Method for producing prednisolone by one-step fermentation with Nocardia

Abstract

The present invention discloses a method for preparing prednisolone by one-step fermentation, using Nocardia simplex as the fermentation strain, and performing C 1,2 -position dehydrogenation and C 21 -position hydrolysis in one step to convert compound I into compound IV. The optimized fermentation process of the present invention has a conversion rate of 95%-98%, few by-products, a product purity greater than 99.5%, and an overall yield of more than 80%. The present invention uses a powder-liquid mixer device that integrates microcrystallization and substrate sterilization, simplifies the pretreatment process of steroid substrates, improves production efficiency, enhances the substrate sterilization effect, does not use cosolvents, and reduces costs.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation method of steroid drug intermediates, and particularly relates to a method for producing prednisolone by one-step fermentation using Nocardia Background Art

[0002] Prednisolone is an adrenocortical hormone drug, which has anti-inflammatory and anti-allergic effects. Clinically, it can be used for various acute severe bacterial infections, severe allergic diseases, thrombocytopenic purpura, acute lymphoblastic leukemia, various adrenocortical insufficiency diseases, etc. Prednisolone is an important intermediate for synthesizing steroid drugs such as 16α-hydroxy prednisolone and triamcinolone acetonide, and is widely used in the steroid drug field.

[0003] Patent Application No. 201510976860.3 reported a preparation method for directly generating prednisolone by one-step biological fermentation using Arthrobacter simplex, and Patent Application No. 201910557820.3 disclosed a method for producing prednisolone by one-step fermentation using Arthrobacter uratoxydans, realizing the dehydrogenation at the C 1,2 position and the hydrolysis at the C 21 position in one step. However, the reported Arthrobacter transformation of this substrate is not complete. In actual operation, the content of by-products is relatively high. In particular, the polarity of some by-products is very close to that of the main product, causing great difficulties in refining and it is difficult to obtain a product with high purity. Moreover, when using Arthrobacter for dehydrogenation and hydrolysis in one step, there is a certain probability of bacterial contamination in actual fermentation, resulting in substrate degradation. The substrate can be directly degraded into carbon dioxide and water, leading to unstable extraction yield. Therefore, how to provide a high-yield and high-purity production method for preparing prednisolone by one-step fermentation using hydrocortisone acetate is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides a method for producing prednisolone by one-step fermentation of hydrocortisone acetate using Nocardia simplex. The conversion rate can reach 95%-98%, with few by-products, simple refining operation, the product purity is greater than 99.5%, the total yield is 80-82%, and the yield is stable, suitable for industrial large-scale production. For the pretreatment of the steroid fermentation substrate, generally, it is first microcrystallized, then put into a disinfection kettle for pulping and disinfection before feeding. The present invention uses a powder-liquid mixer device that integrates microcrystallization and substrate sterilization, simplifies the pretreatment process of the steroid substrate, improves production efficiency, and does not use a cosolvent to reduce costs. The substrate and water are fully contacted by grinding through a homogenizing emulsifying pump, making the disinfection more thorough and reducing the risk of bacterial contamination caused by incomplete substrate disinfection.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for producing prednisolone by one-step fermentation of hydrocortisone acetate using Nocardia simplex, and the reaction formula is as follows:

[0007]

[0008] Compound Ⅰ (hydrocortisone acetate) Compound Ⅳ (prednisolone)

[0009] The structural formula of the by-product generated during the fermentation process is as follows:

[0010]

[0011] Preferably, the specific steps of the above method for producing prednisolone by one-step fermentation are as follows:

[0012] Slant spore preparation → Seed liquid culture → Fermentation broth culture →

[0013] Add fermentation substrate → Substrate conversion → Terminate fermentation → Extraction and purification of the product.

[0014] Preferably, every 100 ml of the slant medium in the slant spore preparation contains the following components by mass: glucose 0.8 - 1.5 g, yeast extract 0.2 - 0.6 g, peptone 0.2 - 0.6 g, disodium hydrogen phosphate 0.1 - 0.5 g, magnesium sulfate heptahydrate 0.05 - 0.2 g, potassium dihydrogen phosphate 0.1 - 0.3 g, agar 2.0 - 2.5 g, with a pH of 7.0 - 7.6, a culture temperature of 28°C - 32°C, and a culture time of 30 - 50 h.

[0015] Preferably, every 1 L of the seed liquid medium in the seed liquid culture contains the following components by mass: beef extract 2 - 5 g, peptone 3 - 7 g, antifoaming agent 0.1 - 0.3 g, with a pH of 7.0 - 7.5, a culture temperature of 28 - 32°C, and shake flask culture at 150 - 200 r / min for 15 h - 24 h.

[0016] Preferably, every 1 L of the fermentation broth medium in the fermentation broth culture contains the following components by mass: glucose 8 - 15 g, corn steep liquor 8 - 20 g, potassium dihydrogen phosphate 2 - 5 g, peptone 2 - 6 g, yeast extract 0.5 - 2 g, antifoaming agent 0.1 - 0.3 g, and the pH value of the fermentation broth is 6.5 - 7.5.

[0017] Preferably, the inoculation amount in the fermentation broth culture is 10% - 25% of the weight of the fermentation broth, the culture temperature is 28 - 32°C, the stirring speed is 280 - 400 rpm, the air flow rate is 0.3 - 0.6 vvm, the pressure in the fermenter is 0.02 - 0.06 MPa, and the culture time is 3 - 10 h.

[0018] Preferably, the weight of the input substrate hydrocortisone acetate is 1% - 6% of the weight of the fermentation broth.

[0019] Preferably, before feeding, the fermentation substrate needs to be pretreated by a feed liquid mixer device integrating microcrystallization and substrate sterilization. The specific operation is as follows:

[0020] Water, defoamer and substrate are added into the disinfection kettle in sequence. The weight of water is 5 - 10 times that of the substrate, and the weight of the defoamer is 0.02% - 0.06% of the weight of the fermentation broth. The substrate is microcrystallized by a self - priming high - shear dispersion emulsifier and a three - stage pipeline homogenizing emulsifier pump. Samples are taken after 1 - 10 h to measure the particle size distribution. The particle size of the material is controlled so that D98 ≤ 30 μm. After passing the qualification, the temperature is slowly raised to 115 - 121 °C for disinfection for 20 min, and then cooled to 25 - 32 °C for standby feeding.

[0021] The mixers involved above include the self - priming high - shear dispersion emulsifier and the 132KW three - stage pipeline homogenizing emulsifier pump produced by Nantong Claire Co., Ltd., the homogenizing emulsifier pump of Deli Shi Pump Industry Co., Ltd., etc.

[0022] Preferably, after adding the substrate, the air flow rate is controlled at 0.1 - 0.3 vvm, the stirring speed is 280 - 400 rpm, and the fermentation temperature is 28 - 32 °C. Continuous fermentation is carried out for 20 - 60 h. Samples are taken every 8 - 12 h during fermentation for HPLC analysis. Fermentation is terminated after the conversion rate is greater than 95%.

[0023] Preferably, the extraction and purification are specifically as follows:

[0024] The pH value of the obtained fermentation broth is adjusted to 2.0 - 3.0 with one of sulfuric acid, hydrochloric acid, phosphoric acid or oxalic acid. The temperature is raised to 70 - 90 °C and kept warm for 2 - 4 h, then cooled to 30 °C - 40 °C for filtration. The filtrate is extracted with ethyl acetate until there is no material left, and then concentrated to a small volume. After phase change with toluene and leaving the mother liquor, it is filtered and dried to obtain the aqueous phase material. After the filter cake of the fermentation broth is dried, it is repeatedly extracted with a mixed solvent of chloroform:methanol = 3:1 with a volume 10 - 20 times the weight until there is no material left in the bacterial residue. Then the aqueous phase material is added to the extraction mixed solvent and concentrated to a thick porridge - like state together. After phase change with toluene and leaving the mother liquor, it is filtered and dried to obtain prednisolone.

[0025] Through the above - mentioned technical solutions, compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] A method for preparing prednisolone by one - step fermentation of hydrocortisone acetate is provided. Using Nocardia simplex for fermentation, it has a high conversion rate, few by - products, high product purity, high and stable yield, and is suitable for industrial production. In addition, a pretreatment method for fermentation substrate integrating microcrystallization and substrate sterilization is provided, which simplifies the pretreatment process of steroid substrate, enhances the substrate sterilization effect, does not use cosolvents, saves costs, and has certain reference significance for the pretreatment of fermentation substrates of other compounds. Description of the Drawings

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0028] Figure 1 It is the transformation map of Compound I Hydrocortisone Acetate in the fermentation broth of Nocardioides simplex in Example 1;

[0029] Figure 2 It is the map after extraction and purification of the aqueous phase after filtration of the fermentation of Compound I Hydrocortisone Acetate by Nocardioides simplex in Example 1;

[0030] Figure 3 It is the map after combined purification of the crude extract with bacteria and the aqueous phase material after filtration of the fermentation of Compound I Hydrocortisone Acetate by Nocardioides simplex in Example 1;

[0031] Figure 4 It is the transformation map of Compound I Hydrocortisone Acetate in the fermentation broth of Nocardioides simplex in Example 2. Detailed implementation manners

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0033] The Nocardioides simplex used in the present invention is Nocardioides simplex, (Beijing Bio-win Biotechnology Co., Ltd., platform number bio-106997, other numbers NBRC12069 = ATCC6946);

[0034] The injection information for HPLC analysis is flow rate: 1.0 ml / min, column: 250 * 4.6 mm C18 water: (methanol / acetonitrile 1:1);

[0035] The self-priming high-shear dispersion emulsifier and the 132KW three-stage pipeline homogenizing emulsifier pump are both purchased from Nantong Claire Mixing Equipment Co., Ltd.

[0036] Example 1

[0037] (1) Strain culture: Pick Nocardia simplex and inoculate it on a slant medium in a 100 ml / 500 ml eggplant bottle. Incubate it upside down at 30 °C for 48 h. After the incubation, store it in a refrigerator at 5 °C for later use. Each 100 ml of the slant medium contains the following mass components: 1.2 g of glucose, 0.35 g of yeast extract, 0.5 g of peptone, 0.3 g of disodium hydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.2 g of potassium dihydrogen phosphate, 2.5 g of agar, and pH 7.5.

[0038] (2) Seed liquid culture: Wash the strain in the above-mentioned eggplant bottle with 100 ml of sterile water. Take 10 ml and inoculate it into the seed liquid. Prepare 1 L of seed liquid medium in a 5 L baffled shake flask. Place it in a shaker at 30 °C and 160 r / min and shake and culture for 20 h. Take a sample to detect the enzyme activity, that is, use the 2,3,5-triphenyltetrazolium chloride method to detect that the seed liquid turns bright red within 1.0 min. Microscopic examination shows no contaminants, the strain is completely divided, and the cell shape is flat, round, and thick, conforming to the growth characteristics in the middle and late logarithmic phase, waiting for transfer. Each 1 L of the seed liquid medium contains the following mass components: 3 g of beef extract, 6 g of peptone, 0.2 g of antifoaming agent, and pH value 7.5.

[0039] (3) Fermentation broth culture: Prepare 5 L of fermentation broth in a 10 L fermenter. After autoclaving at 121 - 123 °C for 20 min, cool it down to about 30 °C and wait for inoculation. Each 1 L of the fermentation broth medium contains the following mass components: 10 g of glucose, 12 g of corn steep liquor, 2.5 g of potassium dihydrogen phosphate, 5 g of peptone, 1.5 g of yeast extract, 0.2 g of antifoaming agent, and the pH value of the fermentation broth is 6.5; After the seed liquid is detected to be qualified, inoculate it into the fermenter by the flame inoculation method. The transfer amount is about 16%. The culture temperature is 30 °C, the stirring speed of the 10 L tank is 300 rpm, the air flow rate is 0.4 vvm, the tank pressure is 0.05 MPa. After culturing for 8 h, take a sample to detect the enzyme activity and sterility qualification, and prepare to add the substrate.

[0040] (4) Pretreatment and feeding of the substrate: Add water, antifoaming agent, and the substrate to the substrate disinfection kettle in sequence. The weight of water is 5 times the weight of the substrate, the weight of the antifoaming agent is 0.05% of the weight of the fermentation broth, and the weight of the substrate hydrocortisone acetate is 5% of the weight of the fermentation broth. Use a self-priming high-shear dispersion emulsifier and a three-stage pipeline homogenizing emulsifier to microcrystallize the substrate. After circulating for 3 h, measure the particle size distribution. The particle size of the material D99 ≤ 30 μm. After passing the qualification, slowly heat it up to 115 - 121 °C for disinfection for 20 min, and cool it down to 25 - 32 °C for standby feeding.

[0041] (5) Conversion of the substrate: Add the substrate to the fermentation broth, adjust the air flow rate to 0.02 vvm, the fermenter pressure to 0.05 MP, and the fermentation temperature to 30 °C. Continuously ferment for 24 h. During the fermentation process, take a sample for HPLC analysis every 12 h. The conversion rate at 60 h is as Figure 1As shown, where 6.7 min is the product compound Ⅳ prednisolone with a relative peak area of 95.29%, so the conversion rate is 95.29% and the conversion is terminated; in addition, from Figure 1 it can be seen that 18.9 min is the by-product compound Ⅱ with a relative peak area of 0.75%, 20.6 min is the compound Ⅰ hydrocortisone acetate with a relative peak area of 2.89%, and there is basically no by-product Ⅲ.

[0042] (6) Extraction and refinement: Adjust the pH value of the fermentation broth to about 2.5 with hydrochloric acid, heat it to about 85 °C, keep it warm for 3 h, and then cool it to about 30 °C for filtration. The aqueous phase filtered from the fermentation broth is extracted with ethyl acetate until there is no material, and then concentrated to a small volume. After changing the phase with toluene and leaving the mother liquor, it is filtered. The filter cake of the aqueous phase material is dried and the purity is detected as shown in the appendix Figure 2 As shown, where 6.5 min is the product compound Ⅳ prednisolone with a relative peak area of 98.5%, so the purity is 98.51%. In addition, from Figure 2 it can be seen that 18.8 min is the by-product compound Ⅱ with a relative peak area of 0.48%. The crude product with bacteria filtered from the fermentation broth is dried and repeatedly extracted with 15 times the weight of a mixed solvent (chloroform: methanol = 3:1) until there is no material in the bacterial residue. The aqueous phase material is put into this extraction mixed solvent, and the two are concentrated to a thick porridge state. After changing the phase with toluene and leaving the mother liquor, it is filtered and dried to obtain the refined product of compound Ⅳ prednisolone. The product purity is as shown in Figure 3 As shown, where 6.4 min is the product compound Ⅳ prednisolone with a relative peak area of 99.8%, so the product purity is 99.8%, and it is qualified for warehousing. The weight yield of the refined product is calculated to be 80.1%, and the total yield of collecting the mother liquor and returning it for feeding is 81.2% (the theoretical yield is 89.6%).

[0043] Example 2

[0044] (1) Slant seed culture: Pick Nocardia simplex and inoculate it on a slant medium in a 100 ml / 500 ml eggplant bottle, and culture it in an inverted position at 30 °C for 48 h. After the culture is completed, store it in a refrigerator at 5 °C for standby. Each 100 ml of the slant medium includes the following mass components: 1.0 g of glucose, 0.4 g of yeast extract, 0.4 g of peptone, 0.4 g of disodium hydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.2 g of potassium dihydrogen phosphate, 2.5 g of agar, and PH 7.5.

[0045] (2) Seed culture: Wash the strain in one eggplant bottle above with 100 ml of sterile water, take 10 ml and inoculate it into the seed culture. Prepare 1.2 L of seed culture medium in a 5 L baffled shake flask, place it in a shaker at 30 °C and 180 r / min for oscillating culture for 18 h. Take samples to detect the enzyme activity, that is, use the 2,3,5-triphenyltetrazolium chloride method to detect that the seed culture turns bright red within 1.0 min. Microscopic examination shows no contaminants, the strain has completely divided, and the cell shape is flat, round and thick, conforming to the growth characteristics in the middle and late logarithmic phase, waiting for transfer. Each 1 L of seed culture medium includes the following mass components: 3 g of beef extract, 5 g of peptone, 0.2 g of antifoaming agent, and the pH value is 7.5.

[0046] (3) Fermentation broth culture: Prepare 4.8 L of fermentation broth in a 10 L fermenter, sterilize it with high-pressure steam at 121 - 123 °C for 20 min, and then cool it to about 30 °C and wait for inoculation. Each 1 L of fermentation broth culture medium includes the following mass components: 12 g of glucose, 12 g of corn steep liquor, 2.5 g of potassium dihydrogen phosphate, 4 g of peptone, 1.0 g of yeast extract, 0.2 g of antifoaming agent, and the pH value of the fermentation broth is 7.0; After the seed culture is detected to be qualified, inoculate it into the fermenter by the flame inoculation method, and the transfer amount is about 20%. The culture temperature is 30 °C, the stirring speed of the 10 L tank is 350 rpm, the air flow rate is 0.4 vvm, the tank pressure is 0.05 MPa, take samples to detect the enzyme activity and sterility qualification after culturing for 5 h, and prepare to add the substrate.

[0047] (4) Pretreatment and feeding of the substrate: Add water, antifoaming agent and the substrate into the substrate disinfection kettle in sequence, where the weight of water is 6 times the weight of the substrate, the weight of the antifoaming agent is 0.05% of the weight of the fermentation broth, and the weight of the substrate hydrocortisone acetate is 5% of the weight of the fermentation broth. Use a self-priming high-shear dispersion emulsifier and a three-stage pipeline homogenizing emulsifier to microcrystallize the substrate, measure the particle size distribution after circulating for 3 h, the particle size of the material D99 ≤ 30 μm, after passing the test, slowly heat it up to 115 - 121 °C for disinfection for 20 min, and cool it to 25 - 32 °C for standby feeding.

[0048] (5) Conversion of the substrate: Add the substrate to the fermentation broth, adjust the air flow rate to 0.025 vvm, the pressure of the fermenter to 0.05 MP, the fermentation temperature to 30 °C, and continuously ferment for 24 h. Take samples for HPLC analysis every 12 h during the fermentation process. The conversion rate at 60 h is as Figure 4 shown, where 6.7 min is the product compound Ⅳ prednisolone, and the relative peak area is 97.48%, which is 97.48%, and terminate the conversion; in addition, as Figure 4 known, 18.9 min is the by-product compound Ⅱ, and the relative peak area is 1.51%.

[0049] (6) Extraction and refinement: Adjust the pH value of the fermentation broth to about 2.0 with hydrochloric acid, heat it up to about 85 °C, keep it warm for 3 h, and then cool it to about 30 °C for filtration. The aqueous phase filtered from the fermentation broth is extracted with ethyl acetate until there is no material left, and then concentrated to a small volume. After replacing the phase with toluene and leaving the mother liquor, the aqueous phase material is filtered out. The crude product with bacteria filtered from the fermentation broth is dried and repeatedly extracted with 20 times its weight of a mixed solvent (chloroform: methanol = 3:1) until there is no material left in the bacterial residue. The aqueous phase material is added to this extraction mixed solvent, and the mixture is concentrated to a thick porridge-like state. After replacing the phase with toluene and leaving the mother liquor, it is filtered and dried to obtain the prednisolone fine product. The product purity is 99.83%, and it is qualified for warehousing. Calculate that the weight yield of the fine product is 80.8%, and the total yield of collecting the mother liquor and recycling it is 81.9%.

[0050] Example 3

[0051] (1) Slant seed culture: Pick Nocardia simplex and inoculate it on a slant medium in a 500 ml eggplant bottle with 100 ml of medium. Incubate it upside down at 32 °C for 40 h. After the incubation, store it in a refrigerator at 5 °C for standby. Each 100 ml of the slant medium contains the following mass components: 1.5 g of glucose, 0.2 g of yeast extract, 0.3 g of peptone, 0.2 g of disodium hydrogen phosphate, 0.05 g of magnesium sulfate heptahydrate, 0.2 g of potassium dihydrogen phosphate, 2.5 g of agar, and pH 7.5.

[0052] (2) Seed liquid culture: Wash the bacteria in one eggplant bottle above with 100 ml of sterile water, take 10 ml and inoculate it into the seed liquid. Prepare 1.2 L of seed liquid medium in a 5 L baffled shaking flask, place it in a shaker at 30 °C and 180 r / min for oscillation culture for 18 h. Take a sample to detect the enzyme activity, that is, use the 2,3,5-triphenyltetrazolium chloride method to detect that the seed liquid turns bright red within 1.0 min. Microscopic examination shows no contaminants, the bacteria are completely divided, and the bacterial shape is flat, round, and thick, conforming to the growth characteristics in the middle and late logarithmic phase, and waiting for transfer. Each 1 L of the seed liquid medium contains the following mass components: 4 g of beef extract, 4 g of peptone, 0.2 g of antifoaming agent, and pH value 7.5.

[0053] (3) Fermentation broth culture: Prepare 4.8 L of fermentation broth in a 10 L fermenter, sterilize it with high-pressure steam at 121 - 123 °C for 20 min, and then cool it to about 30 °C and wait for inoculation. Each 1 L of the fermentation broth medium contains the following mass components: 15 g of glucose, 15 g of corn steep liquor, 3.0 g of potassium dihydrogen phosphate, 3.0 g of peptone, 2.0 g of yeast extract, 0.2 g of antifoaming agent, and the pH value of the fermentation broth is 7.0; After the seed liquid is detected to be qualified, it is inoculated into the fermenter by the flame inoculation method, and the transfer amount is about 20%. The culture temperature is 30 °C, the stirring speed of the 10 L tank is 350 rpm, the air flow rate is 0.4 vvm, the tank pressure is 0.05 MPa. After culturing for 3 h, take a sample to detect the enzyme activity and sterility to be qualified, and prepare to add the substrate.

[0054] (4) Pretreatment and feeding of the substrate: Water, defoamer, and substrate are sequentially added to the substrate disinfection kettle. The weight of water is 5 times that of the substrate, the weight of the defoamer is 0.05% of the weight of the fermentation broth, and the weight of the substrate hydrocortisone acetate is 5% of the weight of the fermentation broth. The substrate is micronized using a self-priming high-shear dispersion emulsifier and a three-stage pipeline homogenizing emulsifier. After circulating for 4 h, the particle size distribution is measured. The particle size D99 of the material is ≤ 30 μm. After passing the test, the temperature is slowly raised to 115 - 121 °C for disinfection for 20 min, and then cooled to 25 - 32 °C for standby feeding.

[0055] (5) Conversion of the substrate: The substrate is added to the fermentation broth, the air flow rate is adjusted to 0.025 vvm, the pressure in the fermenter is 0.05 MP, the fermentation temperature is 30 °C, and continuous fermentation is carried out for 24 h. Samples are taken every 12 h during the fermentation process for HPLC analysis. The conversion rate is 97.02% after 60 h, and the conversion is terminated.

[0056] (6) Extraction and purification: The pH value of the fermentation broth is adjusted to about 2.0 with hydrochloric acid, heated to about 85 °C, held for 3 h, and then cooled to about 30 °C for filtration. The aqueous phase filtered from the fermentation broth is extracted with ethyl acetate until there is no material left, and then concentrated to a small volume. After the toluene phase change and leaving the mother liquor, the aqueous phase material is filtered out. The crude product with bacteria filtered from the fermentation broth is dried and repeatedly extracted with 20 times the weight of a mixed solvent (chloroform: methanol = 3:1) until there is no material left in the bacterial residue. The aqueous phase material is added to this extraction mixed solvent, and the mixture is concentrated to a thick porridge state. After the toluene phase change and leaving the mother liquor, it is filtered and dried to obtain the prednisolone fine product. The product purity is 99.71%, and it is qualified for warehousing. The weight yield of the fine product is calculated to be 80.5%, and the total yield of collecting the mother liquor and returning it for feeding is 81.7%.

[0057] In this specification, the various embodiments are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the embodiments can be referred to each other. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple. For the relevant parts, reference can be made to the description in the method section.

[0058] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for producing prednisolone by one-step fermentation using Nocardia, characterized in that, The specific steps are as follows: Inclined-plane strain preparation → Seed liquor culture → Fermentation broth culture → Feeding of fermentation substrate → Substrate conversion → Termination of fermentation → Extraction and purification of product; Among them: The Nocardia is strain ATCC6946; In the fermentation broth culture, the inoculation amount is 10%-25% of the weight of the fermentation broth, the culture temperature is 28-32°C, the stirring speed is 280-400 rpm, the air flow rate is 0.3-0.6 vvm, the pressure in the fermenter is 0.02-0.06 MPa, and the culture time is 3-10 h; The weight of the fed substrate hydrocortisone acetate is 5%-6% of the weight of the fermentation broth; Before feeding, the fermentation substrate needs to be pretreated by a feed liquid mixing device that combines microcrystallization and substrate sterilization. The specific operation is as follows: In the disinfection kettle, water, antifoaming agent and substrate are added in sequence. The weight of water is 5-10 times the weight of the substrate, and the weight of the antifoaming agent is 0.02%-0.06% of the weight of the fermentation broth; The substrate is microcrystallized with a self-priming high-shear dispersion emulsifier and a three-stage pipeline homogenizing emulsifier. The particle size distribution is measured by sampling after 1-10 h, and the particle size of the material is controlled at D98≤30 μm. After passing the test, it is heated to 115-121°C for disinfection for 20 min, and then cooled to 25-32°C for standby feeding; After feeding the substrate, control the air flow rate at 0.1-0.3 vvm, the stirring speed at 280-400 rpm, and the fermentation temperature at 28-32°C, and continuously ferment for 24-60 h; The extraction and purification are specifically as follows: Adjust the pH value of the obtained fermentation broth to 2.0-3.0 with one of sulfuric acid, hydrochloric acid, phosphoric acid or oxalic acid, heat it to 70-90°C and keep it warm for 2-4 h, then cool it to 30°C-40°C and filter. The filtrate is extracted with ethyl acetate until there is no material, and then concentrated to a small volume. After changing the phase with toluene and leaving the mother liquor, filter and dry to obtain the aqueous phase material; After drying the filter cake of the fermentation broth, extract it repeatedly with a mixed solvent of chloroform:methanol = 3:1 with a volume ratio of 10-20 times the weight until there is no material in the bacterial residue. Then add the aqueous phase material to the extraction mixed solvent and concentrate it to a thick porridge state together. Change the phase with toluene and leave the mother liquor, filter and dry to obtain prednisolone.

2. The method for producing prednisolone by one-step fermentation with Nocardia according to claim 1, characterized in that, In the preparation of the inclined-plane seeds, every 100 mL of the inclined-plane culture medium contains the following components in mass: glucose 0.8-1.5 g, yeast extract 0.2-0.6 g, peptone 0.2-0.6 g, disodium hydrogen phosphate 0.1-0.5 g, magnesium sulfate heptahydrate 0.05-0.2 g, potassium dihydrogen phosphate 0.1-0.3 g, agar 2.0-2.5 g, PH is 7.0-7.6, the culture temperature is 28°C-32°C, and the culture time is 30-50 h.

3. The method for producing prednisolone by one-step fermentation using Nocardia according to claim 1, characterized in that, In the seed liquor culture, every 1 L of the seed liquor culture medium contains the following components in mass: beef extract 2-5 g, peptone 3-7 g, antifoaming agent 0.1-0.3 g, PH is 7.0-7.5, the culture temperature is 28-32°C, and it is shaken and cultured at 150-200 r / min on a shaker for 15-24 h.

4. The method for producing prednisolone by one-step fermentation using Nocardia according to claim 1, characterized in that, In the fermentation broth culture, every 1 L of the fermentation broth culture medium contains the following components in mass: glucose 8-15 g, corn steep liquor 8-20 g, potassium dihydrogen phosphate 2-5 g, peptone 2-6 g, yeast extract 0.5-2 g, antifoaming agent 0.1-0.3 g, and the pH value of the fermentation broth is 6.5-7.

5.

5. The method for producing prednisolone by one-step fermentation using Nocardia according to claim 1, characterized in that, Samples were taken every 8 - 12 h during the fermentation process for HPLC analysis, and the fermentation was terminated after the conversion rate was greater than 95%.

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