Method for preparing high-standard exemestane through microbial conversion

Abstract

The invention belongs to the field of bioengineering pharmacy, and particularly relates to a method for preparing high-standard exemestane through microbial conversion. According to the method, Nocardia simplex Noc (PN)-TS001 is adopted as a fermentation strain, methylenide is efficiently converted by microorganisms to prepare high-standard exemestane, and through optimization and combination of processes of all stages such as methylenide pretreatment, microorganism fermentation conversion and extraction refining, the substrate feeding concentration is increased to 1%-8%, the conversion rate reaches 88%-95%, the total yield reaches 88%-90%, and the yield of exemestane is increased to 90%. The detection content of the prepared exemestane refined product is more than 99.5%, the impurity content is less than 0.1%, and each index meets the USP standard. The method is simple and feasible in process, environment-friendly, low in cost, high in conversion efficiency, high in yield and high in product quality, and has a very good industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering pharmacy, and particularly relates to a method for preparing high-standard exemestane through microbial conversion. BACKGROUND

[0002] Exemestane, English name Exemestane, chemical name 1,4-diene-3,17-dione-6-methylene androstane or 6-methylene androst-1,4-diene-3,17-dione, CAS number is 107868-30-4, is a white or white crystalline powder. Exemestane is the second generation of aromatase inhibitors, and the effect of the drug is stronger than that of the same kind of aromatase inhibitor formestane, and the toxicity is lower. It is clinically used for the treatment of metastatic breast cancer and as an adjuvant therapy for early breast cancer, and has definite curative effect, good tolerance and relatively few side effects.

[0003] At present, there are many synthesis routes of exemestane reported in the literature, and the main synthesis route is to first chemically synthesize the key intermediate 6-methylene androst-4-ene-3,17-dione (referred to as methylene), and then the methylene is prepared into exemestane through chemical method or dehydrogenase method or microbial conversion method. The traditional chemical method often uses a large amount of toxic and harmful solvents and auxiliary materials, which pollutes the environment, produces a large amount of by-products, and it is extremely difficult to separate impurities and has low purification yield. The dehydrogenase method and microbial dehydrogenation conversion method have attracted the attention of researchers due to their strong reaction specificity, green and environmentally friendly production method, among which the dehydrogenase method has high selectivity and high efficiency, but the cost of enzyme preparation is higher than that of microbial method. RU2425052C1 discloses a method for preparing exemestane by using simple nocardia BKM Ac-2033D dehydrogenase, and the conversion yield is 85-87% under the condition of 5-100 g / l initial substrate concentration. However, enzyme inducers need to be added to induce the production of dehydrogenase, and then exogenous electron acceptors are added for enzyme reaction, which is complex and has high preparation cost.

[0004] The microbial method has low production cost and has potential for large-scale industrial production application, but the solubility of the substrate 6-methylene androstene dione in water system is low, which greatly limits the speed and yield of the conversion reaction, and the microbial metabolism is complex, so the fermentation parameters and reaction conditions need to be strictly controlled. Deng Le's research group of Hunan Normal University School of Life Sciences used simple arthrobacter to prepare exemestane by C1,2 microbial dehydrogenation of methylene in a dimethylbenzene / fermentation broth two-phase system, and the conversion rate was only 13.6% in 24 hours. After using the improved hydroxypropyl-β-cyclodextrin-methylene complex for conversion, the conversion rate can reach more than 90% only at a low feeding concentration of 0.2%, and 4.5 times the weight of hydroxypropyl-β-cyclodextrin is needed for complexation, which has high auxiliary material cost and is not conducive to industrial production. SUMMARY

[0005] In view of this, the present invention provides a method for preparing high-standard exemestane through microbial transformation. This method uses Nocardia (PN)-TS001 as the fermentation strain, efficiently transforming methylene compounds to prepare high-standard exemestane. Through the combination and optimization of processes such as methylene compound pretreatment, microbial fermentation transformation, and extraction and purification, the substrate concentration, conversion rate, and product purity are improved.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A simple strain of Nocardia ( Nocardioides simplex Noca(PN)-TS001 was deposited on August 21, 2021, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 23140.

[0007] This strain was obtained through mutagenesis and iterative culture using Nocardioides simplex ATCC6946 (Beijing Bioway Biotechnology Co., Ltd.) as the original strain.

[0008] A method for preparing high-standard exemestane through microbial transformation, wherein the microorganism is Nocardia simplex ( Nocardioides simplex Noca(PN)-TS001, using methylene compounds as substrates, is used to prepare exemestane, comprising the following steps: S1. Culture of Nocardia simplex strain Noca(PN)-TS001 and fermentation broth culture S2. Pretreatment of methylene compounds The methylene compound is added to a first organic solvent and heated to dissolve. It is then added dropwise into water for water precipitation. The mixture is stirred, filtered, and dried to obtain a filter cake. The filter cake is crushed and dissolved, and an antifoaming agent is added. The mixture is then pulped at 75-80°C and kept at that temperature for 3-5 hours. The temperature is then lowered to 28-32°C to obtain the pretreated methylene compound for later use. S3. Feeding and Conversion The pretreated methylene compound was added to a fermentation broth containing Nocardia simplex Noca(PN)-TS001 and placed in a shaker at 28-32°C and 180-200 r / min for 36-72 h to convert the mixture. The reaction was then terminated, and the solid was filtered and dried to obtain the crude product containing the bacteria. The amount of the pretreated methylene compound added is 1-8% of the total mass of the fermentation broth culture medium; S4. Extraction and Refining The bacterial crude product was repeatedly extracted with a second organic solvent until the filter residue was sampled and TLC analysis showed no ultraviolet color. The collected liquid portion was decolorized with activated carbon to obtain the extract. After the extract is concentrated to a paste, phase exchange is performed with ethyl acetate, 1-1.5 times the volume of mother liquor is reserved, and the first filtrate and solid are obtained by filtration, and the solid is dried to obtain the crude exemestane extract; The crude exemestane extract is dissolved in 3-5 times the volume of chloroform or dichloromethane, slowly dripped into petroleum ether, and after beating and cooling to 0-5℃, the solid and the second filtrate are obtained by centrifugation after stirring and crystallization; the solid is refined 1-2 more times according to this method, the filter cake is rinsed with ethyl acetate to replace the residual chloroform or dichloromethane solvent, and the third filtrate and a white solid are obtained, and the white solid is dried to obtain the refined exemestane.

[0009] Preferably, the strain culture in step S1 is a slant culture medium, and the culture medium includes the following components by weight: glucose 3-12 parts, yeast extract 3-12 parts, and pH 7.2-7.6.

[0010] Preferably, the fermentation liquid culture medium in step S1 includes the following components by weight: glucose 8-15 parts, corn syrup 8-20 parts, potassium dihydrogen phosphate 1-3 parts, peptone 2-6 parts, yeast extract 1-3 parts, sodium sulfite 0.1-0.3 parts, and antifoam agent 0.3-0.5 parts, and the pH value is 6.5-7.5. The specific fermentation process is as follows: the slant strain is washed with sterile water, the culture is inoculated into the fermentation liquid culture medium, and the culture is shaken at 28-32℃ and 180-200r / min for 20-24h; the sample is detected for enzyme activity within 1.0min, and the red is large, the microscope shows no foreign bacteria, the strain is completely split, and the growth characteristics of the logarithmic middle and late stages are met, and the preparation is ready for feeding.

[0011] Preferably, the first organic solvent in step S2 is any one of DMF, DMSO, dioxane, and tetrahydrofuran.

[0012] Preferably, in step S2, the filter cake is crushed to D99≤30μm.

[0013] Preferably, in step S2, the filter cake is crushed and then added into 3-8 times the weight of sterile water.

[0014] Preferably, in step S4, the first filtrate, the second filtrate, and the third filtrate are each added with an appropriate amount of activated carbon and neutral alumina for decolorization, and then concentrated to a paste, and the solid is obtained by filtration, and the solid is dried to obtain the extraction mother liquor, the refined mother liquor one, and the refined mother liquor two, respectively. The content of exemestane in the extraction mother liquor, the refined mother liquor one, and the refined mother liquor two is detected, and if the content is ≤50%, the sample is returned to the fermentation liquid in step S3 for conversion, and if the content is >50%, the sample is combined with the crude exemestane in step S4 for refinement.

[0015] Preferably, in step S4, the second organic solvent is one or a mixture of chloroform, dichloromethane, methanol, acetone, etc.

[0016] Preferably, the amount of petroleum ether used in step S4 is 1 to 3 times the volume of the amount of chloroform or dichloroform used.

[0017] Biological Preservation Information

[0018] Nocardia simplex ( Nocardioides simplex Noca(PN)-TS001, accession number: CGMCC No.23140, was deposited on August 21, 2021 at the China General Microbiological Culture Collection Center, located at Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This method uses Nocardia simplex Noca(PN)-TS001 as the dominant fermentation strain to efficiently convert methylene compounds to prepare exemestane. The culture medium formula and fermentation conditions of Nocardia simplex were optimized. After optimization, the conversion rate reached 88%~95% and the total yield reached 88%~90%.

[0020] (2) This method pre-treats the substrate methylene compounds. First, the chemical excipient residues in the substrate methylene compounds are removed by the method of dissolving and precipitating with water, which avoids the adverse effects of chemical excipient residues such as formaldehyde, N-methylaniline, and benzoic acid on the microbial metabolic system. Then, a low-temperature, long-time pulping sterilization method is used to avoid the adverse effects of high-temperature steam sterilization, which can easily lead to changes in crystal form or the generation of impurities. In addition, long-time pulping with water increases the water solubility of the methylene compounds, making it easier to complete the microbial fermentation and transformation. The feed concentration can be increased to 1%~8% after pretreatment.

[0021] (3) This method optimizes the refining process of crude exemestane, effectively removes the methylene compounds that are difficult to refine, and in particular, the unconverted methylene compounds are effectively utilized by collecting the mother liquor and using it for fermentation refeeding or refining. The purity of the refined exemestane obtained is >99.5%, the methylene impurity is <0.1%, and all indicators meet the USP standard.

[0022] (4) This method is simple and easy to implement, environmentally friendly, low in cost, high in conversion efficiency, high in yield, and high in product quality, and has a very good industrial application prospect. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0024] Figure 1 The photo of microscope (1000X) observation of simple Nocardia strain Noca(PN)-TS001 of the application; Figure 2 The photo of colony morphology plate of simple Nocardia strain Noca(PN)-TS001 of the application; Figure 3 The phylogenetic tree of simple Nocardia strain Noca(PN)-TS001 of the application based on 16S rRNA gene sequence; Figure 4 The detection result of methylenecyclohexane after fermentation conversion by simple Nocardia Noca(PN)-TS001 in Example 1 of the application; Figure 5 The detection result of methylenecyclohexane after fermentation conversion by simple Nocardia Noca(PN)-TS001 in Example 2 of the application; Figure 6 The detection result of methylenecyclohexane after fermentation conversion by simple Nocardia Noca(PN)-TS001 in Example 3 of the application; Figure 7 The detection result of methylenecyclohexane after fermentation conversion by simple Nocardia Noca(PN)-TS001 in Example 3 of the application; Figure 8 The detection result of methylenecyclohexane after fermentation conversion by simple Nocardia Noca(PN)-TS001 in Example 3 of the application; Figure 9 The detection result of methylenecyclohexane after fermentation conversion by simple Nocardia Noca(PN)-TS001 in Example 4 of the application; Figure 10 The detection result of methylenecyclohexane after fermentation conversion by simple Nocardia Noca(PN)-TS001 in Example 4 of the application; Figure 11 The detection result of methylenecyclohexane after fermentation conversion by simple Nocardia Noca(PN)-TS001 in Example 4 of the application; DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0026] Unless otherwise specified, all raw materials and reagents used in this invention are commercially available products.

[0027] The simple Nocardia used in this invention ( Nocardioides simplex Noca(PN)-TS001 was prepared using Nocardioides simplex ATCC6946 (purchased from Beijing Biowell Biotechnology Co., Ltd.) as the starting strain, following the procedure: (1) Weigh 6 mg of nitrosoguanidine (NTG, purchased from Sigma) into a sterile centrifuge tube, add 0.05 ml of acetone to aid dissolution, and add 1 ml of 0.2 mM pH 6.0 phosphate buffer to dissolve it completely; (2) Prepare the original strain of Nocardia simplex at a concentration of 10 8 ~10 9 A bacterial suspension of 1 cell / ml was prepared by mixing 5 ml of the bacterial suspension with the above nitrosoguanidine solution. (3) Immediately place the mixture in a 30°C water bath and shake for 10 min to 1 h. Collect the bacterial cells by centrifugation, then wash the bacterial cells twice with 5 ml of the above phosphate buffer to terminate the mutagenic effect of NTG. Finally, add 5 ml of physiological saline to the centrifuge tube and shake well. (4) Dilute the mutagenic bacterial suspension 10 times and spread it on a slant plate containing 0.1% methylene compound for initial screening and culture for 3-6 days to obtain single colonies; (5) Select single colonies for expansion culture, inoculate them into shake flasks for rescreening, and select single colonies with high conversion rate and high extraction yield for preservation. Finally, conduct subculture stability experiments to determine the dominant strain.

[0028] Microscopic images of the obtained dominant strains are as follows: Figure 1 As shown, the colony morphology is as follows Figure 2 As shown, sequencing was performed by Wuhan Tianyi Huayu Gene Technology Co., Ltd., and its 16S rRNA is shown in SEQ ID No. 1. The phylogenetic tree construction is as follows. Figure 3 As shown, it was identified as Nocardia simplex (… Nocardioides simplex It was deposited on August 21, 2021, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 23140.

[0029] The 16S rRNA sequence is shown below: Preparation of methylene: androst-4-ene-3, 17-dione (4AD) as the initial substrate, add anhydrous methanol, original acetic acid trimethyl ester and p-toluene sulfonic acid to carry out enol etherization reaction; the treated crude product is dissolved in tetrahydrofuran and anhydrous methanol, N-methyl aniline, formaldehyde and p-toluene sulfonic acid are added to carry out Mannich reaction; the obtained reaction solution is added dropwise to hydrolyze and refine with concentrated hydrochloric acid to obtain the methylene, and the purity of the obtained methylene is ≥99.0%.

[0030] Example 1

[0031] (1) Strain culture: pick up Nocardia simplex Noca (PN)-TS001 and inoculate on slant medium, and culture at 30°C for 72h, and then store in refrigerator at 5°C for standby. Each 100ml of slant medium includes the following mass components: glucose 0.5g, yeast extract 0.5g, pH 7.4~7.6; (2) Fermentation liquid culture: wash one slant strain with 100ml sterile water, and take 15ml to inoculate in 2L fermentation liquid medium. 5L baffled flask is filled with 2L liquid, and is placed in a shaker at 30°C and 180r / min for 20~24h. Take sample to detect enzyme activity, that is, use 2, 3, 5 chlorinated triphenyl tetrazolium method to detect the enzyme activity of fermentation liquid, which shows bright red color within 1.0min, and no miscellaneous bacteria are observed under microscope, the strain is completely split, robust, and meets the growth characteristics of logarithmic middle and late stage, and is ready for feeding. Each 1L of fermentation liquid medium includes the following mass components: glucose 10g, corn syrup 12g, potassium dihydrogen phosphate 2g, peptone 5g, yeast extract 1g, sodium sulfite 0.1g, defoaming agent 0.3g, and pH value is 7.0~7.5.

[0032] (3) Feeding conversion: the prepared methylene is micronized to D99≦30μm, and then is fed into the qualified fermentation liquid, and the feeding concentration is 1%, and is placed in a shaker at 30°C and 180r / min for 48h. Take sample to detect, and the liquid chromatography detection result is shown in Table 1, and the conversion rate is 88.96%. Figure 4

[0033] As can be seen from Example 1, for the methylene with lower feeding concentration (1%), even without pretreatment, the microbial fermentation conversion of Nocardia simplex Noca (PN)-TS001 can reach 88.96% conversion rate, and has very good conversion capacity, which is an advantageous strain for microbial fermentation conversion to prepare exemestane.

[0034] Example 2

[0035] ​(1) Strain culture: pick up Nocardia simplex Noca(PN)-TS001 and inoculate on slant medium, and culture at 30°C for 72h, then store in refrigerator at 5°C for standby. Each 100ml slant medium includes the following mass components: glucose 0.5g, yeast extract 0.5g, pH 7.4-7.6; (2) Fermentation liquid culture: wash one slant strain with 100ml sterile water, and take 15ml to inoculate in 2L fermentation liquid medium. 5L baffled flask is filled with 2L liquid, and is placed in a shaker at 30°C and 180r / min for 20-24h. Take sample to detect enzyme activity, i.e. use 2,3,5 chlorinated triphenyl tetrazolium method to detect the enzyme activity of fermentation liquid in 1.0min, and the result is red, no miscellaneous bacteria are observed under microscope, the strain is completely split, robust, and meets the growth characteristics of logarithmic middle and late stage, and is ready for feeding. Each 1L fermentation liquid medium includes the following mass components: glucose 10g, corn syrup 12g, potassium dihydrogen phosphate 2g, peptone 5g, yeast extract 1g, sodium sulfite 0.1g, antifoam agent 0.3g, and pH value is 7.0-7.5.

[0036] (3) Feeding conversion: the prepared methylene compound is micronized to D99≦30μm, and then is fed into the qualified fermentation liquid, and the feeding concentration is 5%, and is placed in a shaker at 30°C and 180r / min for 48-60h. Take sample to detect, and the detection result is shown in Table 1, and the conversion rate is about 30%. Figure 5

[0037] As can be seen from comparative example 2 and example 1, when the feeding concentration is increased from 1% to 5%, the unpretreated methylene compound is obviously decreased in the microbial fermentation conversion effect by Nocardia simplex Noca-TS001; when the feeding concentration of methylene compound is increased to 5%, the conversion rate is reduced to 30%, which indicates that the microbial fermentation conversion has limited effect on the methylene compound with high feeding concentration.

[0038] Example 3

[0039] (1) Strain culture: pick up Nocardia simplex Noca(PN)-TS001 and inoculate on slant medium, and culture at 30°C for 72h, then store in refrigerator at 5°C for standby. Each 100ml slant medium includes the following mass components: glucose 0.5g, yeast extract 0.5g, pH 7.4-7.6; ​(2) Fermentation liquid culture: using 100 ml sterile water, washing down a slope strain, taking 15 ml to inoculate in 2 L fermentation liquid medium. 5 L baffled flask liquid volume is 2 L, placed in 30℃, 180 r / min shaking table vibration culture 20 h~24 h. Sampling detection enzyme activity, that is, using 2,3,5 chlorinated triphenyl tetrazolium method detects the enzyme activity of fermentation liquid in 1.0 min big red, microscopic examination no miscellaneous bacteria, strain complete split, robust, meet the logarithmic middle and late growth characteristics, ready to feed. Including the following mass components in each 1 L fermentation liquid medium: glucose 10 g, corn syrup 12 g, potassium dihydrogen phosphate 2 g, peptone 5 g, yeast extract 1 g, sodium sulfite 0.1 g, defoaming agent 0.3 g, pH value is 7.0~7.5.

[0040] (3) Methylene compound pretreatment: taking the prepared methylene compound, using 3V DMF to warm to 80~90℃ to dissolve clear, slowly added into 30 volume times of water, water analysis, stirring for 30 min, then filtering, drying the filter cake. The dried filter cake is crushed to D99≦30μm by a pulverizer. Taking 100 g into 350 ml sterile water, adding 0.8 g of composite silicone type defoaming agent, 75~80℃ beating and keeping warm for 3~5 h, then cooling to 28~32℃ for feeding.

[0041] (4) Feeding conversion: the pretreated methylene compound is fed into the fermentation liquid that has passed the detection, the feeding concentration is 5%, placed in 30℃, 180 r / min shaking table vibration conversion 48 h~60 h, sampling detection, liquid chromatography detection results are shown in Figure 6 , 61 h conversion rate 91%. After adjusting the pH value of the fermentation liquid to 2.0~3.0, keeping warm at 55~70℃ for 1~2 h, then cooling to 30~40℃, terminating the reaction, filtering to obtain the crude product with bacteria 104.5 g.

[0042] (5) Extraction: adding the crude product with bacteria into 10 volume times of chloroform, 5 volume times of methanol mixed solvent, then adding 3 g of activated carbon, warming up and extracting three times, the filtrate is concentrated to paste, then using ethyl acetate to exchange phase, leaving about 1 volume times of mother liquor, filtering and drying to obtain the crude product of exemestane 86.8 g. The liquid chromatography detection results of the extraction crude product are shown in Figure 7 , the conversion rate is 94.66%.

[0043] (6) Purification: the extract was dissolved in 3 volumes of chloroform, slowly dropped into 4.5 volumes of petroleum ether, and then stirred for 2 hours. The temperature was lowered to 0-5°C, and the mixture was stirred for 3 hours to precipitate crystals. The crystals were centrifuged. The process was repeated once, and the cake was washed with ethyl acetate to replace the residual chloroform. A white solid was obtained, which was dried to obtain 75.6 g of exemestane. The total yield was 89.6% after the extract and the purification mother liquor were refined into a solid and then returned to the fermentation step or the purification step according to the content. The liquid chromatography detection results of the refined product are shown in Table 1, and the purity was 99.89%. The other impurities were less than 0.1%, and the total detection met the USP standard. Figure 8

[0044] As shown in Table 1, the methylene compound was pretreated, and even if the concentration was increased to 5%, the microbial fermentation conversion effect was still very good. Figure 3 Figure 6 Figure 7 Figure 8 As shown in Table 1, after further extraction and purification, the purity of the exemestane product was greatly improved, and there was basically no other impurities.

[0045] Example 4

[0046] (1) Bacterial culture: a simple nocardia Noca (PN)-TS001 was inoculated on a slope culture medium, and cultured at 30°C for 72 hours. The culture was stored in a refrigerator at 5°C for standby use. Each 100 ml of the slope culture medium included the following components: glucose 0.4 g, yeast extract 0.4 g, and pH 7.4-7.6. (2) Fermentation liquid culture: 15 ml of the slope culture medium was inoculated in 2 L of the fermentation liquid medium. The 5 L baffled flask was filled with 2 L of liquid, and was placed in a shaker at 30°C and 180 r / min for 20-24 hours. The enzyme activity was detected by using the 2,3,5 chlorinated triphenyl tetrazolium method. The fermentation liquid enzyme activity was red within 1.0 min, and there was no mixed bacteria under microscopic examination. The strain was completely split, and was thick and strong, which met the growth characteristics of the logarithmic middle and late stages, and was ready for use. Each 1 L of the fermentation liquid medium included the following components: glucose 10 g, corn syrup 12 g, potassium dihydrogen phosphate 2 g, peptone 5 g, yeast extract 1 g, sodium sulfite 0.1 g, and defoaming agent 0.3 g, and the pH value was 7.0-7.5.

[0047] (3) Methylene compound pretreatment: the prepared methylene compound was dissolved in 3 V of DMF at 80-90°C, and was slowly dropped into 30 volumes of water for water precipitation. After stirring for 30 min, the filter cake was dried and crushed to D99≦30 μm. 160 g of the dried filter cake was added into 500 ml of sterile water, 0.8 g of a composite silicone defoaming agent was added, and the mixture was stirred at 75-80°C for 3-5 hours. The temperature was lowered to 28-32°C for use.​​​​

[0048] (4) Substrate conversion: the pretreated methylene compound was put into the qualified fermentation broth with a concentration of 8%, and was placed in a shaking bed at 30°C and 180 r / min for 48-60 h. Sampling and liquid chromatography detection showed that the conversion rate was 89.91% at 72 h, as shown in Table 2. Figure 9

[0049] As can be seen from Table 2, after the pretreatment of the methylene compound, even if the substrate concentration is increased to 8%, the microbial fermentation conversion effect is still good, and the conversion rate reaches 89.91% at 72 h. Figure 9 Comparative Example 1 (1) Bacterial culture: the simple nocardia Noca (PN)-TS001 was inoculated on a slant medium, and was cultured at 30°C for 72 h, and was stored in a refrigerator at 5°C for standby. Each 100 ml of the slant medium included the following components: glucose 0.5 g, yeast extract 0.5 g, and pH 7.4-7.6. (2) Fermentation broth culture: 15 ml of the slant strain was inoculated in 2 L of the fermentation broth medium with 100 ml of sterile water. The 5 L baffled flask was filled with 2 L of liquid, and was placed in a shaking bed at 30°C and 180 r / min for 16-18 h. Sampling and enzyme activity detection showed that the fermentation broth was bright red after 3.0 min of 2,3,5-chlorinated triphenyl tetrazolium detection, and no miscellaneous bacteria were observed under a microscope. The substrate was ready for use. Each 1 L of the fermentation broth medium included the following components: glucose 8 g, corn syrup 10 g, potassium dihydrogen phosphate 2 g, protein peptone 5 g, and antifoaming agent 0.3 g, and the pH value was 7.0-7.5.

[0050] (3) Methylene compound pretreatment: the prepared methylene compound was heated to 80-90°C with 3V of DMF until it was dissolved, and was slowly added into 30 times of water for water separation. After stirring for 30 min, the filter cake was dried. The dried filter cake was crushed to D99≦30 μm with a pulverizer. 100 g of the crushed filter cake was put into 350 ml of sterile water, 0.8 g of a composite organosilicon antifoaming agent was added, and the mixture was beaten at 75-80°C for 3-5 h, and then was cooled to 28-32°C for substrate conversion.

[0051] (4) Substrate conversion: the pretreated methylene compound was put into the qualified fermentation broth with a concentration of 5%, and was placed in a shaking bed at 30°C and 180 r / min for 61 h. Sampling and detection showed that the conversion rate was about 15%, as shown in Table 3. Figure 10 ​​​

[0052] As can be seen from Comparative Example 3 and Comparative Example 1, the different strain culture medium formulations and fermentation conditions also greatly affect the microbial fermentation conversion effect.

[0053] Comparative Example 2 (1) Strain culture: pick up Nocardia simplex ATCC6946 (commercially available) and inoculate on a slope culture medium, and incubate at 30°C for 72h, then store in a refrigerator at 5°C for standby use. Each 100ml of the slope culture medium includes the following mass components: glucose 0.4g, yeast extract 0.4g, pH 7.4~7.6; (2) Fermentation liquid culture: wash down one slope strain with 100ml of sterile water, take 15ml and inoculate in 2L of fermentation liquid medium. 5L of baffled flask is filled with 2L of liquid, and is placed in a shaker at 30°C and 180r / min for 20h~24h of vibration culture. Take sample for enzyme activity detection, i.e. use 2,3,5 chlorinated triphenyl tetrazolium method to detect the fermentation liquid enzyme activity within 1.0min, and the result is large red, no miscellaneous bacteria are observed under microscope, the strain is completely split, is robust, and meets the logarithmic mid-late growth characteristics, and is ready for feeding. Each 1L of the fermentation liquid medium includes the following mass components: glucose 10g, corn syrup 12g, potassium dihydrogen phosphate 2g, proteose peptone 5g, yeast extract 1g, sodium sulfite 0.1g, antifoaming agent 0.3g, and pH value is 7.0~7.5.

[0054] (3) Methylene compound pretreatment: take prepared methylene compound with purity ≥99%. Warm up to 80~90°C with 3V of DMF to dissolve, slowly drop into 30 times volume of water for water separation, stir for 30min, then filter, and dry the filter cake. Dry the filter cake and crush to D99≦30μm with a pulverizer. Take 100g and put into 350ml of sterile water, add 0.8g of composite silicone antifoaming agent, and beat the pulp at 75~80°C for 3~5h, then cool to 28~32°C for feeding.

[0055] (4) Feeding conversion: put the pretreated methylene compound into the fermentation liquid which has passed detection, and the feeding concentration is 5%, and place in a shaker at 30°C and 180r / min for 48h~60h of vibration conversion, take sample for detection, and the detection result is shown in Table 1, and the conversion rate is about 50%. Figure 11

[0056] As can be seen from Comparative Example 3 and Comparative Example 2, under the same fermentation conditions, the conversion rates of different strains also greatly differ, which may be related to different enzyme system space conformations of different strains leading to different substrate binding degrees or different substrate tolerances of different strains, thereby further confirming that Nocardia simplex Noca(PN)-TS001 is an advantageous strain for microbial fermentation conversion preparation of exemestane.

[0057] ​The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A simple strain of Nocardia ( Nocardioides simplex Noca(PN)-TS001 was deposited on August 21, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 23140. Its 16S rRNA is shown in SEQ ID No.

1.

2. A method for preparing high-standard exemestane through microbial transformation, wherein the microorganism is Nocardia simplex (… Nocardioides simplex Noca(PN)-TS001, which prepares exemestane using methylene compounds as substrates, is characterized by, The method comprises the following steps: S1. Simple Nocardia Noca (PN)-TS001 strain culture and fermentation liquor preparation; S2. Pretreatment of methylene compound The methylene compound is added into a first organic solvent to be dissolved at a high temperature, and then added dropwise into water to be precipitated, stirred, filtered, and dried to obtain a filter cake; the filter cake is crushed, dissolved, and added with an antifoaming agent, and then beaten at 75-80℃ for 3-5h, and then cooled to 28-32℃ to obtain the pretreated methylene compound, which is ready for use; S3. Feeding and conversion The pretreated methylene compound is put into the fermentation liquor containing the simple Nocardia Noca (PN)-TS001, and placed in a shaking bed at 28-32℃ and 180-200r / min to be vibrated and converted for 36-72h; the reaction is terminated, and the solid is filtered and dried to obtain a crude product with bacteria; The feeding amount of the pretreated methylene compound is 1-8% of the total mass of the fermentation liquor medium; S4. Extraction and refinement The crude product with bacteria is repeatedly extracted with a second organic solvent until no ultraviolet coloring substance is detected by TLC sampling of the filter residue, and the liquid part is collected and decolorized with activated carbon to obtain an extraction liquid; The extraction liquid is concentrated to a paste, and then replaced with ethyl acetate to obtain a mother liquor, and then filtered to obtain a first filtrate and a solid, and the solid is dried to obtain an exemestane extraction crude product; and the crude product is refined to obtain an exemestane fine product.

3. The process for the preparation of high label Exemestane by high efficient transformation of Nocardia simplex Noca (PN)-TS001 as claimed in claim 2, wherein, The strain culture in step S1 is a slant culture medium, and the culture medium comprises the following components in parts by weight: glucose 3-12 parts, and yeast extract 3-12 parts, and the pH is 7.2-7.

6.

4. The process for the preparation of high label Exemestane by high efficient transformation of Nocardia simplex (PN)-TS001 as claimed in claim 2, wherein, The fermentation liquor medium in step S1 comprises the following components in parts by weight: glucose 8-15 parts, corn syrup 8-20 parts, potassium dihydrogen phosphate 1-3 parts, peptone 2-6 parts, yeast extract 1-3 parts, sodium sulfite 0.1-0.3 parts, and antifoaming agent 0.3-0.5 parts, and the pH value is 6.5-7.

5.

5. The process for the preparation of high label Exemestane by high efficient transformation of Nocardia simplex Noca (PN)-TS001 as claimed in claim 2, wherein, The fermentation liquor preparation process is as follows: the slant strain is washed with sterile water, and the cultured strain is inoculated into the fermentation liquor medium, and then vibrated and cultured in a shaking bed at 28-32℃ and 180-200r / min for 20-24h; sampling detection shows that the enzyme activity is red within 1.0min, and there is no foreign bacteria under microscope, and the strain is completely split, robust, and meets the growth characteristics of the logarithmic middle and late stages, and is ready for feeding.

6. The process for the preparation of high-purity exemestane by microbial conversion as claimed in claim 2 wherein, The first organic solvent in step S2 is any one of DMF, DMSO, dioxane, and tetrahydrofuran.

7. The process for the preparation of high-strength exemestane by microbial conversion as claimed in claim 2 wherein, The crude product refinement process in step S4 is as follows: the exemestane extraction crude product is dissolved in 3-5 times the volume of chloroform or dichloromethane, slowly dripped into petroleum ether, cooled to 0-5℃ after beating, and then centrifuged to obtain a solid and a second filtrate; the solid is refined again for 1-2 times according to the method, and then the filter cake is rinsed with ethyl acetate to replace the residual chloroform or dichloromethane solvent to obtain a third filtrate and a white solid, and the white solid is dried to obtain the exemestane fine product.

8. The process for the preparation of high-strength exemestane by microbial conversion as claimed in claim 2 wherein, The first filtrate, the second filtrate and the third filtrate in step S4 are respectively added with an appropriate amount of activated carbon and neutral alumina for decolorization, and then concentrated to a paste, filtered to obtain a solid, and the solid is dried to obtain an extraction mother liquor, a refined mother liquor I and a refined mother liquor II, respectively; The extraction mother liquor, the refined mother liquor I and the refined mother liquor II are respectively detected for the content of exemestane, if the content is ≤50%, the extraction mother liquor is returned to the fermentation liquid in step S3 for conversion, if the content is >50%, the extraction mother liquor is incorporated into the crude exemestane in step S4 for refining.

9. The process for the preparation of high label Exemestane by Nocardia simplex Noca (PN)-TS001 high efficiency transformation as claimed in claim 2, wherein, The second organic solvent in step S4 is one or a mixture of more than one of trichloromethane, dichloromethane, methanol and acetone.

10. The process for the preparation of high label Exemestane by Nocardia simplex Noca (PN)-TS001 high efficiency transformation as claimed in claim 2, wherein, The amount of the petroleum ether in step S4 is 1-3 times of the amount of the trichloromethane or dichloromethane.

Images