green and environment-friendly; the carbonyl side chain of the cholic acid compound is constructed on the phytosterol side chain in one step by a Mycobacterium sp.NRRL B-3805 mutant strain biological fermentation method, the operation is simple, the yield is high, the isomer impurities are few, and the usage amount of an organic solvent is small; the deoxycholic acid compound prepared by the method has the advantages of simple and easily-obtained reaction reagent, mild reaction conditions and high reaction yield, and is suitable for industrial mass production.)

1. The preservation number is CCTCC NO: use of a mutagenized species of M2020987 in the preparation of deoxycholic acid.

2. The preservation number is CCTCC NO: use of M2020987 mutant strain in combination with Aspergillus ochraceus ATCC 18500 strain for the preparation of deoxycholic acid.

3. The preservation number is CCTCC NO: the application of M2020987 mutant strain Mycobacterium smegmatis NK-XHX-118 with combined preservation number of M2020987 mutant strain CCTCC NO: M2013544 in preparing deoxycholic acid.

4. A method for preparing deoxycholic acid from phytosterol, comprising the steps of:

A) the preservation number is CCTCC NO: inoculating a mutagenic strain M2020987 to a first transformation culture medium after culturing, and performing first fermentation transformation on phytosterol shown in the formula I to obtain a compound 1;

B) performing second slant culture and second seed culture on Aspergillus ochraceus ATCC 18500 strain, inoculating to a second transformation culture medium, and performing second fermentation transformation on the compound 1 to obtain a compound 2;

or culturing Mycobacterium smegmatis NK-XHX-118 strain with the preservation number of CCTCC NO of M2013544 by a third seed, inoculating the strain to a third transformation culture medium, and performing third fermentation transformation on the compound 1 to obtain a compound 2';

C) reacting the compound 2 or the compound 2' under the action of acetic anhydride and concentrated sulfuric acid to generate a compound 3;

D) reacting the compound 3 with air under the action of a catalyst to generate a compound 4;

E) reacting the compound 4 with hydrogen under the action of a catalyst to generate a compound 5;

F) dissolving the compound 5 in tetrahydrofuran and water, and reacting with sodium borohydride to generate deoxycholic acid shown in a formula II;

wherein R is- (CH)₂)₃CH(C₂H₅)CH(CH₃)₂、-CH₂CH＝CHCH(C₂H₅)CH(CH₃)₂、-(CH₂)₃CH(CH₃)CH(CH₃)₂or-CH₂CH＝CHCH(CH₃)CH(CH₃)₂。

5. The method of claim 4, wherein the first fermentative conversion process is: the preservation number is CCTCC NO: the M2020987 mutation strain is added into a transformation medium after primary seed culture and secondary seed culture, the inoculation amount is 10-40%, the temperature is 20-30 ℃, the rotating speed is 200-600 rpm, and the air flow is 0.1-0.4 Nm³The pressure is 0.01-0.1 MPa, and the conversion time is 72-180 h.

6. The method of claim 5, wherein the primary seed culture and the secondary seed culture are formulated in a medium comprising: 10-20 g/L of yeast extract powder, 10-20 g/L of glucose, 2-6 g/L of sodium nitrate, 0.1-1.0 g/L of diammonium hydrogen phosphate and 7.5 of pH;

the transformation medium formula is as follows: 10-40 g/L of phytosterol, 10-20 g/L of soybean oil, 30-80 g/L of corn steep liquor, 2-6 g/L of sodium nitrate and 0.1-1.0 g/L of diammonium phosphate, and adjusting the pH value to 8.0.

7. The process according to claim 4, characterized in that after the first fermentative conversion a post-treatment is carried out to obtain compound 1;

the post-treatment specifically comprises the following steps:

adjusting the pH value of the system to 5.0-6.0, heating and stirring, standing and layering; extracting the oil layer with methanol, concentrating to remove solvent, pulping with ethyl acetate under reflux, cooling to separate out solid, and filtering; concentrating the filtrate to remove solvent, stirring with chloroform and water at room temperature under the condition of pH12-13, standing for layering; adjusting the pH value of the aqueous phase to 1.0, and standing for layering; the oil obtained after separation was taken out as compound 1.

8. The method of claim 4, wherein the conditions of the second fermentative conversion are: the inoculation amount is 5-10%, the room temperature, the rotating speed is 200-300 rpm, and the air flow is 0.2-0.4 Nm³H, pressure 0.01-0.1 MPa, conversionThe time is 72-120 h;

the culture medium for the second slant culture comprises: 100-300 g/L of potato blocks, 10-30 g/L of glucose and 10-20 g/L of agar, and adjusting the pH value to 6.5;

the culture medium for the second seed culture is as follows: 5-20 g/L of corn steep liquor and 10-40 g/L of glucose, and adjusting the pH value to 7.2 +/-0.2;

the second transformation medium is: 5-20 g/L of corn steep liquor and 10-40 g/L of glucose, and adjusting the pH value to 7.2 +/-0.2;

after the second fermentation conversion, carrying out post-treatment to obtain a compound 2;

the post-treatment comprises the following steps:

after the second fermentation conversion is finished, heating to 80 ℃ for inactivation, cooling to normal temperature, adjusting the pH value to about 3.0, and performing suction filtration; washing the filter cake with dichloromethane, collecting the filtrate, concentrating the dry solvent, adding water, stirring uniformly, and filtering to obtain the compound 2.

9. The method of claim 4, wherein the conditions of the third fermentative conversion are: the inoculation amount is 10-30%, the temperature is 28-32 ℃, the rotating speed is 300-600 rpm, and the air flow is 0.1-0.4 Nm³The reaction time is 48-72h, the pressure is 0.02-0.1 MPa;

the culture medium for the third seed culture is as follows: 5-20 g/L of yeast extract powder, 10-20 g/L of glucose, 2-6 g/L of sodium nitrate and 0.01-0.1% of diammonium phosphate, and adjusting the pH to 7.5;

the third transformation medium is: 5-20 g/L of phytosterol, 10-80 g/L of corn steep liquor, 2-6 g/L of sodium nitrate and 0.1-0.8 g/L of diammonium hydrogen phosphate, and adjusting the pH to 8.0;

after the third fermentation and transformation, carrying out post-treatment to obtain a compound 2';

the post-treatment comprises the following steps:

adjusting the pH value of a water layer to 1-2, extracting with dichloromethane, collecting a dichloromethane layer, concentrating the solid, adding water into the solid, stirring, and filtering to obtain a solid, namely a compound 2'.

10. The process according to claim 4, wherein the catalyst in step D) is N-hydroxyphthalimide, benzoylperoxide and cobalt acetate tetrahydrate;

the catalyst in the step E) is palladium carbon.

Technical Field

The invention relates to the technical field of chemical pharmacy, in particular to a method for preparing deoxycholic acid from phytosterol, and specifically relates to a method for preparing a deoxycholic acid compound by taking the phytosterol as a raw material in a manner of combining biological fermentation and chemical synthesis.

Background

Deoxycholic Acid (DCA) also known as Deoxycholic Acid (3 α,12 α -dihydroxy-5 β -cholanic Acid) is a free bile Acid obtained by cholic Acid without an oxygen atom, and exists in bile mainly in the form of taurine and glycine. Deoxycholic acid and its salt have surface activity, are safe and effective emulsifiers in cosmetics and medicaments, have antifungal and anti-inflammatory effects, and can be used for treating tooth root diseases. Can be used for treating sebaceous gland hypersecretion in skin surgery. The application of the product in incense powder can remove excessive sebum and sweat stain without causing dry skin feeling. Studies have shown that deoxycholate injected into adipose tissue degrades adipocytes via a cytolytic mechanism. Because deoxycholate infused with fat is rapidly inactivated by exposure to protein and then rapidly returned to the intestinal contents, its action is limited spatially. As a result of this attenuation, which confers clinical safety, lipolysis therapy typically requires 4-6 courses. This surgical-free local fat removal not only facilitates therapeutic treatment associated with pathological local fat deposits (e.g., dyslipidemia susceptible to medical intervention in HIV treatment), but also facilitates cosmetic fat removal without the attendant risks inherent in surgical procedures (e.g., liposuction). In the united states, DCA has been FDA approved for the reduction of submental moderate to severe fat.

At present, two main methods for preparing deoxycholic acid are available, one is extracted and separated from organisms, but deoxycholic acid obtained from animal sources may contain pathogens; the other method is to adopt a synthetic method to obtain deoxycholic acid, and the existing synthetic method has the disadvantages of complex process, environmental pollution and relatively high cost. In recent years, there are reports on the synthesis of deoxycholic acid from phytosterol as a raw material, and patent CN106146594A reports a method for synthesizing DCA from stigmasterol and ergosterol as raw materials, wherein the method adopts a total synthesis method, has the problems of long reaction steps, low yield, expensive reagents and the like, and is not suitable for industrial production.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for preparing deoxycholic acid from phytosterol, which is simple to operate and has high yield.

In order to achieve the aim, the invention provides a culture medium with a preservation number of CCTCC NO: use of a mutagenized species of M2020987 in the preparation of deoxycholic acid.

The invention also provides a preservation number of CCTCC NO: use of M2020987 mutant strain in combination with Aspergillus ochraceus ATCC 18500 strain for the preparation of deoxycholic acid.

The Aspergillus ochraceus ATCC 18500 strain is derived from American type strain Collection (ATCC).

The invention also provides a preservation number of CCTCC NO: the application of M2020987 mutant strain Mycobacterium smegmatis NK-XHX-118 with combined preservation number of M2020987 mutant strain CCTCC NO: M2013544 in preparing deoxycholic acid.

The preservation number is CCTCC NO: the M2020987 mutant is a mutant of Mycobacterium sp.NRRL B-3805.

The mutant strain is obtained by mutagenesis by taking mycobacterium NRRL B-3805 as an initial strain.

The original strain of mycobacterium NRRL B-3805 is derived from the American agricultural research culture Collection (NRRL).

The invention provides a mutagenesis method of mycobacterium mutagenic strain for producing 4-cholenic acid-3-ketone, comprising the following steps:

the method comprises the steps of taking mycobacteria NRRL B-3805 as a starting strain, performing slant culture and secondary seed culture, collecting bacteria to prepare a bacterial suspension, adding nitrosoguanidine for mutagenesis, diluting the bacterial suspension after mutagenesis, coating the bacterial suspension on a solid plate culture medium, selecting a single bacterial colony for phytosterol fermentation, detecting the yield of 4-cholenic acid-3-ketone, obtaining a target mutagenic strain capable of producing 4-cholenic acid-3-ketone, and separating and storing the strain.

Preferably, the formula of the culture medium for slant culture is as follows: 10-20 g/L of yeast extract powder, and more preferably 10 g/L; 15-30 g/L of glucose, and more preferably 15 g/L; 2-6 g/L of sodium nitrate, more preferably 5.4 g/L; 0.1-1 g/L diammonium hydrogen phosphate, and more preferably 0.6 g/L; agar powder 20g/L, pH 7.5.

In a preferred embodiment of the present invention, the culture medium for seed culture comprises: 10-20 g/L of yeast extract powder, and more preferably 10 g/L; 15-30 g/L of glucose, and more preferably 15 g/L; 2-6 g/L of sodium nitrate, more preferably 5.4 g/L; diammonium phosphate is 0.1-1 g/L, more preferably 0.6g/L, and pH is 7.5.

In a preferred embodiment of the present invention, the solid medium comprises: 10-20 g/L of yeast extract powder, more preferably 10g/L, and 15-30 g/L of glucose, more preferably 15 g/L; 2-6 g/L of sodium nitrate, more preferably 5.4 g/L; 0.1-1 g/L diammonium hydrogen phosphate, and more preferably 0.6 g/L; agar powder 20g/L, pH 7.5.

The time for the mutagenesis treatment is preferably 30 min.

Specifically, the invention provides a method for preparing deoxycholic acid from phytosterol, which comprises the following steps:

A) the preservation number is CCTCC NO: inoculating a mutagenic strain M2020987 to a first transformation culture medium after culturing, and performing first fermentation transformation on phytosterol shown in the formula I to obtain a compound 1;

B) performing second slant culture and second seed culture on Aspergillus ochraceus ATCC 18500 strain, inoculating to a second transformation culture medium, and performing second fermentation transformation on the compound 1 to obtain a compound 2;

or culturing Mycobacterium smegmatis NK-XHX-118 strain with the preservation number of CCTCC NO of M2013544 by a third seed, inoculating the strain to a third transformation culture medium, and performing third fermentation transformation on the compound 1 to obtain a compound 2';

C) reacting the compound 2 or the compound 2' under the action of acetic anhydride and concentrated sulfuric acid to generate a compound 3;

D) reacting the compound 3 with air under the action of a catalyst to generate a compound 4;

E) reacting the compound 4 with hydrogen under the action of a catalyst to generate a compound 5;

F) dissolving the compound 5 in tetrahydrofuran and water, and reacting with sodium borohydride to generate deoxycholic acid shown in a formula II;

wherein R is- (CH)₂)₃CH(C₂H₅)CH(CH₃)₂、-CH₂CH＝CHCH(C₂H₅)CH(CH₃)₂、-(CH₂)₃CH(CH₃)CH(CH₃)₂or-CH₂CH＝CHCH(CH₃)CH(CH₃)₂。

The route of the above reaction is as follows:

route one:

the deoxycholic acid compound is prepared from phytosterol by means of combining biological fermentation and chemical synthesis. Compared with animal-derived raw materials, the phytosterol is widely present in roots, stems, leaves and fruits of plants, has a remarkable storage amount in nature, and waste residues in the production of modern commercial products contain high-content phytosterol, such as residues or distillates after squeezing in the grease industry, beet residues and bagasse in the sugar industry, and waste liquid and waste water of papermaking all contain a certain content of phytosterol. According to the technical means, the method for synthesizing the deoxycholic acid by combining microbial fermentation and chemical synthesis is adopted, and a microbial conversion method is adopted for side chain construction and 9 or 11-site alpha hydroxyl introduction in 2 key steps of deoxycholic acid synthesis, so that the method has the advantages of simplicity in operation, high yield, less isomer impurities, less organic solvent consumption and the like.

Preferably, the first fermentation conversion method specifically comprises the following steps: the preservation number is CCTCC NO: the M2020987 mutation strain is added into a transformation medium after primary seed culture and secondary seed culture, the inoculation amount is 10-40%, the temperature is 20-30 ℃, the rotating speed is 200-600 rpm, and the air flow is 0.1-0.4 Nm³The pressure is 0.01-0.1 MPa, and the conversion time is 72-180 h.

In a preferred embodiment of the present invention, the culture medium formula for the first-stage seed culture and the second-stage seed culture is: 10-20 g/L of yeast extract powder, 10-20 g/L of glucose, 2-6 g/L of sodium nitrate, 0.1-1.0 g/L of diammonium hydrogen phosphate and pH of 7.5.

According to the invention, the formula of the transformation medium is preferably as follows: 10-40 g/L of phytosterol, 10-20 g/L of soybean oil, 30-80 g/L of corn steep liquor, 2-6 g/L of sodium nitrate and 0.1-1.0 g/L of diammonium phosphate, and adjusting the pH value to 8.0.

Preferably, the first fermentation conversion is followed by a post-treatment to yield compound 1.

The post-treatment is preferably as follows:

adjusting the pH value of the system to 5.0-6.0, heating and stirring, standing and layering; extracting the oil layer with methanol, concentrating to remove solvent, pulping with ethyl acetate under reflux, cooling to separate out solid, and filtering; concentrating the filtrate to remove solvent, stirring with chloroform and water at room temperature under the condition of pH12-13, standing for layering; adjusting the pH value of the aqueous phase to 1.0, and standing for layering; the oil obtained after separation was taken out as compound 1.

Preferably, the method further comprises the following steps of after the oil obtained after the layering is taken: adding ethyl acetate and active carbon into the oily substance, stirring at normal temperature, filtering and concentrating to obtain a compound 1.

The time for stirring at normal temperature is preferably 2 hours.

Preferably, the conditions of the second fermentation conversion are as follows: the inoculation amount is 5-10%, the room temperature, the rotating speed is 200-300 rpm, and the air flow is 0.2-0.4 Nm³The pressure is 0.01-0.1 MPa, and the conversion time is 72-120 h.

Preferably, the culture medium for the second slant culture is: 100-300 g/L of potato blocks, 10-30 g/L of glucose and 10-20 g/L of agar, and adjusting the pH value to 6.5.

Preferably, the culture medium for the second seed culture is: 5-20 g/L of corn steep liquor and 10-40 g/L of glucose, and adjusting the pH value to 7.2 +/-0.2.

Preferably, the second transformation medium is: 5-20 g/L of corn steep liquor and 10-40 g/L of glucose, and adjusting the pH value to 7.2 +/-0.2.

Preferably, after the second fermentation conversion, the post-treatment is carried out to obtain a compound 2;

the post-treatment comprises the following steps:

after the second fermentation conversion is finished, heating to 80 ℃ for inactivation, cooling to normal temperature, adjusting the pH value to about 3.0, and performing suction filtration; washing the filter cake with dichloromethane, collecting the filtrate, concentrating the dry solvent, adding water, stirring uniformly, and filtering to obtain the compound 2.

Preferably, the conditions of the third fermentation conversion are as follows: the inoculation amount is 10-30%, more preferably 20%, the temperature is 28-32 ℃, more preferably 30 ℃, the rotating speed is 300-600 rpm, more preferably 400rpm, and the air flow is 0.1-0.4 Nm³H, more preferably 0.2Nm³The pressure is 0.02-0.1 MPa, the more preferable pressure is 0.05MPa, and the conversion time is 48-72 h.

Preferably, the culture medium for the third seed culture is: 5-20 g/L of yeast extract powder, more preferably 10g/L, 10-20 g/L of glucose, more preferably 15g/L, 2-6 g/L of sodium nitrate, more preferably 5.4g/L, 0.01-0.1%, more preferably 0.06% of diammonium phosphate, and the pH is adjusted to 7.5.

Preferably, the third transformation medium is: 5-20 g/L of phytosterol, more preferably 10g/L, 10-80 g/L of corn steep liquor, more preferably 60g/L, 2-6 g/L of sodium nitrate, more preferably 5.4g/L, 0.1-0.8 g/L of diammonium hydrogen phosphate, more preferably 0.6g/L, and the pH is adjusted to 8.0.

Preferably, the third fermentative conversion is followed by a post-treatment to yield compound 2'.

The post-treatment is preferably:

adjusting the pH value of a water layer to 1-2, extracting with dichloromethane, collecting a dichloromethane layer, concentrating the solid, adding water into the solid, stirring, and filtering to obtain a solid, namely a compound 2'.

In a preferred embodiment of the present invention, after collecting the dichloromethane layer, the method further comprises: adding activated carbon for decolorization, filtering, collecting filtrate, concentrating the filtrate, adding water into the solid, and stirring to obtain compound 2'.

In the preferred embodiment of the present invention, the catalyst in step D) is N-hydroxyphthalimide, benzoylperoxide and cobalt acetate tetrahydrate.

Preferably, the catalyst in step E) is palladium on carbon.

Compared with the prior art, the invention provides a CCTCC NO: use of a mutagenized species of M2020987 in the preparation of deoxycholic acid. The invention provides a novel method for preparing deoxycholic acid by taking phytosterol as a raw material through a means of combining biological fermentation and chemical synthesis, and the method does not use animal source extraction, can take industrial wastewater and waste residues as raw materials, and is green and environment-friendly; the carbonyl side chain of the cholic acid compound is constructed on the phytosterol side chain in one step by a Mycobacterium sp.NRRL B-3805 mutant strain biological fermentation method, the operation is simple, the yield is high, the isomer impurities are few, and the usage amount of an organic solvent is small; the deoxycholic acid compound prepared by the method has the advantages of simple and easily-obtained reaction reagent, mild reaction conditions and high reaction yield, and is suitable for industrial mass production.

Drawings

FIG. 1 is a carbon spectrum of Compound 1 prepared in example 1 of the present invention;

FIG. 2 is a hydrogen spectrum of Compound 1 prepared in example 1 of the present invention;

FIG. 3 is a carbon spectrum of deoxycholic acid prepared in example 7 according to the present invention;

FIG. 4 is a hydrogen spectrum of deoxycholic acid prepared in example 7 according to the present invention.

Biological preservation Instructions

And (3) classification and naming: mycobacterium sp.mt-0265, deposited at the chinese type culture collection on 30 months 12/2020, address: china, Wuhan university, the preservation number is CCTCC NO: m2020987.

Detailed Description

To further illustrate the present invention, the method for preparing deoxycholic acid from phytosterol provided by the present invention is described in detail below with reference to examples.

The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

The raw materials and other chemical reagents adopted in the embodiment of the invention are all commercial products.

EXAMPLE 1 preparation of Compound 1

Transformation of primary strain

1 bacterial species

Mycobacterium sp.NRRL B-3805

2 transformation Process

2.1 seed culture

First-stage seed: 10g/L of yeast extract powder, 15g/L of glucose, 5.4g/L of sodium nitrate and 0.06 percent of diammonium phosphate, wherein the pH value is 7.5,100 ml of culture medium is put into a 500ml shake flask and sterilized for 30 minutes at 121 ℃. After cooling, the cells were inoculated from the slant and incubated at 200rpm and 30 ℃ for 48 hours.

Secondary seeds: 10g/L of yeast extract powder, 15g/L of glucose, 5.4g/L of sodium nitrate and 0.06 percent of diammonium phosphate, wherein the culture medium with the pH value of 7.5,500 ml is put into a 2000 ml shake flask and sterilized for 30 minutes at 121 ℃. After cooling, the mixture was inoculated from a first-stage shake flask with an inoculum size of 10% and cultured at 200rpm for 48 hours at 30 ℃.

2.2 transformation

10L fermenter, load 7L, culture medium: 20g/L of stigmasterol, 160g/L of soybean oil, 60g/L of corn steep liquor, 5.4g/L of sodium nitrate and 0.6g/L of diammonium phosphate, adjusting the pH value to 8.0,121 ℃, sterilizing for 30 minutes, cooling to 30 ℃, and inoculating, wherein the inoculation amount is 20%.

And (3) conversion control: rotation speed 400rpm, air flow 0.2Nm³H, pot pressure 0.05MPa。

And (3) after the conversion is finished for about 120 hours, adjusting the pH value to 5.0-6.0 by using phosphoric acid, heating to 80 ℃, stirring for 1 hour, standing for 2 hours, and layering.

2.3 working-up

Extracting the oil layer with methanol for three times, concentrating the dry solvent under reduced pressure with 3 times of methanol, adding 0.4 times of ethyl acetate, pulping at 50 deg.C under reflux for 2 hr, cooling to 30 deg.C, and filtering.

Concentrating the filtrate at 50 ℃ under reduced pressure, adding 100ml of chloroform and 500ml of water, adjusting the pH value to 12-13 with sodium hydroxide, rapidly stirring at normal temperature for reacting for 2 hours, standing for 4 hours, and layering.

The aqueous phase obtained by separation was adjusted to pH 1.0 with hydrochloric acid, stirred for 30 minutes and separated. A yellow oil was obtained.

The resulting oil was stirred at room temperature for 2 hours with 100ml of ethyl acetate and 5g of activated carbon, filtered to remove the carbon, and concentrated under reduced pressure to give 1.5 g of a pale yellow oil containing 11.2% of Compound 1.

Two-strain mutagenesis and transformation

1.1 mutation Breeding of cells

1.2 Strain culture

Seed culture medium: 10g/L of yeast extract powder, 15g/L of glucose, 5.4g/L of sodium nitrate, 0.6g/L of diammonium hydrogen phosphate and 7.5 of pH.

Solid medium: 10g/L of yeast extract powder, 15g/L of glucose, 5.4g/L of sodium nitrate, 0.6g/L of diammonium hydrogen phosphate, 20g/L of agar powder and 7.5 of pH.

Inoculating a ring of well-grown slant seeds, activating in a 500ml triangular flask filled with 100ml liquid seed culture medium, and shake culturing at 30 ℃ 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.

Preparing a 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, and adding sterile potassium phosphate buffer solution(pH6.0) preparing a bacterial suspension, and diluting the concentration to 10⁸-10⁹One per ml. 1.3 Nitroguanidine (NTG) mutagenesis treatment

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, immediately placing the mixture into a water bath at 30 ℃ for vibration (in a dark condition) for 0, 10, 15, 20, 25, 30 and 35 minutes respectively, centrifugally collecting thalli, washing the thalli for 3 times by PBS (phosphate buffer solution) to remove NTG (nitroglycerin) residues, finally adding 5ml of sterile physiological saline into the centrifuge tube, shaking the mixture uniformly, taking out a certain bacterial suspension, and diluting the bacterial suspension to a certain concentration by the physiological saline for later use. 100 mu L of the bacterial suspension is coated on a solid plate culture medium, and is cultured in a dark place at 30 ℃, and the lethality rate is 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 of 0, 10, 15, 20, 25, 30 and 35 minutes are respectively 0, 17.2%, 30.4%, 45.1%, 68.1%, 89.7% and 95.3%. When the lethality rate is 80%, the bacterial mutagenesis effect is best, so 30min is the optimal treatment time.

And (3) performing mutagenesis treatment on the bacterial suspension of the starting strain by the method by taking 30min as the optimal mutagenesis time in the test, selecting single colonies with good growth from 1200 single colonies, performing phytosterol biotransformation on the selected mutant strain and the starting strain, and detecting the generation amount of the compound 1.

According to the transformation process in the 'transformation of the first and original strains', the strain Mycobacterium sp-091 (CCTCC NO: M2020987) obtained by mutagenesis and 140 g of stigmasterol are used for feeding, and 5.1 g of light yellow paste is obtained, wherein the content of the compound 1 is 71.2%.

The carbon spectrum and the hydrogen spectrum of compound 1 are shown in fig. 1 and fig. 2.

¹³C NMR(101MHz,DMSO)δ198.44,171.48,123.60,55.81,53.65,42.45,38.60,35.60,35.41,35.26,34.07,32.49,32.15,28.05,24.23,21.05,18.58,17.35,12.24.

¹H NMR(400MHz,DMSO)δ11.94(s,1H),5.62(s,1H),3.32(s,1H),2.50(dt,J＝3.5,1.7Hz,2H),2.47–2.32(m,2H),2.32–2.03(m,4H),1.97(dd,J＝22.1,11.8Hz,2H),1.79(d,J＝2.8Hz,2H),1.72–1.21(m,11H),1.14(s,3H),0.99(ddd,J＝16.9,12.1,7.3Hz,2H),0.88(d,J＝6.2Hz,3H),0.68(s,3H).

EXAMPLE 2 preparation of Compound 2

1. Seed culture

(1) Slant culture

Aspergillus ochraceus ATCC 18500 is used as a production strain, the preserved strain is subjected to streak inoculation on a slant, and the slant is cultured for 6-7 days at 30 ℃, wherein the slant culture medium is a potato culture medium: potato pieces 200g/L (boiled for 30 minutes, filtrate from four layers of gauze), glucose 20g/L, agar 20g/L, pH6.5.

(2) Seed culture

Preparing spore suspension: taking a fresh slant for culturing for 6-7 days, washing off slant spores with 0.05% Tween-80 sterile water to prepare spore suspension, and performing microscopic examination to count the concentration of spores to be 2-3 × 10⁷Per ml;

and (3) seed culture in a shaking flask: the inoculation amount is 10%, the culture is carried out for 36-48 h at 30 ℃ and 180 rpm.

The seed culture medium comprises the following components: 10g/L of corn steep liquor and 30g/L of glucose; the pH value is 7.2 +/-0.2.

Sterilizing with high pressure steam at 121 deg.C for 30min, and cooling to room temperature.

2. Transformation of

(1) The formula of the culture medium is as follows: 10g/L of corn steep liquor and 30g/L of glucose; the pH value is 7.2 +/-0.2.

(2) Pre-culturing thalli: 10L fermentation tank, 7L fermentation liquor volume, 121 deg.C sterilization for 30 minutes, cooling to room temperature. Inoculating with 5% of inoculum size, and culturing for 16 hr.

(3) And (3) transformation: under the protection of a fire circle, 70 g of the pretreated substrate is added into the cultured bacterial liquid to start conversion. Conversion parameters: air flow rate 0.4m³The rotation speed is 300rpm, and the tank pressure is 0.05 MPa. Conversion time: 72 hours, conversion 90.22%.

The substrate pretreatment method comprises the following steps: 70 g of Compound 1, 200 ml of sterile water are added, heated to 80 ℃ and inactivated for 1 hour.

3. Post-treatment

After fermentation and conversion, inactivating at 80 deg.C, cooling to 25 deg.C, and vacuum filtering. Adding dichloromethane with 5 times volume of the substrate into the filter cake layer, stirring for 30 minutes at normal temperature, filtering, extracting the filter cake with dichloromethane with 2 times volume of the filter cake layer under stirring, and performing suction filtration to dry the filter cake layer. Collecting dichloromethane filtrate, concentrating dry solvent under reduced pressure, adding a small amount of water, stirring at normal temperature, performing suction filtration, removing filtrate, collecting filter cake, and drying the filter cake at 50 ℃ to obtain light yellow crude product.

Refining of crude product

Adding 2 times volume of toluene into the crude product, heating to 60 deg.C, stirring for 2 hr, filtering while hot to obtain about 40.2 g of white solid containing compound 2 as main component, wherein the content of compound 2 is 96.75% and the content of compound 1 is 1.22% by liquid phase detection.

EXAMPLE 3 preparation of Compound 2

1 bacterial species

The strain name is as follows: the Mycobacterium smegmatis NK-XHX-118 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2013544.

2 transformation Process

2.1 seed culture

First-stage seed: 10g/L of yeast extract powder, 15g/L of glucose, 5.4g/L of sodium nitrate and 0.06 percent of diammonium phosphate, wherein the pH value is 7.5,100 ml of culture medium is put into a 500ml shake flask and sterilized for 30 minutes at 121 ℃. After cooling, the cells were inoculated from the slant and incubated at 200rpm and 30 ℃ for 48 hours.

Secondary seeds: 10g/L of yeast extract powder, 15g/L of glucose, 5.4g/L of sodium nitrate and 0.06 percent of diammonium phosphate, wherein the culture medium with the pH value of 7.5,500 ml is put into a 2000 ml shake flask and sterilized for 30 minutes at 121 ℃. After cooling, the mixture was inoculated from a first-stage shake flask with an inoculum size of 10% and cultured at 200rpm for 48 hours at 30 ℃.

2.2 transformation

10L fermenter, load 7L, culture medium: 10g/L of phytosterol, 60g/L of corn steep liquor, 5.4g/L of sodium nitrate and 0.6g/L of diammonium hydrogen phosphate, wherein the pH value is adjusted to 8.0,121 ℃, the mixture is sterilized for 30 minutes, cooled to 30 ℃, and inoculated, and the inoculation amount is 20%.

And (3) conversion control: rotation speed 400rpm, air flow 0.2Nm³H, the tank pressure is 0.05 MPa.

And (3) converting for about 48-72 hours, heating to 80 ℃ after the conversion is finished, keeping the temperature for 30 minutes for inactivation, and cooling to 30 ℃.

2.3 working-up

Adjusting the pH value of a water layer to 1-2 by using hydrochloric acid, adding dichloromethane for extraction for 2 times, adding 0.5 volume of dichloromethane for each time, stirring and extracting for 1 hour, standing for 2 hours, and layering. Separating the lower dichloromethane layer, adding 0.1W of activated carbon, stirring and decoloring at normal temperature for 2 hours, filtering, concentrating the dry solvent under reduced pressure, adding a small amount of water, stirring uniformly, and performing suction filtration to obtain 71 g of light yellow solid, wherein the main component is a compound 2', and the normalization content of the target product is 96.1% through liquid phase detection.

EXAMPLE 4 preparation of Compound 3

Under the protection of nitrogen, 20g of compound 2' is added into a reaction bottle, 80g of acetic anhydride is added, the temperature is raised to 40 ℃, 2g of concentrated sulfuric acid is added, the reaction lasts for about 0.5 to 1 hour, the raw materials are monitored to react completely, the temperature is reduced, the reaction solution is added and transferred to 400ml of ice water for elutriation, the mixture is stirred for 1 to 2 hours and filtered, and a filter cake is dried to obtain the compound 3, wherein the yield is 94.3 percent, and the purity is 98.6 percent.

EXAMPLE 5 preparation of Compound 4

Adding 15g of compound 3, 100ml of cyclohexanone, 4.5g of N-hydroxyphthalimide, 0.0375g of benzoylperoxide and 0.0375g of cobalt acetate tetrahydrate into a reaction bottle, heating to 50-60 ℃, blowing air for reaction, concentrating to remove cyclohexanone after the reaction is completed, adding 300ml of dichloromethane, filtering to remove a catalyst, adding triethylamine and acetic anhydride, reacting for 10 hours at 20-30 ℃, completely adding methanol for quenching, concentrating, adding ethyl acetate for replacing discharge, and drying to obtain compound 4, wherein the yield is 53.6%, and the purity is 99.2%.

EXAMPLE 6 preparation of Compound 5

Under nitrogen protection, 8g of compound 4, 40ml of tetrahydrofuran and 40ml of absolute ethanol, 0.8g of pyridine were added to a reaction flask. Dissolve clear with stirring and then add 0.8g of 10% Pd/C. The system is firstly replaced by nitrogen, then replaced by hydrogen, and finally hydrogenated to complete reaction under normal pressure. After the reaction is finished, the system is completely replaced by nitrogen, and palladium-carbon is filtered. Transferring the filtrate to a reaction kettle, concentrating to be in a viscous state, performing silica gel column chromatography, performing gradient elution, and performing dichloromethane: and (3) combining the pure products with methanol of 30: 1-15: 1, and drying to obtain the compound 5 with the yield of 91.1% and the purity of 99.6%.

EXAMPLE 7 preparation of deoxycholic acid

Under the protection of nitrogen, 5g of compound 5,50 ml of tetrahydrofuran and 5ml of water are added into a reaction bottle, 1.4g of sodium borohydride is added in portions, and the reaction is finished. Dripping 10% hydrochloric acid to dissolve, concentrating tetrahydrofuran, cooling, filtering, drying filter cake, adding mixed solvent of acetonitrile and tetrahydrofuran, recrystallizing, cooling, filtering, and drying to obtain deoxycholic acid with yield of 85% and purity of 99.1%.

The carbon spectrum and hydrogen spectrum of deoxycholic acid are shown in FIGS. 3 and 4.

¹³C NMR(101MHz,MeOD)δ176.95,72.64,71.17,46.75,46.20,42.26,36.08,35.83,35.34,35.11,33.94,33.44,30.97,30.75,29.69,26.11,23.52,22.40,16.25,11.89.

¹H NMR(400MHz,MeOD)δ3.95(s,1H),3.52(s,1H),2.51–2.11(m,2H),2.03–1.69(m,7H),1.70–1.22(m,14H),1.13(dd,J＝27.3,11.5Hz,2H),1.00(d,J＝5.6Hz,4H),0.93(s,3H),0.71(s,3H).

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.