A method for producing dexamethasone epoxy hydrolysate by microbial fermentation

Abstract

The present invention provides a method for producing dexamethasone epoxy hydrolysate by microbial fermentation. The method uses Glutamicibacter sp., which has a preservation number of CGMCC No. 30439, and uses compound 1 as a raw material to convert it into dexamethasone epoxy hydrolysate in one go. The present invention also optimizes the carbon source and dosage of the fermentation medium used in the method, the timing and amount of substrate addition, and the pH control range of the fermentation system after substrate addition. The method for producing dexamethasone epoxy hydrolysate by microbial fermentation of the present invention has high feed amount, high substrate conversion rate, and few impurities.

Description

Technical Field

[0001] The invention relates to a method for producing dexamethasone epoxide hydrolyzate by microbial fermentation. Background Art

[0002] Dexamethasone epoxy hydrolysate is a key intermediate in the production process of dexamethasone. It plays an important role in the synthesis of dexamethasone, a synthetic glucocorticoid widely used for anti-inflammatory and immunosuppression. With the widespread use of dexamethasone in the medical field, the market demand for compound 8DM has also increased. In addition, in addition to being used to synthesize dexamethasone, compound 8DM is also used in the synthesis of high-end glucocorticoid drugs such as flumethasone and mometasone furoate.

[0003] With the development of glucocorticoid drugs, the research and development and production process of compound 8DM has undergone continuous optimization and improvement. At present, it has gradually shifted from traditional chemical synthesis methods to more efficient and environmentally friendly biological fermentation methods.

[0004] Compound 8DM tradition Production The method route is as follows:

[0005]

[0006] At present, the microbial fermentation production method of compound 8DM generally dehydrogenates and hydrolyzes the acetate ester of the substrate at the same time, and usually uses simple Bacillus as the production strain of the biological fermentation method. During the fermentation process, the generation and purity of compound 8DM are affected by many factors, such as culture temperature, pH value, raw material quality, culture time, solvent selection and other factors, which may affect the generation efficiency and final purity of compound 8DM. At present, the microbial fermentation production method of compound 8DM still has many problems in cost and conversion process. The most important problem is that there are many impurities in the conversion process, which mainly include substrates, substrate hydrolysates and acetate esters of dexamethasone epoxy hydrolysates.

[0007] There are other problems in the preparation of compound 8DM by biological fermentation. For example, the prior art CN106520896A discloses a method for preparing dexamethasone intermediates by one-step fermentation conversion of microorganisms. The method uses simple arthrobacter and Bacillus megaterium to produce compound 8DM by mixed fermentation. The method requires the preparation of two production strains, and the process is lengthy and the operation is complicated. The prior art CN110066846A and the prior art CN110157764A both disclose a method for obtaining compound 8DM by one-step fermentation of simple arthrobacter. The fermentation process of the former uses a solubilizer (any one of Tween-80, methanol, ethanol, acetone, DMF, and DMSO) to dissolve the substrate for feeding. Although this method improves the conversion rate of the target product, the substrate feeding amount is only 1 to 6 g / L relative to the fermentation broth. The latter introduces 0.1 to 0.2% coenzyme A for feeding while using methanol to dissolve the substrate. Although this strategy also successfully improves the conversion rate, the high cost of coenzyme A limits its application in industrial production.

[0008] Therefore, further research is needed on the relevant factors in the transformation process of microbial fermentation to produce compound 8DM in order to obtain a fermentation production method with lower cost and higher yield. Summary of the invention

[0009] In view of the problems existing in the process of producing dexamethasone epoxy hydrolysate (compound 8DM) by microbial fermentation in the prior art, the present invention provides a method for producing dexamethasone epoxy hydrolysate (compound 8DM) by microbial fermentation. The method has a high substrate feed amount, a high conversion rate, and few impurities. The method of producing dexamethasone epoxy hydrolysate by microbial fermentation of the present invention comprises the steps of:

[0010] Using Glutamicibacter sp., with a collection number of CGMCC No. 30439, compound 1 was used as a raw material to transform it into compound 8DM in one step:

[0011] The conversion process is:

[0012]

[0013] In another preferred embodiment, the carbon source of the fermentation medium is glucose.

[0014] In another preferred embodiment, the fermentation medium, based on its total volume, comprises the following components:

[0015] Glucose 10-20 g / L, yeast extract 4-8 g / L, potassium dihydrogen phosphate 0.5-3.5 g / L, magnesium sulfate 0.1-0.3 g / L.

[0016] In another preferred embodiment, the yeast extract is Angel FM802.

[0017] In another preferred embodiment, after the Glutamicibacter sp. is fermented and cultured for 2 to 5 hours, Compound 1 is added to the fermentation culture solution for the first time; after the Glutamicibacter sp. is fermented and cultured for 5 to 15 hours, Compound 1 is added to the fermentation culture solution for the second time.

[0018] In another preferred embodiment, the volume ratio of the compound 1 added to the fermentation broth for the first time is 3-10 g / L, and the volume ratio of the compound 1 added to the fermentation broth for the second time is 80-110 g / L.

[0019] In another preferred embodiment, the second addition of compound 1 to the fermentation broth is carried out in three batches, and the amount of compound 1 added in each batch is 1 / 2 to 1 / 3 of the total amount of compound 1 added to the fermentation broth for the second time, wherein the interval between the first batch of compound 1 and the second batch of compound 1 is 3 to 5 hours, the interval between the second batch of compound 1 and the third batch of compound 1 is 2 to 4 hours, and the interval between the first batch of compound 1 and the first batch of compound 1 added to the fermentation broth is 2 to 25 hours. In another more preferred embodiment, the interval between the first batch of compound 1 and the first batch of compound 1 added to the fermentation broth is 2 to 5 hours.

[0020] In another preferred embodiment, before adding compound 1 to the fermentation broth for the second time, the pH of the fermentation system is regulated to 6.8-7.2, and after adding compound 1 to the fermentation broth for the second time, the pH of the fermentation system is regulated to 8.8-9.2.

[0021] In another preferred embodiment, during the fermentation culture of the Glutamicibacter sp., the fermentation culture temperature is 25-35° C., the tank pressure is 0.02-0.07 MPa, the flow rate is 0.5-2.5 vvm, the rotation speed is 100-750 rpm, and the dissolved oxygen is >30%.

[0022] In another preferred embodiment, the method for producing dexamethasone epoxide hydrolyzate (compound 8DM) by microbial fermentation provided by the present invention comprises the steps of:

[0023]

[0024] Glutamicibacter sp., with a deposit number of CGMCC No.30439, is cultured to obtain mature seed liquid, which is then inoculated into a fermentation medium. After 4 to 5 hours of fermentation, compound 1 is added to the culture liquid for the first time. After 5 to 15 hours of fermentation, compound 1 is added to the culture liquid for the second time.

[0025] The fermentation medium, based on its total volume, contains 10-20 g / L of glucose, 4-8 g / L of yeast extract, 0.5-3.5 g / L of potassium dihydrogen phosphate, 0.1-0.3 g / L of magnesium sulfate and water.

[0026] Before adding compound 1 to the fermentation broth for the second time, the pH of the fermentation system is regulated to be 6.8-7.2, and after adding compound 1 to the fermentation broth for the second time, the pH of the fermentation system is regulated to be 8.8-9.2.

[0027] The volume ratio of the compound 1 added to the fermentation broth for the first time to the fermentation broth is 3 to 10 g / L, and the volume ratio of the compound 1 added to the fermentation broth for the second time to the fermentation broth is 80 to 110 g / L.

[0028] The second addition of compound 1 to the culture medium is carried out in three batches, and the amount of compound 1 added in each batch is 1 / 2 to 1 / 3 of the total amount of compound 1 added to the culture medium for the second time, wherein the interval between the first batch of compound 1 and the second batch of compound 1 is 3 to 5 hours, the interval between the second batch of compound 1 and the third batch of compound 1 is 2 to 4 hours, and the interval between the first batch of compound 1 and the first addition of compound 1 to the fermentation culture medium is 2 to 5 hours, and

[0029] The fermentation culture temperature is 25-35° C., the tank pressure is 0.02-0.07 MPa, the flow rate is 0.5-2.5 vvm, the rotation speed is 100-750 rpm, and the dissolved oxygen is >30%.

[0030] In another preferred embodiment, in the fermentation broth obtained by the method for producing dexamethasone epoxy hydrolyzate by microbial fermentation of the present invention, the conversion rate of compound 1 is 98.23%, impurity 2 is not contained, and the total content of impurities 1 and impurity 3 is 1.77% (determined by HPLC).

[0031]

[0032] The invention also provides a Glutamicibacter sp., which is preserved in the China General Microbiological Culture Collection Center, and its preservation number is CGMCC No.30439.

[0033] The present invention also provides the use of the Glutamicibacter sp., with a preservation number of CGMCC No. 30439, which converts compound 1 into compound 8DM in one step.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the HPLC spectrum of the final fermentation broth obtained in Example 1, wherein the content of the product (compound 8DM) is 75.68%, the contents of impurity 1, impurity 2 and impurity 3 are all obvious, especially the content of impurity 2 is 11.49%, and the content of impurity 3 is 9.00%.

[0036] Figure 2 This is the HPLC spectrum of the final fermentation broth obtained in Example 18, wherein the content of the product (Compound 8DM) is 98.23%, there is no impurity 2, and the contents of impurity 1 and impurity 3 are both relatively low, at 0.34% and 1.43%, respectively. DETAILED DESCRIPTION

[0037] In view of the problems existing in the process of producing dexamethasone epoxy hydrolysate, compound 8DM, by microbial fermentation in the prior art, the inventors of the present application used Glutamicibacter sp., whose deposit number is CGMCC No. 30439, and used compound 1 as a raw material to convert it into the target product compound 8DM in one go. By adjusting the components and ratios of the culture medium and optimizing the fermentation conditions, the substrate feed amount was increased to 83-120 g / L (the weight-to-volume ratio of the substrate to the fermentation liquid) without using any solubilizer or expensive coenzyme, and the total impurity content was reduced to less than 2% while achieving a high conversion rate (98%), thereby achieving low-cost and high-yield fermentation production of compound 8DM.

[0038] As described herein, "yeast extract" can be prepared using methods well known in the art, or can be obtained through commercial channels, for example, including but not limited to Angel FM802.

[0039] As described herein, "mature seeds" refer to the above-mentioned Glutamicibacter sp. obtained by culturing in a seed culture medium, with a maturity standard pH of 7.0-7.5 and an OD of 1.5-2.0. In a specific embodiment of the present invention, the preparation process of "mature seeds" is as follows: the above-mentioned Glutamicibacter sp. is spread on a slant culture medium (the slant culture medium is prepared with purified water, comprising components: soluble starch 15-21 g / L, yeast extract 3-7 g / L, peptone 8-12 g / L, agar powder 18-22 g / L, pH 6.8-7.2, and a 10-30% NaOH solution is used before sterilization to adjust the pH to 6.8-7.2, and sterilize at 120-125°C with high-pressure steam for 15-25 min), and the mature bacteria are obtained by culturing at 28-32°C for 2-4 days. The mature bacteria are cultured in a seed culture medium (the seed culture medium is prepared with tap water, comprising the following components: 3-7 g / L yeast extract powder, 8-12 g / L peptone, 8-12 g / L sodium chloride, adjusted to pH 6.8-7.2 with a 10-30% NaOH solution before sterilization, and sterilized with high-pressure steam at 120-125° C. for 15-25 min) at 28-32° C. and 220-260 rpm for 2-4 days to obtain mature seeds.

[0040] After the fermentation broth in which the substrate (Compound 1) is completely converted is obtained by the method of the present invention, it can be processed by conventional methods in the art to obtain the target compound. For example, the fermentation broth is centrifuged or filtered to obtain a precipitate, the precipitate is dissolved in an organic solvent, the target compound is dissolved in the organic solvent, and the solution containing the target compound is further processed to obtain the target compound 8DM.

[0041] The batch addition method of substrate can reduce the inhibition of bacterial activity caused by excessive one-time addition of substrate; at the same time, optimizing and controlling the pH of the fermentation liquid after the second batch of substrate addition is beneficial to the stability of the pH of the system during the bacterial transformation process and promotes the transformation process.

[0042] Compared with the prior art, the method for producing dexamethasone epoxide hydrolysate (compound 8DM) by microbial fermentation of the present invention has the following advantages:

[0043] 1. The substrate feed amount is as high as 83-120 g / L (weight volume ratio of substrate to fermentation liquid), the substrate conversion rate is as high as 98%, and the impurities are less than 2%.

[0044] 2. Only one microorganism is needed, the culture medium composition is simple, no additional coenzymes and solubilizers need to be added, and the production cost is low.

[0045] The present invention is further described in detail below in conjunction with the examples, which are explanations of the present invention and are not limited to the following examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0046] The fermentation broth treatment process in the following examples is as follows: 4 mL of the fermentation broth is transferred to a 5 mL centrifuge tube and centrifuged at 10000 r / min for 5 min; the supernatant is decanted, 25 mg of the precipitate is placed in a 50 mL volumetric flask, dissolved and diluted to scale with a diluent, and shaken well; filtered, and the supernatant is taken for HPLC analysis. The diluent is acetonitrile: water = 50:50.

[0047] The contents of the product, impurity 1, impurity 2 and impurity 3 in the following examples were detected by high performance liquid chromatography, wherein the chromatographic column was ZORBAX Eclipse Plus C18 (4.6×100 mm, 3.5 μm), the mobile phase was acetonitrile: water = 30:70; the flow rate was 1.5 mL / min, the column temperature was 40° C., the detection wavelength was 240 nm; and the injection volume was 20 μL.

[0048] The strain used in the following examples is Glutamicibacter sp., which is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.30439.

[0049] Example 1

[0050] In this embodiment, the production of dexamethasone epoxy hydrolysate, compound 8DM, by fermentation with Bacillus glutamicum includes the following steps:

[0051] ① Slant culture: First inoculate glutamicum bacteria into the slant culture medium, spread it evenly with a spreading stick, and culture it in a 30℃ biochemical incubator for 3 days. When the slant is completely covered with light yellow smooth colonies, the slant strain is obtained and can be used immediately or placed in a 4℃ environment for later use.

[0052] The slant culture medium of this example comprises the following components: 18 g / L soluble starch, 5 g / L yeast extract, 10 g / L peptone, 20 g / L agar powder, pH 6.8-7.2, prepared with purified water, and sterilized with high pressure steam at 120-125° C. for 15-25 min.

[0053] ② Seed culture: Use an inoculation spatula to scrape about 0.5 to 1.5 cm from the cultured slant medium. 2 The strain was inoculated into a 1L conical flask containing 200mL of the primary seed culture medium and cultured at 28-32°C and 220-260rpm for 2-4 days.

[0054] The shake flask seed culture medium of this embodiment comprises the following components: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, prepared with tap water, pH 6.8-7.2, adjusted to pH 6.8-7.2 with 10-30% NaOH solution before sterilization, and sterilized with high pressure steam at 120-125° C. for 15-25 min.

[0055] ③ Fermentation culture: The cultured seed liquid is inoculated at a rate of 10% (v / v) into a 15L fermentation tank containing 8L fermentation medium, with a temperature of 28-32°C, a tank pressure of 0.02-0.07Mpa, a flow rate of 0.5-2.5vvm, a rotation speed of 100-750rpm, and a dissolved oxygen >30%.

[0056] Substrate addition process: culture for 3 hours, first substrate (Compound 1) 5 g / L (based on the volume of the fermentation medium, the amount of Compound 1 mentioned below in this article is based on the volume of the fermentation medium), culture for 15-25 hours, second substrate addition 95 g / L.

[0057] pH control process: During the fermentation culture process, the pH of the fermentation system decreases before the second substrate addition, and ammonia water is used to control the pH of the fermentation system to 6.8-7.2. After the second substrate addition, the pH of the fermentation system increases, and the pH of the fermentation system is not controlled.

[0058] The fermentation medium of this embodiment includes: 10 g / L glycerol, 6 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate, and tap water. Before sterilization, the pH is adjusted to 6.8-7.2 with a 20% NaOH solution, and sterilized with high pressure steam at 121°C for 30 minutes.

[0059] During the conversion process, the conversion of the substrate was detected by HPLC. The fermentation was cultured for about 72 hours, and the HPLC detection spectrum of the fermentation broth was shown in Figure 1 .

[0060] The abbreviations and contents of various substances in the fermentation broth (determined by HPLC) are shown in Table 1 below.

[0061] Table 1

[0062]

[0063] Embodiments 2 to 5

[0064] The preparation steps of Examples 2 to 5 are basically the same as those of Example 1, except that the carbon source types of the fermentation tank culture medium in step ③ of these examples are different, and the carbon source concentration is 10 g / L. The results of the carbon source types and the contents of products and impurities in the fermentation broth (determined by HPLC) are shown in Table 2 below.

[0065] Table 2

[0066]

[0067]

[0068] It can be seen from Table 2 that the product conversion rate of Example 2 is the highest, indicating that glucose is most suitable as the carbon source of the fermentation medium.

[0069] Embodiments 6 to 9

[0070] The preparation steps of Examples 6 to 9 are basically the same as those of Example 2, except that the glucose concentration of the fermentation medium in step ③ of these Examples is different. The glucose concentration and the content of the product and impurities in the fermentation broth (determined by HPLC) are shown in Table 3 below.

[0071] Table 3

[0072]

[0073] It can be seen from Table 3 that the product conversion rate of Example 8 is the highest, indicating that the glucose concentration in the fermentation medium is the most suitable when it is 1.5%.

[0074] Embodiments 10 to 11

[0075] The preparation steps of Examples 10 to 11 are basically the same as those of Example 8, except that the time point of the second substrate addition in step ③ of these Examples, the time point of the second substrate addition and the results of the content of the product and impurities in the fermentation broth (determined by HPLC) are shown in Table 4 below.

[0076] Table 4

[0077]

[0078]

[0079] It can be seen from Table 4 that the product conversion rate of Example 10 is the highest, which indicates that the second substrate addition time point of 5 to 15 hours is the most appropriate feeding time point during the fermentation culture process.

[0080] Embodiments 12 to 14

[0081] The preparation steps of Examples 12 to 14 are basically the same as those of Example 10, except that in step ③ of these examples, the number of batches of the second substrate addition and the amount of substrate added in each batch, the number of batches of the second substrate addition and the amount of substrate added in each batch, and the results of the content of the product and impurities in the fermentation broth (determined by HPLC) are shown in Table 5 below.

[0082] Table 5

[0083]

[0084] It can be seen from Table 5 that the product conversion rate of Example 13 is the highest, indicating that it is most appropriate to add the substrate to the culture medium for the second time in three batches.

[0085] Embodiments 15 to 19

[0086] The preparation steps of Examples 15 to 19 are basically the same as those of Example 13, except that in step ③ of these examples, the pH of the fermentation system is regulated after the second addition of substrate. The pH value of the fermentation system and the content of the product and impurities therein (determined by HPLC) are shown in Table 6 below.

[0087] Table 6

[0088]

[0089] From Table 6, it can be seen that the conversion rate of the product in Example 18 is as high as 98.23% (see HPLC detection spectrum). Figure 2 ), indicating that it is most appropriate to control the pH of the fermentation system to 8.8-9.2 after the second addition of substrate.

[0090] The present invention optimizes the method of producing compound 8DM by microbial fermentation, especially studies the carbon source and dosage of the fermentation medium, the timing and dosage of substrate addition, and the pH control range of the fermentation system after substrate addition, and finally achieves that the impurities in the conversion process are controlled to less than 2% based on 10% feed, and the substrate conversion rate is as high as 98.23%. The operating parameters of the present invention are clearly controlled, suitable for industrial production, and can greatly improve economic benefits.

[0091] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for producing dexamethasone epoxy hydrolysate by microbial fermentation, wherein the dexamethasone epoxy hydrolysate is compound 8DM, characterized in that: The method comprises the steps of: Using Glutamicibacter sp., whose deposit number is CGMCC No.30439, compound 1 was used as a raw material to transform it into compound 8DM in one go. The conversion process is:

2. The method for producing dexamethasone epoxy hydrolysate by microbial fermentation according to claim 1, characterized in that: The carbon source of the fermentation medium was glucose.

3. The method for producing dexamethasone epoxy hydrolysate by microbial fermentation according to claim 2, characterized in that: The fermentation medium, based on its total volume, comprises the following components: Glucose 10-20 g / L, yeast extract 4-8 g / L, potassium dihydrogen phosphate 0.5-3.5 g / L, magnesium sulfate 0.1-0.3 g / L.

4. The method for producing dexamethasone epoxy hydrolysate by microbial fermentation according to claim 3, characterized in that: After the Glutamicibacter sp. is fermented and cultured in the fermentation medium for 2 to 5 hours, compound 1 is added to the fermentation medium for the first time; after the Glutamicibacter sp. is fermented and cultured for 5 to 15 hours, compound 1 is added to the fermentation medium for the second time.

5. The method for producing dexamethasone epoxy hydrolysate by microbial fermentation according to claim 4, characterized in that: The volume ratio of the compound 1 added to the fermentation broth for the first time is 3-10 g / L, and the volume ratio of the compound 1 added to the fermentation broth for the second time is 80-110 g / L.

6. The method for producing dexamethasone epoxy hydrolysate by microbial fermentation according to claim 4, characterized in that: Before adding compound 1 to the fermentation broth for the second time, the pH of the fermentation system was adjusted to 6.8-7.

2. After adding compound 1 to the fermentation broth for the second time, the pH of the fermentation system was adjusted to 8.8-9.

2.

7. A Glutamicibacter sp., characterized in that: The Glutamicibacter sp. is deposited in the China General Microbiological Culture Collection Center, and its deposit number is CGMCC No.30439.

8. The use of Glutamicibacter sp. according to claim 7, which converts compound 1 into dexamethasone epoxy hydrolysate, compound 8DM, at one time.

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