Exemestane dehydrogenase, gene, strain and exemestane preparation method

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to exemestane dehydrogenase, a gene, a strain and a preparation method of exemestane. The sequence of the exemestane dehydrogenase is SEQ ID NO.1. The exemestane dehydrogenase is higher in activity, higher in conversion speed and higher in conversion rate, can be used for treating substrates with larger feeding amount.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to exemestane dehydrogenase, genes, strains and a method for preparing exemestane. Background Art

[0002] The growth of breast cancer cells depends on the presence of estrogen. The estrogen in the circulation of postmenopausal women is mainly converted from androgens by aromatase. Blocking estrogen production by inhibiting aromatase is an effective and selective treatment for postmenopausal hormone-dependent breast cancer.

[0003] Exemestane is an irreversible steroidal aromatase inactivator that can inactivate aromatase by irreversibly binding to the active site of aromatase, thereby significantly reducing the level of estrogen in the blood circulation of postmenopausal women to achieve the purpose of treating hormone-dependent breast cancer. It has the advantages of good tolerance, no cross-resistance, and no obvious side effects, and has become a first-line drug for the treatment of advanced breast cancer.

[0004] Exemestane was originally chemically synthesized using androst-4-ene-3,17-dione as the raw material. Exemestane was obtained through condensation reaction, bromination and elimination. The yield of this method was only 28.8%. Later, an enzymatic method was used to prepare exemestane, using dehydrogenase to directly obtain exemestane from the precursor in one step, which improved efficiency and increased the yield.

[0005] Patent application CN117265055A discloses a method for preparing exemestane by an enzymatic method, in which the reaction substrate is synthesized under the action of dehydrogenase TQ-0008. The feed concentration of this patent application is low, the feed concentration of the raw material is only 2%, and the production efficiency is low; if the feed amount of the substrate is increased, the substrate cannot be fully converted. Its enzyme activity is low, the enzyme dosage is large, and the total amount of dehydrogenase bacteria for the second conversion is 12%-18%. The amount of bacteria is extremely large, which will make the later extraction and refining more difficult and affect the final yield. The conversion time is long. Although the enzymatic method is used for conversion, the conversion time reaches 96-120h, and the production efficiency is low; if the time is shortened to 72h, the conversion rate will drop sharply to below 79%. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an exemestane dehydrogenase, a gene, a strain and a method for preparing exemestane. The exemestane dehydrogenase has stronger activity and faster conversion speed, can process a larger amount of substrate, and has a higher conversion rate.

[0007] An embodiment of the present invention provides an exemestane dehydrogenase, and the sequence of the exemestane dehydrogenase is SEQ ID NO.1.

[0008] The embodiment of the present invention provides a gene encoding the exemestane dehydrogenase, and the sequence is SEQ ID NO.2.

[0009] The embodiment of the present invention provides a strain, in which the gene is inserted into the chassis strain, or a plasmid containing the gene is inserted into the chassis strain.

[0010] The embodiment of the present invention provides a method for preparing exemestane, comprising mixing an enzyme solution containing the exemestane dehydrogenase, a buffer solvent, a cosolvent and a substrate, adjusting the pH value to 8-9, adding an electron acceptor, performing conversion, and after the conversion is completed, processing to obtain exemestane.

[0011] Optionally, the buffer solvent is Tris-HCl buffer, PBS phosphate buffer or sodium acetate buffer solution, and the final concentration of the buffer solvent is 20-100 mmol / L.

[0012] Optionally, the co-solvent is methanol, ethanol, isopropanol or acetone.

[0013] Optionally, the structural formula of the substrate is: .

[0014] Optionally, the feed concentration of the enzyme solution is 10-30%, and the electron acceptor is phenazine methyl sulfate.

[0015] Optionally, the substance for adjusting the pH value to 8-9 is alkali solution or hydrochloric acid.

[0016] Optionally, the conversion temperature is 25-35°C, the time is generally 8-16h, air or oxygen is introduced during the conversion, and the treatment operation is to filter, add methanol to the filter residue (the added volume of methanol is 8-12 times the weight of the filter residue, that is, when the weight of the filter residue is 1g, the added volume of methanol is 8-12mL), raise the temperature to 50-70°C for slurrying, filter, concentrate the filtrate, add water, concentrate, and dry to obtain exemestane.

[0017] Preferably, the final concentration of the buffer solvent is 50 mmol / L, the cosolvent is isopropanol, the feed concentration of the enzyme solution is 20-30%, the electron acceptor is phenazine methyl sulfate, the pH value is adjusted to 8.5-9, air or oxygen is introduced during the conversion, and the conversion time is more than 12 hours.

[0018] The preparation method of the enzyme solution containing the exemestane dehydrogenase of the present invention is: Take a preserved TQ-01 glycerol strain and inoculate 0.1~1ml into 50~100ml liquid LB medium. The components of the liquid LB medium are as follows:

[0019] Before inoculation, kanamycin with a final concentration of 10-50 mg / L should be added to the liquid culture medium. After inoculation, it should be placed in a constant temperature shaker for cultivation. The culture conditions are 37°C, 100-200 rpm, and 8-24 hours. After the seeds are cultivated, they are inoculated into the sterilized fermentation medium for fermentation. The fermentation medium components are as follows:

[0020] In the early stage of fermentation, the temperature should be controlled at 37°C, and the pH should be controlled at 6.8~7.5 with (ammonia / phosphoric acid). When the OD600 of the fermentation system reaches 5~10, the temperature should be lowered to 25~30°C. After the temperature drops to the target temperature, the IPTG solution that has been sterilized by a membrane can be added for induction. The final concentration of IPTG induction is 0.1~1mmol / L.

[0021] After induction, continue fermentation for 12 to 24 hours to complete the fermentation. Collect the bacteria by centrifugation in the tank, prepare the collected bacteria into 300 g / L bacterial solution with clean water, add 1% mass fraction of dipotassium hydrogen phosphate, adjust the pH to 8.0-9.0 with 20% liquid alkali, and then use a homogenizer to homogenize and crush. The homogenization pressure is 800 MPa. During the averaging process, the temperature is controlled between 10 and 30 ° C. The prepared TQ-01 enzyme solution can be obtained by homogenizing three times.

[0022] The reaction route of the enzyme solution catalyzing the conversion of substrate to obtain exemestane is: .

[0023] The beneficial effects of the present invention are that the dehydrogenase of the present invention has higher activity and faster conversion speed, and the conversion can be completed in 24h-36h, which saves time and greatly increases production efficiency. Moreover, due to the high enzyme activity, the amount of raw materials can be increased. The raw material feeding amount of the present invention can reach 10-15%, which greatly improves the yield and saves production costs compared with the 2% feeding amount of the prior art. In this scheme, the bacterial cells obtained by fermentation are subjected to cell rupture treatment to release enzyme activity, thereby achieving the purpose of reducing enzyme dosage and saving costs. Moreover, reducing the enzyme dosage can effectively reduce the amount of protein during extraction, reduce the difficulty of extraction, reduce the amount of solvent used, and improve the yield. DETAILED DESCRIPTION

[0024] Example 1 Take a preserved TQ-01 glycerol strain.

[0025] The construction method of TQ-01 glycerol bacteria is as follows: 1. The nucleotide sequence of exemestane dehydrogenase is SEQ ID NO: 2, and the sequence of exemestane dehydrogenase is SEQ ID NO: 1. The SEQ ID NO: 2 sequence was sent to Shanghai Qingke Biotechnology for whole gene synthesis and constructed into a pET-28a plasmid expression vector to obtain a recombinant plasmid.

[0026] 2. Introduce the recombinant plasmid into 100 μL by heat shock method Escherichia Coli BL21(DE3) competent cells were plated on LB solid medium containing kanamycin resistance and cultured at 37°C overnight (about 16 hours). The larger single clones were selected and amplified with universal primers (T7, T7ter) for colony PCR (polymerase chain reaction) to screen positive clones, thereby obtaining the engineered bacteria (PET-KstD).

[0027] 3. Inoculate the engineered strain into LB medium at a 0.1% inoculation rate, culture at 37°C for 12-16 hours to activate the strain, add the activated bacterial solution to the prepared sterile glycerol tube. The ratio of the solution in the glycerol tube is 60% glycerol plus 40% deionized water. Add 0.8 ml of solution to each glycerol tube, sterilize at 121°C for 30 minutes before use, add 1 ml of activated bacterial solution when using, and then preserve the strain at -80°C.

[0028] Take 0.1 ml of TQ-01 glycerol bacteria and inoculate it into 100 ml of liquid LB medium. The components of liquid LB medium are as follows:

[0029] Before inoculation, kanamycin with a final concentration of 50 mg / L should be added to the liquid culture medium. After inoculation, it should be placed in a constant temperature shaker for cultivation. The culture conditions are 37°C, 200 rpm, and 12 hours. After the seeds are cultivated, they are inoculated into a sterilized 5L fermenter for fermentation. The fermentation volume is 2.5L. The fermentation medium components are as follows:

[0030] The temperature should be controlled at 37℃ in the early stage of fermentation, and pH should be controlled at 7.0 with (ammonia / phosphoric acid). 600 When the temperature reaches 6, start cooling down to 25°C. After the temperature drops to the target temperature, add the sterilized IPTG solution for induction. The final concentration of IPTG induction is 0.2mmol / L.

[0031] After induction, the fermentation was continued for another 22 hours to complete the fermentation. The bacterial concentration in the tank was 60 g / L, and the bacteria were collected by centrifugation. The collected bacteria were prepared into 300 g / L bacterial solution with clean water, and 1% of dipotassium hydrogen phosphate was added. The pH was adjusted to 8.0 with 20% liquid alkali, and then homogenized and crushed with a homogenizer. The homogenization pressure was 800 MPa. The temperature was controlled between 10 and 30 °C during the averaging process. The prepared TQ-01 enzyme solution was obtained by homogenization three times, and the enzyme solution pH was 7.5.

[0032] The prepared TQ-01 enzyme solution was used for feed conversion, and the conversion was carried out in a shake flask. A 500ml shake flask was filled with 100ml of the conversion system. A buffer substance was first added to the system. The buffer substance was Tris-Hcl buffer, and the final concentration of Tris was 50mmol / L. Then, 2% isopropanol was added to the system as a solubilizing solvent, and then a conversion substrate compound (a) was added at a final concentration of 10%. Then, 25% of the prepared TQ-01 enzyme solution was added to the system, and the pH was adjusted to 9.0 with (liquid alkali / hydrochloric acid). Finally, 0.02% of phenazine methyl sulfate was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 200 rpm, the conversion time was 16 h, and the reaction was terminated after TLC detection confirmed that there was no substrate residue. The residue was collected by filtration, and 12 v of methanol was used to heat to 60 °C for 2 h. The filtrate was collected by filtration, concentrated to a small volume under negative pressure, and 1 v of water was added. The material was further concentrated and discharged, and the solid was collected and dried to obtain the target product, exemestane. 10 g of raw material (a) was added, and 9.2 g of the product exemestane was finally collected. The liquid phase detection showed that the product purity was 99.8% and the yield was 92%.

[0033] Example 2 The TQ-01 enzyme solution prepared in Example 1 was used for feed conversion. The system was filled with 600 ml of the conversion system in a 1L conversion tank. A buffer substance was first added to the system. The buffer substance was Tris-Hcl buffer solution. The final concentration of Tris was 50 mmol / L. Then, 2% isopropanol was added to the system as a solubilizing solvent. Then, a conversion substrate compound (a) was added with a final concentration of 10%. Then, 25% of the prepared TQ-01 enzyme solution was added to the system. The pH was adjusted to 9.0 with (liquid alkali / hydrochloric acid). Finally, 0.02% of phenazine methyl sulfate was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 800 rpm, the ventilation volume was 0.1 L / min, the tank pressure was 0.02 Mpa, the conversion time was 12 h, and the reaction was terminated after TLC detection confirmed that there was no substrate residue. The residue was collected by filtration, and 12 v of methanol was used to heat to 60 °C for 2 h. The filtrate was collected by filtration, concentrated under negative pressure to a small volume, 1 v of water was added, and the material was continued to be concentrated. The solid was collected and dried to obtain the target product, exemestane. 60 g of raw material (a) was added, and 55.8 g of the product exemestane was finally collected. The liquid phase detection showed that the product purity was 99.7% and the yield was 93%.

[0034] Example 3 The TQ-01 enzyme solution prepared in Example 1 was used for feed conversion. The conversion was carried out in a shake flask. A 500 ml shake flask was filled with 100 ml of the conversion system. A buffer substance was first added to the system. The buffer substance was sodium acetate buffer. The final concentration of sodium acetate was 50 mmol / L. Then, 2% isopropanol was added to the system as a solubilizing solvent. Then, a conversion substrate compound (a) was added at a final concentration of 10%. Then, 25% of the prepared TQ-01 enzyme solution was added to the system. Then, the pH was adjusted to 9.0 with (liquid alkali / hydrochloric acid). Finally, 0.02% of phenazine methyl sulfate was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 200 rpm, the conversion time was 24 h, and the reaction was terminated after TLC detection confirmed that there was no substrate residue. The residue was collected by filtration, and 12 v of methanol was used to heat to 60 °C for 2 h. The filtrate was collected by filtration, concentrated to a small volume under negative pressure, and 1 v of water was added. The material was further concentrated and discharged. The solid was collected and dried to obtain the target product, exemestane. 10 g of raw material (a) was added, and 9.1 g of the product exemestane was finally collected. The liquid phase detection showed that the product purity was 98.5% and the yield was 91%.

[0035] Example 4 The TQ-01 enzyme solution prepared in Example 1 was used for feed conversion. The conversion was carried out in a shake flask. A 500 ml shake flask was filled with 100 ml of the conversion system. A buffer substance was first added to the system. The buffer substance was PBS buffer solution. The final concentration of PBS was 50 mmol / L. Then, 2% isopropanol was added to the system as a solubilizing solvent. Then, a conversion substrate compound (a) was added at a final concentration of 10%. Then, 25% of the prepared TQ-01 enzyme solution was added to the system. Then, the pH was adjusted to 9.0 with (liquid alkali / hydrochloric acid). Finally, 0.02% of phenazine methyl sulfate was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 200 rpm, the conversion time was 30 h, and the reaction was terminated after TLC detection confirmed that there was no substrate residue. The residue was collected by filtration, and slurried with 12v methanol at 60°C for 2h. The filtrate was collected by filtration, concentrated under negative pressure to a small volume, 1v water was added, and the material was further concentrated. The solid was collected and dried to obtain the target product, exemestane. 10 g of raw material (a) was added, and 9.1 g of the product exemestane was finally collected. The liquid phase detection showed that the product purity was 97.3% and the yield was 91%.

[0036] Example 5 The TQ-01 enzyme solution prepared in Example 1 was used for feed conversion. The conversion was carried out in a shake flask. A 500 ml shake flask was filled with 100 ml of the conversion system. A buffer substance was first added to the system. The buffer substance was Tris-Hcl buffer. The final concentration of Tris was 50 mmol / L. Methanol with a concentration of 2% was then added to the system as a solubilizing solvent. A conversion substrate compound (a) with a final concentration of 10% was then added. Then, the prepared TQ-01 enzyme solution with a concentration of 25% was added to the system. The pH was then adjusted to 9.0 with (liquid alkali / hydrochloric acid). Finally, phenazine methyl sulfate with a final concentration of 0.02% was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 200 rpm, the conversion time was 30 h, and the reaction endpoint was reached. The residue was collected by filtration, and the mixture was slurried with 12 v methanol at 60°C for 2 h. The filtrate was collected by filtration, concentrated under negative pressure to a small volume, 1 v of water was added, and the concentration was continued. The solid was collected and dried to obtain the target product, exemestane. 10 g of raw material (a) was added, and 8.8 g of the product exemestane was finally collected. The liquid phase detection showed that the product purity was 93.1% and the yield was 88%.

[0037] Example 6 The TQ-01 enzyme solution prepared in Example 1 was used for feed conversion. The conversion was carried out in a shake flask. A 500 ml shake flask was filled with 100 ml of the conversion system. A buffer substance was first added to the system. The buffer substance was Tris-Hcl buffer. The final concentration of Tris was 50 mmol / L. Then, 2% ethanol was added to the system as a solubilizing solvent. Then, a conversion substrate compound (a) with a final concentration of 10% was added. Then, 25% of the prepared TQ-01 enzyme solution was added to the system. Then, the pH was adjusted to 9.0 with (liquid alkali / hydrochloric acid). Finally, 0.02% of phenazine methyl sulfate was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 200 rpm, the conversion time was 30 h, and the reaction endpoint was reached. The residue was collected by filtration, and the mixture was slurried with 12 v methanol at 60°C for 2 h. The filtrate was collected by filtration, concentrated under negative pressure to a small volume, 1 v water was added, and the concentration was continued. The solid was collected and dried to obtain the target product, exemestane. 10 g of raw material (a) was added, and 8.9 g of the product exemestane was finally collected. The liquid phase detection showed that the product purity was 92.5% and the yield was 89%.

[0038] Example 7 The prepared TQ-01 enzyme solution was used for feed conversion, and the conversion was carried out in a tank. A 1L conversion tank contained 600ml of the conversion system. A buffer substance was first added to the system. The buffer substance was Tris-Hcl buffer solution. The final concentration of Tris was 50mmol / L. Then, 5% isopropanol was added to the system as a solubilizing solvent. Then, a conversion substrate compound (a) was added at a final concentration of 10%. Then, 25% of the prepared TQ-01 enzyme solution was added to the system. Then, the pH was adjusted to 9.0 with (liquid alkali / hydrochloric acid). Finally, 0.02% of phenazine methyl sulfate was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 800 rpm, the ventilation volume was 0.1 L / min, the tank pressure was 0.02 Mpa, the conversion time was 12 h, and the reaction was terminated after TLC detection confirmed that there was no substrate residue. The residue was collected by filtration, and 12 v of methanol was used to heat to 60 °C for 2 h. The filtrate was collected by filtration, concentrated under negative pressure to a small volume, 1 v of water was added, and the material was continued to be concentrated. The solid was collected and dried to obtain the target product, exemestane. 60 g of raw material (a) was added, and 55.4 g of the product exemestane was finally collected. The liquid phase detection showed that the product purity was 99.8%, and the yield was 92.3%.

[0039] Example 8 The prepared TQ-01 enzyme solution was used for feed conversion, and the conversion was carried out in a tank. A 1L conversion tank contained 600ml of the conversion system. A buffer substance was first added to the system. The buffer substance was Tris-Hcl buffer solution, and the final concentration of Tris was 50mmol / L. Then, 2% isopropanol was added to the system as a solubilizing solvent, and then a conversion substrate compound (a) was added with a final concentration of 20%. Then, 25% of the prepared TQ-01 enzyme solution was added to the system, and the pH was adjusted to 9.0 with (liquid alkali / hydrochloric acid). Finally, 0.02% of phenazine methyl sulfate was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 800 rpm, the ventilation volume was 0.1 L / min, the tank pressure was 0.02 Mpa, the conversion time was 24 h, and the reaction was terminated after reaching the reaction endpoint. The residue was collected by filtration, and 12 v of methanol was used to heat the temperature to 60 °C for 2 h. The filtrate was collected by filtration, concentrated under negative pressure to a small volume, 1 v of water was added, and the material was continued to be concentrated. The solid was collected and dried to obtain the target product, exemestane. 60 g of raw material (a) was added, and 54.1 g of the product exemestane was finally collected. The liquid phase detection showed that the product purity was 93.5% and the yield was 90.2%.

[0040] Example 9 The prepared TQ-01 enzyme solution was used for feed conversion, and the conversion was carried out in a tank. A 1L conversion tank contained 600ml of the conversion system. A buffer substance was first added to the system. The buffer substance was Tris-Hcl buffer solution, and the final concentration of Tris was 50mmol / L. Then, 2% isopropanol was added to the system as a solubilizing solvent, and then a conversion substrate compound (a) was added at a final concentration of 10%. Then, 25% of the prepared TQ-01 enzyme solution was added to the system, and the pH was adjusted to 8.0 with (liquid alkali / hydrochloric acid). Finally, 0.02% of phenazine methyl sulfate was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 800 rpm, the ventilation volume was 0.1 L / min, the tank pressure was 0.02 Mpa, the conversion time was 24 h, and the reaction was terminated after reaching the reaction endpoint. The residue was collected by filtration, and 12 v of methanol was used to heat the temperature to 60 °C for 2 h. The filtrate was collected by filtration, concentrated under negative pressure to a small volume, 1 v of water was added, and the material was continued to be concentrated. The solid was collected and dried to obtain the target product, exemestane. 60 g of raw material (a) was added, and 54.6 g of the product exemestane was finally collected. The liquid phase detection showed that the product purity was 75.3% and the yield was 91%.

[0041] Example 10 The prepared TQ-01 enzyme solution was used for feed conversion, and the conversion was carried out in a tank. A 1L conversion tank contained 600ml of the conversion system. A buffer substance was first added to the system. The buffer substance was Tris-Hcl buffer solution. The final concentration of Tris was 50mmol / L. Then, 2% isopropanol was added to the system as a solubilizing solvent. Then, a conversion substrate compound (a) was added at a final concentration of 10%. Then, 25% of the prepared TQ-01 enzyme solution was added to the system. Then, the pH was adjusted to 9.0 with (liquid alkali / hydrochloric acid). Finally, 0.02% of phenazine methyl sulfate was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 800 rpm, the ventilation volume was 1 L / min, the tank pressure was 0.02 Mpa, and the foaming was severe. 1% polyether defoamer was added. The conversion time was 12 h. The reaction was terminated after TLC detection confirmed that there was no substrate residue. The residue was collected by filtration, and 12 v methanol was heated to 60 °C for 2 h. The filtrate was collected by filtration. The negative pressure was concentrated to a small volume, 1 v water was added, and the material was continued to be concentrated. The solid was collected and dried to obtain the target product exemestane. 60 g of raw material (a) was added, and 55.5 g of the product exemestane was finally collected. The liquid phase detection showed that the product purity was 99.8%, and the yield was 92.5%.

[0042] Embodiment 11 The prepared TQ-01 enzyme solution was used for feed conversion, and the conversion was carried out in a tank. A 1L conversion tank contained 600ml of the conversion system. A buffer substance was first added to the system. The buffer substance was Tris-Hcl buffer solution, and the final concentration of Tris was 50mmol / L. Then, 2% isopropanol was added to the system as a solubilizing solvent, and then a conversion substrate compound (a) was added at a final concentration of 10%. Then, 25% of the prepared TQ-01 enzyme solution was added to the system, and the pH was adjusted to 9.0 with (liquid alkali / hydrochloric acid). Finally, 0.05% of phenazine methyl sulfate was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 800 rpm, the ventilation volume was 1 L / min, the tank pressure was 0.02 Mpa, and the foaming was serious. 1% polyether defoamer was added. The conversion time was 24 h. The reaction was terminated after the reaction endpoint was reached. The residue was collected by filtration, and 12 v methanol was used to heat to 60 °C for 2 h. The filtrate was collected by filtration. The negative pressure was concentrated to a small volume, 1 v water was added, and the material was continued to be concentrated. The solid was collected and dried to obtain the target product exemestane. 60 g of raw material (a) was added, and 55.3 g of the product exemestane was finally collected. The liquid phase detection product purity was 97.6%, and the yield was 92.2%.

[0043] Example 12 The prepared TQ-01 enzyme solution was used for feed conversion, and the conversion was carried out in a tank. A 1L conversion tank contained 600ml of the conversion system. A buffer substance was first added to the system. The buffer substance was Tris-Hcl buffer solution, and the final concentration of Tris was 50mmol / L. Then, 2% isopropanol was added to the system as a solubilizing solvent, and then a conversion substrate compound (a) was added at a final concentration of 10%. Then, 10% of the prepared TQ-01 enzyme solution was added to the system, and the pH was adjusted to 9.0 with (liquid alkali / hydrochloric acid). Finally, 0.02% of phenazine methyl sulfate was added to start the conversion. The conversion temperature was 30°C, the shaking speed was 800 rpm, the ventilation volume was 1 L / min, the tank pressure was 0.02 Mpa, and the foaming was serious. 1% polyether defoamer was added. The conversion time was 24 h. The reaction was terminated after the reaction endpoint was reached. The residue was collected by filtration, and 12 v methanol was used to heat to 60 °C for 2 h. The filtrate was collected by filtration. The negative pressure was concentrated to a small volume, 1 v water was added, and the material was continued to be concentrated. The solid was collected and dried to obtain the target product exemestane. 60 g of raw material (a) was added, and 55.2 g of the product exemestane was finally collected. The liquid phase detection product purity was 75.3%, and the yield was 92%.

[0044] By comparing the experimental results of various embodiments, it can be seen that: 1. When sodium acetate or PBS is used as a buffer substance, it will affect the activity of the enzyme in the system, prolong the conversion time, and reduce the final conversion rate. Therefore, Tris is preferred as the buffer substance of the conversion system; 2. When methanol or ethanol is used as a solubilizing solvent, the enzyme activity will be inhibited or inactivated, resulting in a prolonged conversion time and a reduced final conversion rate. Therefore, isopropanol is preferably used as the solubilizing substance of the conversion system; 3. Increasing the concentration of isopropanol by a small amount will not significantly inhibit enzyme activity, but it will reduce the yield during extraction. The preferred concentration is 2% isopropanol to achieve a solubilizing effect while ensuring the yield; 4. Increasing the substrate concentration or reducing the amount of enzyme added will result in insufficient enzyme activity in the system, resulting in incomplete conversion; 5. The optimal conversion pH of TQ-01 enzyme should be around 9.0. Lowering the conversion pH can accelerate the reaction rate in the early stage, but it will inactivate the enzyme and affect the final conversion rate. 6. Increasing the dosage of phenazine methyl sulfate will inhibit the activity of the enzyme in the conversion system to a certain extent and will also increase the cost. The preferred concentration is 0.02%; 7. Increasing ventilation will speed up the reaction rate, but serious foaming will occur, making it difficult to carry out production. It is best to choose low ventilation to increase the tank pressure to increase the oxygen concentration.

[0045] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0046] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.

Claims

1. An exemestane dehydrogenase, characterized in that: The sequence of the exemestane dehydrogenase is SEQ ID NO.

1.

2. A gene characterized by Encodes the exemestane dehydrogenase as described in claim 1.

3. A strain characterized by: The gene as claimed in claim 2 is inserted into the chassis strain, or a plasmid containing the gene as claimed in claim 2 is contained in the chassis strain.

4. A method for preparing exemestane, characterized in that: An enzyme solution containing the exemestane dehydrogenase as claimed in claim 1, a buffer solvent, a cosolvent and a substrate are mixed, the pH value is adjusted to 8-9, an electron acceptor is added, and conversion is carried out. After the conversion is completed, treatment is performed to obtain exemestane.

5. The preparation method according to claim 4, characterized in that: The buffer solvent is Tris-HCl buffer, PBS phosphate buffer or sodium acetate buffer solution, and the final concentration of the buffer solvent is 20-100 mmol / L.

6. The preparation method according to claim 4, characterized in that: The co-solvent is methanol, ethanol, isopropanol or acetone.

7. The preparation method according to claim 4, characterized in that: The structural formula of the substrate is: The feed concentration of the enzyme solution is 10-30%, and the electron acceptor is phenazine methyl sulfate.

8. The preparation method according to claim 4, characterized in that: The substance that adjusts the pH value to 8-9 is lye or hydrochloric acid.

9. The preparation method according to claim 4, characterized in that: The conversion temperature is 25-35°C, air or oxygen is introduced during the conversion, and the treatment operation is filtering, adding methanol to the filter residue and heating it to 50-70°C for pulping, filtering, concentrating the filtrate, adding water, concentrating, and drying to obtain exemestane.

10. The preparation method according to claim 4, characterized in that: The buffer solvent is Tris-Hcl buffer, the final concentration of the buffer solvent is 50mmol / L, the cosolvent is isopropanol, the feed concentration of the enzyme solution is 20-30%, the electron acceptor is phenazine methyl sulfate, the pH value is adjusted to 8.5-9, air or oxygen is introduced during the conversion, and the conversion time is more than 12h.

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