阅读说明：本技术 一种甾体中间体的生物转化制备方法 (Biotransformation preparation method of steroid intermediate ) 是由 杜艳 刘辉 杜江涛 王鹏辉 单东奇 白傲雪 刘娟玲 于 2019-12-13 设计创作，主要内容包括：本发明涉及一种甾体中间体的生物转化制备方法,属于生物制药技术领域。本发明先制备中间体I的甾体混悬液,然后将混悬液投入含有霉菌种子的发酵培养基中,在霉菌的作用下发生C<Sub>11</Sub>位羟化反应,并同时水解C<Sub>21</Sub>位醋酸酯,得到中间体Ⅱ。本发明采用乳化投料法取代了有机溶媒溶解底物投料的操作,投入底物的浓度可以提高到15g/L以上,生物转化率提高到90％以上。采用本方法在乳化的同时加入氮源营养物质,在提高底物投料浓度的情况下使生物转化更彻底,生产成本显著降低。(The invention relates to a biotransformation preparation method of a steroid intermediate, belonging to the technical field of biological pharmacy. The invention firstly prepares steroid suspension of an intermediate I, then puts the suspension into a fermentation medium containing mould seeds, and generates C under the action of the mould 11 By hydroxylation reaction with simultaneous hydrolysis of C 21 And (4) performing ester position reaction to obtain an intermediate II. The invention adopts an emulsification feeding method to replace the operation of dissolving the substrate feeding by an organic solvent, the concentration of the fed substrate can be improved to more than 15g/L, and the biotransformation rate is improved to more than 90%. The method is adopted to add the nitrogen source while emulsifyingThe nutrient substances enable the biotransformation to be more thorough under the condition of improving the concentration of the fed substrate, and the production cost is obviously reduced.)

1. The biotransformation preparation method of the steroid intermediate is characterized in that steroid suspension of the intermediate I is prepared firstly, and then the steroid suspension is put into a fermentation medium containing mould seeds, and the intermediate I generates C under the action of the mould₁₁By hydroxylation reaction with simultaneous hydrolysis of C₂₁And (4) performing ester position reaction to obtain an intermediate II.

2. The method for preparing steroid intermediate by biotransformation according to claim 1, wherein the steroid suspension of intermediate I is prepared by:

(1) dissolving a first nitrogen source substance and a surfactant in a proper amount of water, and heating for sterilization;

(2) and adding the intermediate I, stirring and emulsifying to obtain steroid suspension of the intermediate I.

3. The method for preparing steroid intermediates through biotransformation according to claim 2, wherein the first nitrogen source substance in step (1) comprises one or more of yeast extract powder, peptone, corn steep liquor, yeast powder, and yeast extract; the concentration of the first nitrogen source substance in the fermentation liquor is 2-5 g/L.

4. The method for preparing a steroid intermediate through biotransformation according to claim 2, wherein the surfactant in step (1) comprises one or more of tween-80, span-80, tween-40, tween-20, sodium dodecyl sulfate and sodium dodecyl benzene sulfonate, and the initial concentration of the surfactant in the fermentation broth is 0.5-1.0 g/L.

5. The process for the bioconversion preparation of a steroid intermediate as claimed in claim 1, wherein said intermediate I is:

6. The method for preparing steroid intermediates through biotransformation according to claim 1, wherein the mold is one or more of Metarhizium anisopliae, Rhizopus stolonifer, and Aspergillus ochraceus.

7. The method for preparing steroid intermediates through biotransformation according to claim 1, wherein the fermentation medium comprises: the second nitrogen source material is 20-45 g/L, the glucose is 20-40 g/L, KH₂PO₄1 to 3g/L, MgSO₄0.5-1.5 g/L; the second nitrogen source substance is one or more of yeast extract powder, peptone, corn steep liquor, yeast powder, yeast extract, ammonium sulfate and diammonium hydrogen phosphate.

8. The method for producing a steroid intermediate through biotransformation according to claim 1, wherein the culture temperature is 25 to 32 ℃ and the aeration rate is 0.025 to 0.75vvm after the steroid suspension is introduced into the fermentation broth.

9. The method for preparing steroid intermediates through biotransformation according to claim 1, wherein after the fermentation, the final culture broth is inactivated and cooled to room temperature, and extracted with an organic solvent; the organic solvent is one or more of ethyl acetate, butyl acetate, trichloromethane, dichloromethane, dichloroethane, hexane and heptane.

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a biotransformation preparation method of a steroid intermediate.

Background

Steroid hormone substances are main components of cortical hormone medicaments, betamethasone, dexamethasone and the like are steroid hormone substances, and the steroid hormone substances have pharmacological effects of resisting inflammation, resisting infection, resisting shock, inhibiting immunity and the like. Steroid C₁₁The site α -hydroxylation is an indispensable step for the synthesis of various corticosteroids, and compared with a chemical synthesis method, biotransformation has the outstanding advantages of high transformation efficiency, good specificity and the like, so that the current steroid C₁₁Position α -hydroxylation is usually accomplished by microorganisms through a fermentation process.

The enzyme of the microorganism catalyzing hydroxylation reaction is intracellular enzyme, the steroid substrate crystal must be firstly dispersed and gradually dissolved in water, then absorbed by the microorganism and converted by enzyme catalysis in the cell, and finally the product is discharged out of the cell to form new crystal, thus completing the whole conversion process. It can be seen that in order to increase the feed concentration and the conversion rate, two conditions must be satisfied, firstly, the migration rate of the material between the fermentation broth and the microbial cells is fast, and secondly, the enzymes in the microbial cells can be kept in a high-activity state for a longer time.

Since steroid intermediates are generally less hydrophilic, in C on steroids₁₁In the reaction of α -hydroxylation, in the prior art, a water-soluble organic solvent is used for dissolving a substrate, and then the substrate is put into fermentation liquor, so that the speed of the substrate migrating to the inside of microbial cells is improved by means of the cosolvent effect of the organic solvent while the substrate forms ultrafine microcrystals, and the conversion efficiency is further improved.

C commonly used for steroids₁₁The α -hydroxylation fermentation organic solvent comprises methanol, ethanol, propanol, propylene glycol and the like, although the use of the solvents can improve the conversion efficiency to a certain extent, the organic solvent is harmful to microorganisms, and the microorganism cells can be rapidly aged and lose the conversion capacity to the substrate after being contacted with the organic solvent for a long time, so that the use amount of the organic solvent is limited, the feeding concentration of the process can be naturally bottleneck, meanwhile, the use of the organic solvent can bring serious safety and environmental protection problems, and the current increasingly strict requirements on safety and environmental protection are not beneficial to the industrial popularization of the conversion method.

Chinese patent with publication number CN103146793B reports C added with biocatalyst₁₁α -hydroxylation biotransformation method, but still adopts the mode of ethanol dissolving substrate to feed, the highest concentration of the substrate in the transformation process only reaches 8g/L, and the highest biotransformation rate is only 80%.

Disclosure of Invention

The invention provides a biotransformation preparation method of a steroid intermediate, which can solve one or more of the problems in the prior art.

The present invention is directed to existing C₁₁Preliminary studies have found that the steroid substrate intermediate I is fully emulsified after being mixed with a certain amount of emulsifier, and the method can be used for realizing C in a manner of replacing an organic solvent to dissolve the steroid substrate₁₁α -hydroxylating to obtain a biotransformation rate not lower than that of the prior art under the condition of the same feeding concentrationThe experiment shows that after the steroid is emulsified, if a certain amount of nitrogen source substances are synchronously added during feeding, the biotransformation rate of the steroid is improved to a certain extent.

The final research finds that the best technical scheme is as follows: the nitrogen source substance and the surfactant are mixed and then added into the intermediate I for emulsification, and then the mixture is put into a fermentation medium containing mould seeds for fermentation, so that the biological conversion rate of the steroid is greatly improved. C under the condition that the feeding concentration is increased to 15g/L₁₁The biotransformation rate of α -hydroxylation reaction reaches over 90 percent.

According to one aspect of the invention, a biotransformation preparation method of a steroid intermediate is provided, a steroid suspension of an intermediate I (betamethasone debrominate or dexamethasone debrominate) is prepared firstly, and then is put into a mold preculture solution, wherein the initial concentration of the intermediate I in a fermentation culture medium can reach more than 15 g/L. C generation of intermediate I under action of mould₁₁By hydroxylation reaction with simultaneous hydrolysis of C₂₁Performing acetic ester precipitation to obtain an intermediate II (betamethasone hydroxide or dexamethasone hydroxide), wherein the intermediate II is an important intermediate for producing dexamethasone or betamethasone, and the main reaction is as follows:

the mould used in the biological fermentation process is one or more of metarhizium anisopliae, rhizopus stolonifer and aspergillus ochraceus. The method comprises the following specific steps:

(1) strain culture

Preparing a mold slant culture, inoculating the slant culture into a seed culture medium, and culturing to obtain a mold seed solution; transferring the seed liquid into a fermentation culture medium for fermentation pre-culture to obtain a pre-culture liquid; and (5) standby.

(2) Preparation of steroid suspensions

Preparing a mixed aqueous solution of a surfactant and a first nitrogen source substance, sterilizing the mixed aqueous solution, adding the intermediate I, and fully stirring and emulsifying to obtain steroid suspension for later use.

(3) Biotransformation

And (3) transferring the steroid suspension obtained in the step (2) into the pre-culture solution obtained in the step (1) to obtain initial fermentation liquor, and continuing fermentation culture.

(4) Post-treatment

And (3) after biotransformation is finished, inactivating the final culture solution, cooling to room temperature, extracting with an organic solvent, and concentrating the extract to obtain a product intermediate II.

In some embodiments, to accommodate larger scale production, seed solutions can be divided into shake flask seed solutions, primary seed solutions, and secondary seed solutions. Firstly obtaining shake flask seed liquid through slant strains; then inoculating the shake flask seed solution into a seed culture medium with a larger volume, and culturing to obtain a first-stage seed solution; the first-stage seed liquid is proportionally inoculated into a seed culture medium with a larger volume, and a second-stage seed liquid is obtained after culture and is used for fermentation inoculation.

In some embodiments, the shake flask seed solution is inoculated with a primary seed medium in an amount of 1% to 5%; the inoculation amount of the first-stage seed liquid inoculated second-stage seed culture medium is 1-10 percent; the inoculation amount of the second-stage seed liquid inoculated fermentation culture medium is 5-15%.

In some embodiments, the first seed liquid and the second seed liquid can be omitted, and the shake flask seed liquid is directly inoculated for fermentation, wherein the inoculation amount is 5-15%.

In some embodiments, the secondary seed solution may be omitted, and the primary seed solution is inoculated for fermentation, wherein the inoculation amount is 5-15%.

In some embodiments, the seed medium ratio is: 10-15 g/L of a second nitrogen source substance and 10-15 g/L, KH of glucose₂PO₄1 to 3g/L, KNO₃0.5-1 g/L; the fermentation medium proportion is as follows: the second nitrogen source material is 20-45 g/L, the glucose is 20-40 g/L, KH₂PO₄1 to 3g/L, MgSO₄Is 0.5 to 1.5 g/L.

In some embodiments, the second nitrogen source material in the seed medium and the fermentation medium may be one or more of yeast extract, peptone, corn steep liquor, yeast powder, yeast extract, ammonium sulfate, diammonium phosphate.

In some embodiments, the steroid suspension is prepared by: adding a surfactant and a first nitrogen source substance into a feeding tank, adding water to dissolve, and sterilizing the mixed aqueous solution. Sterilizing the materials in the feeding tank, cooling to below 29 ℃, adding a certain amount of the intermediate I, and stirring for at least 1 hour to fully emulsify the intermediate I to obtain steroid suspension.

In some embodiments, the first nitrogen source material used in the preparation of the steroid suspension comprises one or more of yeast extract, peptone, corn steep liquor, yeast powder, and yeast extract. The concentration of the first nitrogen source substance in the fermentation liquor is 2-5 g/L, and the first nitrogen source substance plays a role in supplementing a nitrogen source in the fermentation process.

In some embodiments, in the preparation process of the steroid suspension, the surfactant used comprises one or more of tween-80, span-80, tween-40, tween-20, sodium dodecyl sulfate and sodium dodecyl benzene sulfonate, the initial concentration of the surfactant in the fermentation liquor is 0.5-1.0 g/L, and the surfactant plays a role in emulsification and dispersion in the preparation process of the steroid suspension.

In some embodiments, the prepared steroid suspension is transferred to a pre-culture solution to obtain an initial fermentation broth, wherein the initial concentration of the surfactant is 0.5-1.0 g/L, the initial concentration of the intermediate I is more than 15g/L, and the concentration of the nitrogen source substance is increased by 2-5 g/L.

In some embodiments, the fermentation pre-culture in step (1) is performed at a temperature of 25-32 ℃ and a ventilation rate of 0.025-0.75 vvm for 8-12 hours. This allows the microbial cells to be sufficiently propagated before the biotransformation, greatly increasing the biomass of the mold, and contributing to the biotransformation.

In some embodiments, the biotransformation in step (3) is carried out at a temperature of 25-32 ℃, a ventilation rate of 0.025-0.75 vvm, and a transformation period of 70-76 hours.

In some embodiments, the post-processing mode is: after fermentation conversion, inactivating the final culture solution, cooling to room temperature, and extracting with organic solvent selected from one or more of ethyl acetate, butyl acetate, chloroform, dichloromethane, dichloroethane, hexane, and heptane. The organic solvent can be selected more, and the quality of the product is not influenced.

The invention has the beneficial effects that:

the mode of preparing steroid suspension and then feeding is adopted to replace the operation of feeding after dissolving a substrate by an organic solvent in the prior art.

In the preparation and fermentation processes of the steroid suspension, the surfactant plays a role in emulsification and dispersion, so that the substrate is dispersed in the fermentation liquor more uniformly, the affinity between the substrate and water is increased, meanwhile, the surfactant can increase the permeability of mould cell walls, and the speed of the mould for absorbing the substrate and discharging products is further increased; in the feeding process, nutrient substances are supplemented for the fermentation strains, and the activity period of microbial cells is prolonged. Thus, under the condition of increasing the feeding concentration, the conversion rate is greatly increased.

The new feeding mode does not use organic solvent at all, avoids the stimulation and damage of the organic solvent to the fermentation strains, is safer and more environment-friendly, and can reduce the investment in explosion-proof equipment and plant construction and obviously reduce the cost.

The feeding concentration of the substrate is improved to more than 15g/L (the highest is 21.4g/L), the conversion rate is improved to more than 90 percent, the production efficiency is improved, and the method is more favorable for industrial production.

Drawings

FIG. 1 is a schematic diagram of the reaction of converting intermediate I into intermediate II according to the present invention.

FIG. 2 is a HPLC detection profile of conversion in example 7 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to examples.