Specific high-activity hydrolase, gene, engineering bacterium and application

Abstract

The invention belongs to the field of organic chemistry and biosynthesis, and particularly relates to a specific high-activity hydrolase, a gene, an engineering bacterium and application, the specific high-activity hydrolase is obtained by mutation of an original hydrolase, the sequence of the original hydrolase is SEQ ID NO.1, the mutation is as follows: the 160th site is mutated from A to D, the 254th site is mutated from V to S, or the 160th site is mutated from A to D, and the 254th site is mutated from V to S; the 160th site is mutated from A to D, and the 254th site is mutated from V to S; the method is used for catalyzing hydrolysis of a substrate, and the substrate is a steroid 21-hydroxy acetate compound; the conversion condition is mild, and no organic solvent needs to be added; the conversion specificity is high, and no by-product is generated; and the product purity and yield are high.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of organic chemistry and biosynthesis, and specifically relates to a specific high-activity hydrolytic enzyme, a gene, an engineered bacterium and a use. BACKGROUND

[0002] Steroid glucocorticoid drugs have anti-inflammatory, immunosuppressive, anti-toxic and anti-shock pharmacological activities, and are widely used in the treatment of autoimmune diseases, allergic diseases and critical conditions. Glucocorticoids are a class of steroid hormones with cyclopentane polyhydrogenophenyl as the mother nucleus, and the key functional groups include C3 carbonyl, C4-C5 double bond, C11 hydroxyl, C17 hydroxyl and C21 hydroxyl ketone. These structural characteristics determine their core pharmacological activities such as anti-inflammatory and immunosuppressive.

[0003] For example, the classic process for preparing hydrocortisone from hydrocortisone acetate adopts chemical hydrolysis method. Chinese Patent Application Publication No. CN102367262A discloses a preparation method of hydrocortisone: hydrocortisone acetate is dissolved in methanol, a 2% sodium hydroxide methanol solution is added, and stirring is carried out at 0-8°C for more than 4 hours. After the reaction is completed, neutralization is carried out with acetic acid, the solvent is recovered by reduced pressure concentration, and hydrocortisone is obtained after crystallization, filtration, methanol washing and drying. Chinese Patent Application Publication No. CN113583071A discloses a preparation method of hydrocortisone crude product. The method improves the solubility of the substrate hydrocortisone acetate by using a mixed solvent of dichloromethane and methanol in a specific ratio. At the same time, a mixed solution of sodium hydroxide and potassium carbonate is used as the hydrolysis liquid to improve the hydrolysis efficiency. In addition, the method also optimizes the hydrolysis reaction steps, controls the conversion time by monitoring the substrate residue by thin layer chromatography to improve the product purity. Finally, dichloromethane and methanol are removed in turn by reduced pressure concentration to obtain the hydrocortisone product.

[0004] The above-mentioned methods have problems such as long reaction time, low yield, and solvent cannot be directly reused. The reaction conditions are relatively harsh: it needs to be carried out at low temperature of 0-10°C, and strong alkaline reagents (such as NaOH or KOH) and complex organic solvent systems (tetrahydrofuran, dichloromethane and methanol mixed solution) are used. The environmental and safety problems are prominent: the excessive organic solvent (used to dissolve the substrate and stabilize the intermediate) increases the cost and environmental pressure.

[0005] In addition, the 21-hydroxyl ketone structure of the D-ring-17-side chain of glucocorticoid drugs is a key pharmacophore, but it is easy to undergo intramolecular rearrangement and oxidative cleavage in an alkaline environment, and finally converted into inactive 17-ketone by-product, affecting the product quality. The chemical synthesis route of 21-hydroxyl ester strong base hydrolysis (taking prednisolone as an example) is as follows: . SUMMARY

[0006] The technical problem solved by the present application is to provide a specific high-activity hydrolytic enzyme, a gene, an engineered bacterium and a use, which has mild transformation conditions and does not need to add an organic solvent, has strong transformation specificity and no by-product is generated, and has high product purity and yield.

[0007] The embodiment of the present application provides a specific high-activity hydrolytic enzyme, which is obtained by mutating an original hydrolytic enzyme, wherein the sequence of the original hydrolytic enzyme is SEQ ID NO. 1, and the mutation is that the 160th site is mutated from A to D, the 254th site is mutated from V to S, or the 160th site is mutated from A to D and the 254th site is mutated from V to S.

[0008] Preferably, the mutation is that the 160th site is mutated from A to D and the 254th site is mutated from V to S.

[0009] The embodiment of the present application provides a gene encoding the specific high-activity hydrolytic enzyme.

[0010] The embodiment of the present application provides an engineered bacterium, which expresses the specific high-activity hydrolytic enzyme or contains the gene.

[0011] Preferably, the host microorganism of the engineered bacterium is Escherichia coli.

[0012] The embodiment of the present application provides a use of the specific high-activity hydrolytic enzyme or the engineered bacterium in hydrolysis of a steroid hormone.

[0013] Preferably, the hydrolysis substrate is a steroid 21-hydroxyacetate compound, the hydrolysis system comprises a buffer and a cosolvent, the pH value of the system is controlled to be 7-9 (the pH value is adjusted by sodium hydroxide), and the hydrolysis temperature is 20-45 DEG C. The reaction route is as follows: ; The dashed line position represents a single bond or a double bond, R1 is H, CH3, Cl or F; R2 is H, F, C1, Br, OH or no group, R3 is a carbonyl group, OH or H, or R2 and R3 are an epoxy group; R4 is H, alpha-OH, alpha-CH3 or beta-CH3.

[0014] Preferably, the buffer is Tris-HCl buffer (the concentration is 0.1-0.5 M, the pH value is 6-9, and the pH value is preferably 7.5), the cosolvent is methanol, ethanol or acetone, the pH value of the system is controlled to be 7.3-7.7, and the hydrolysis temperature is 25-35 DEG C, preferably 30 DEG C.

[0015] Preferably, the volume concentration of the Tris-HCl buffer is 5-30%, preferably 10%, and the volume concentration of the cosolvent is 2-30%, preferably 10%.

[0016] Preferably, the substrate is added in an amount of 5-20 wt%, preferably 12 wt%, and the specific high-activity hydrolytic enzyme is added in an amount of 5-30%, preferably 20%.

[0017] The specific high-activity hydrolytic enzyme of the present application has a higher affinity for the substrate, and has a higher conversion purity and yield when performing substrate hydrolysis. The conversion conditions of the present application are mild, do not require corrosion-resistant, high-pressure equipment, have lower energy consumption, do not require the addition of organic solvents, and significantly reduce the COD of wastewater. The conversion specificity is strong, no by-products are generated, and the subsequent purification steps are simpler. Prolonging the conversion time does not reduce the product purity and yield, the operation is simple, and the production cost can be significantly reduced. DETAILED DESCRIPTION

[0018] Example 1 The original non-mutated genome (i.e., SEQ ID NO. 2, and the corresponding amino acid sequence is SEQ ID NO. 1) was connected to the vector pET28a(+) plasmid to obtain plasmid 1. The obtained plasmid 1 was transformed into plasmid amplification Escherichia coli DH5α by heat shock method. After adding 500 uL of antibiotic-free LB medium to the transformed recombinant bacteria, 37℃, 200 rpm shaking incubation for 1 h, the bacteria were taken out and inoculated on kanamycin-resistant LB plates, and incubated at 37℃ for 16 h. The grown single colonies were selected for positive identification, and the positive clone strains were selected and inoculated into LB shake flasks with kanamycin, and incubated at 37℃, 200 rpm for 12 h. The plasmid was extracted using a GenScript kit.

[0019] SEQ ID NO. 1 is: GGGPHRRPDSVTGVFSCSKGVSGLVIALLVQDGFLDLDAEVVKYWPEFGAEGKATITVAQ LLSHQAGLLGVEGGLTLAEYNNSELAAAKLAQMRPLWKPGTAFGYHALTIGVFMEELCRR ITGSTLQEIYEQRIRSVTGAHFFLGLPESEEPRYATLRWAADPSQPWIDPASHFGLSANS AVGDILDLPNLREVRAAGLSSAAGVASAEGMARVYAAALTGLAANGDRAAVAPLLSEETI QTVTAEQVFGIDRVFGETSCFGTVFMKSHARSPYGSYRAFGHDGASASLGFADPVYELAF GYVPQQAEPGGAGCRNLELSAAVRKAVTELAQ.

[0020] SEQ ID NO. 2 is: GGAGGGGGACCCCACCGTCGCCCGGACTCGGTGACAGGCGTATTCAGTTGCTCAAAGGGCGTGAGTGGTCTGGTTATCGCTTTATTGGTACAAGATGGTTTTCTTGATCTGGACGCGGAAGTGGTGAAATATTGGCCTGAGTTCGGAGCCGAGGGGAAAGCGACAATAACGGTGGCGCAGCTTTTATCGCATCAGGCGGGGCTGCTAGGGGTCGAGGGAGGTTTGACGCTCGCCGAGTACAACAACAGTGAACTAGCCGCTGCCAAACTCGCACAGATGCGCCCCCTCTGGAAGCCGGGGACCGCTTTTGGGTATCATGCTTTGACGATAGGTGTCTTCATGGAAGAGCTGTGCCGGCGTATCACTGGTTCAACGCTACAAGAGATTTATGAACAACGTATCCGCTCGGTCACAGGTGCACACTTCTTTTTAGGACTGCCTGAATCGGAAGAACCACGTTACGCCACTCTTCGATGGGCGGCGGATCCCTCGCAGCCGTGGATAGATCCTGCATCGCACTTTGGCCTGAGCGCAAACTCGGCAGTAGGTGACATCTTAGACTTGCCAAATTTAAGAGAGGTGCGGGCCGCCGGGTTAAGCAGCGCCGCGGGCGTTGCTTCGGCCGAAGGTATGGCCCGAGTTTACGCGGCCGCTTTGACGGGCTTGGCAGCGAACGGAGATCGGGCAGCAGTGGCTCCATTGCTCTCGGAAGAGACTATACAGACGGTCACGGCTGAGCAAGTGTTCGGGATTGATCGGGTCTTTGGAGAAACTTCCTGTTTCGGAACCGTATTTATGAAATCACACGCTCGTAGCCCATATGGTTCCTATCGCGCGTTCGGCCACGACGGTGCGTCGGCGAGCCTAGGATTTGCGGATCCTGTGTACGAACTCGCTTTTGGGTATGTCCCCCAGCAAGCTGAACCGGGTGGCGCTGGATGTCGCAATCTAGAACTTTCCGCAGCGGTGCGAAAGGCAGTCACGGAGCTAGCGCAA

[0021] The obtained plasmid was subjected to point mutation by overlap extension PCR method to obtain plasmid 2 (A160D), plasmid 3 (V254S), and plasmid 4 (A160D, V254S). The mutation of plasmid 2 (A160D) used primers 160F and 160F; the mutation of plasmid 3 (V254S) used primers 254F and 254F; and the mutation of plasmid 4 (A160D, V254S) used primers 160F, 160F, 254F, and 254F.

[0022] The primers used are as follows:

[0023] The system used is as follows:

[0024] The PCR program was set as follows: 94°C pre-denaturation for 2 min; [98°C denaturation for 10 s, 58°C annealing for 15 s, 72°C extension for 1 min] 35 cycles; 72°C final extension for 10 min, to obtain the PCR amplification product. The PCR amplification product was purified by using a kit to obtain the target plasmid.

[0025] The hydrolytic enzymes in plasmid 2 (A160D), plasmid 3 (V254S), and plasmid 4 (A160D, V254S) are SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9, respectively.

[0026] SEQ ID NO. 7 is as follows: GGGPHRRPDSVTGVFSCSKGVSGLVIALLVQDGFLDLDAEVVKYWPEFGAEGKATITVAQ LLSHQAGLLGVEGGLTLAEYNNSELAAAKLAQMRPLWKPGTAFGYHALTIGVFMEELCRR ITGSTLQEIYEQRIRSVTGAHFFLGLPESEEPRYATLRWDADPSQPWIDPASHFGLSANS AVGDILDLPNLREVRAAGLSSAAGVASAEGMARVYAAALTGLAANGDRAAVAPLLSEETI QTVTAEQVFGIDRVFGETSCFGTVFMKSHARSPYGSYRAFGHDGASASLGFADPVYELAF GYVPQQAEPGGAGCRNLELSAAVRKAVTELAQ.

[0027] SEQ ID NO. 8 is: GGGPHRRPDSVTGVFSCSKGVSGLVIALLVQDGFLDLDAEVVKYWPEFGAEGKATITVAQ LLSHQAGLLGVEGGLTLAEYNNSELAAAKLAQMRPLWKPGTAFGYHALTIGVFMEELCRR ITGSTLQEIYEQRIRSVTGAHFFLGLPESEEPRYATLRWAADPSQPWIDPASHFGLSANS AVGDILDLPNLREVRAAGLSSAAGVASAEGMARVYAAALTGLAANGDRAAVAPLLSEETI QTVTAEQVFGIDRSFGETSCFGTVFMKSHARSPYGSYRAFGHDGASASLGFADPVYELAF GYVPQQAEPGGAGCRNLELSAAVRKAVTELAQ.

[0028] SEQ ID NO. 9 is: GGGPHRRPDSVTGVFSCSKGVSGLVIALLVQDGFLDLDAEVVKYWPEFGAEGKATITVAQ LLSHQAGLLGVEGGLTLAEYNNSELAAAKLAQMRPLWKPGTAFGYHALTIGVFMEELCRR ITGSTLQEIYEQRIRSVTGAHFFLGLPESEEPRYATLRWDADPSQPWIDPASHFGLSANS AVGDILDLPNLREVRAAGLSSAAGVASAEGMARVYAAALTGLAANGDRAAVAPLLSEETI QTVTAEQVFGIDRSFGETSCFGTVFMKSHARSPYGSYRAFGHDGASASLGFADPVYELAF GYVPQQAEPGGAGCRNLELSAAVRKAVTELAQ.

[0029] The obtained plasmid was transformed into E. coli DH5α by heat shock method. E. coliIn the BL21 (DE3) engineering bacteria, 500ul of LB medium without resistance was added to the transformed recombinant bacteria, which was cultured at 37℃, 200rpm for 1h, then taken out, and inoculated on LB plate with kanamycin resistance (50mg / L), and cultured at 37℃ for 16h. The grown single colonies were selected for positive identification, and the positive clone strains were selected and inoculated into LB flask with kanamycin resistance (50mg / L), and cultured at 37℃, 200rpm for 8h, then induced by adding membrane-permeated IPTG, and the induction concentration was 0.2mM. After induction, the culture condition was changed to 25℃, 200rpm, and cultured for 10h. Then the bacterial cells were collected by centrifugation, weighed, and diluted with water to 300g / L. 1% potassium phosphate dibasic was added as buffer, and then stirred and mixed to obtain four kinds of broken enzyme solutions.

[0030] The obtained four kinds of enzyme solutions were used for feeding conversion, and the conversion system was as follows: 0.5M Tris-HCl (pH7.5) volume concentration 10%, isopropyl alcohol volume concentration 10%, ultrafine grinding substrate compound (a) mass concentration 5%, broken enzyme solution volume concentration 10%. The conversion was carried out by magnetic stirring in a water bath, and the conversion condition was controlled at 30℃. During the conversion process, 2M NaOH solution was used to adjust the pH, and the pH was controlled between 7.5±0.2. When the pH of the conversion system no longer changed, it was judged that the conversion endpoint was reached. The sample was taken for liquid phase detection, and the liquid phase results were as follows:

[0031] From the conversion results, it can be seen that the conversion time of recombinant bacteria 2 and original recombinant bacteria 1 is the same, but the substrate conversion rate is obviously higher. The time to reach the endpoint of recombinant bacteria 3 and recombinant bacteria 4 is obviously shorter, and the conversion efficiency is obviously higher. Although the conversion time of recombinant bacteria 4 is slightly longer than that of recombinant bacteria 3, the substrate conversion rate is obviously higher.

[0032] Example 2 The recombinant bacteria 4 obtained in Example 1 was cultured, and 100ul of glycerol bacteria was taken from the glycerol tube and inoculated into 100ml of liquid LB medium. The composition of the liquid LB medium was as follows:

[0033] Before inoculation, 50mg / L kanamycin was added to the liquid medium to a final concentration. After inoculation, the culture was placed in a constant temperature shaker and cultured at 37℃, 200rpm for 12h. After seed culture, the culture was inoculated into a sterilized 5L fermenter for fermentation, and the fermentation volume was 2.5L. The composition of the fermentation medium was as follows:

[0034] The temperature before fermentation should be controlled at 37℃, the pH should be controlled at 7.0 by (ammonia / phosphoric acid), and when the OD600 of the fermentation system reaches 6, the temperature should be reduced to 25℃, and then IPTG solution after membrane sterilization should be added for induction, and the final concentration of IPTG is 0.2mmol / L.

[0035] After induction, continue to ferment for 22h to complete fermentation, and the bacterial concentration is 48g / L. The collected bacteria are configured into a 300g / L bacterial solution with water, 1% potassium phosphate is added, the pH is adjusted to 8.0 with 20% lye, and then a homogenizer is used for homogenization and crushing. The homogenization pressure is 800Mpa, and the temperature is controlled between 10~30℃ during the homogenization process. The prepared enzyme solution is obtained by homogenizing three times, and the pH of the enzyme solution is 7.5.

[0036] The prepared enzyme solution is used for feeding conversion, and the conversion is carried out by magnetic stirring in a water bath. The system is filled with 300ml of conversion system in a 500ml three-neck flask. 50% of the conversion volume of water is added to the system, followed by 10% of the volume of buffer solution, which is 0.5M Tris-HCl (pH 7.5) buffer solution. Then 10% of the volume concentration of isopropyl alcohol is added as a cosolvent, and then the final mass concentration of the conversion substrate compound (hydrocortisone acetate) is 15%. Then 10% of the volume of the prepared enzyme solution is added, and the final volume is adjusted with water. The pH is adjusted to 7.5 with (lye / hydrochloric acid), and the conversion starts. The conversion temperature is selected as 30℃, the pH is adjusted with 2M NaOH solution, the pH is controlled at 7.5±0.2 during the conversion process, the conversion time is 18h, and after the pH is unchanged, the filter residue is collected by filtration. The sample is detected by liquid chromatography, and the product purity is 98.1%. After dissolving in dichloromethane-methanol mixed solvent (dichloromethane and methanol, volume ratio 1:1), the filtrate is filtered, and the dichloromethane is removed by vacuum concentration. Then the methanol is concentrated, and after the small volume is added with water, the remaining methanol is concentrated. The product crystal is collected by filtration, and the comprehensive yield is 97.3%.

[0037] Example 3 The enzyme solution prepared in Example 2 was used for feeding conversion. The conversion was carried out in a 1L conversion tank with a loading of 600ml of the conversion system. 50% of the conversion volume of water was first added to the system, followed by 10% of the volume of buffer solution, which was 0.5M Tris-HCl (pH 7.5) buffer solution. Then 10% of isopropyl alcohol with a concentration of 15% was added as a cosolvent to the system, followed by the addition of 20% of the volume of the prepared enzyme solution. The system was then made up to the final volume with water, and the pH was adjusted to 7.5 with (liquid alkali / hydrochloric acid) to start the conversion. The conversion temperature was selected to be 30°C, and the pH was adjusted with 2M NaOH solution. The pH was controlled at 7.5±0.2 during the conversion process, and the conversion time was 24h. After the pH remained unchanged, the filter residue was collected by filtration, and the sample was subjected to liquid phase detection. The liquid phase detection showed a product purity of 98.8%. The product crystal was collected by filtration after the solvent was dissolved in dichloromethane-methanol mixed solvent (dichloromethane and methanol, volume ratio 1:1). The dichloromethane was first concentrated under reduced pressure, followed by the concentration of methanol. Water was added to replace the remaining methanol, and the product crystal was collected by filtration. The product was detected by nuclear magnetic resonance to be hydrocortisone, with a molar yield of 97.5%.

[0038] The reaction formula is as follows: .

[0039] Hydrocortisone acetate (self-made): 1 H NMR (500 MHz, CDCl3) δ 5.56 (d, J = 2.1 Hz,1H), 4.97 – 4.76 (m, 2H), 4.38 – 4.27 (m, 1H), 2.66 – 2.52 (m, 1H), 2.37(ddd, J = 16.1, 13.8, 5.1 Hz, 2H), 2.29 – 2.19 (m, 1H), 2.18 – 2.09 (m, 2H),2.07 (d, J = 6.7 Hz, 3H), 2.01 – 1.86 (m, 3H), 1.83 – 1.58 (m, 4H), 1.41 –1.35 (m, 1H), 1.34 (d, J = 5.1 Hz, 3H), 1.30 (dd, J = 11.2, 6.2 Hz, 1H), 1.08– 0.95 (m, 1H), 0.89 (dd, J = 11.2, 3.3 Hz, 1H), 0.78 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 206.1, 201.1, 174.5, 171.5, 121.7, 89.4, 68.4, 67.7, 56.0,52.0, 49.4, 49.2, 49.0, 48.9, 48.7, 48.5, 48.4, 47.3, 39.3, 39.2, 34.6, 33.8,33.6, 32.8, 32.2, 31.4, 23.6, 20.7, 20.4, 16.6.

[0040] Hydrocortisone: 1 H NMR (400 MHz, DMSO- d 6) δ 5.56 (d, J = 1.5 Hz, 1H), 5.17(s, 1H), 4.65 (t, J = 5.8 Hz, 1H), 4.50 (dd, J = 19.1, 5.9 Hz, 1H), 4.31 –4.22 (m, 2H), 4.07 (dd, J = 19.1, 5.8 Hz, 1H), 2.56 (ddd, J = 14.1, 11.5, 2.6Hz, 1H), 2.48 – 2.31 (m, 2H), 2.18 (dt, J = 16.7, 4.2 Hz, 2H), 2.08 (dt, J =13.3, 4.6 Hz, 1H), 1.98 – 1.84 (m, 3H), 1.78 (td, J = 13.5, 4.4 Hz, 1H), 1.65(h, J = 7.4 Hz, 2H), 1.53 (dd, J = 13.5, 2.5 Hz, 1H), 1.45 – 1.38 (m, 1H),1.36 (s, 3H), 1.25 (tt, J = 13.9, 6.9 Hz, 1H), 0.98 (qd, J = 13.2, 4.5 Hz,1H), 0.85 (dd, J = 10.9, 3.2 Hz, 1H), 0.74 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6)δ 211.7, 198.1, 172.4, 121.5, 88.5, 66.5, 65.9, 55.5, 51.6, 46.3, 38.9, 34.0,33.5, 33.0, 32.8, 31.4, 31.2, 23.4, 20.5, 17.0。

[0041] Example 4 The enzyme solution prepared in Example 2 was used for feeding conversion, 600 ml of the conversion system was loaded into a 1 L conversion tank, 50% of the conversion volume of water was first added to the system, then 10% of the volume of the buffer solution was added, the buffer solution was 0.5M Tris-HCl (pH 7.5) buffer solution, then 10% of the concentration of isopropyl alcohol was added to the system as a cosolvent, then the final concentration of 12% of the conversion substrate compound (prednisolone acetate) was added, then 20% of the volume of the prepared enzyme solution was added to the system, and water was added to the final volume, then (liquid alkali / hydrochloric acid) was used to adjust the pH to 7.5, and the conversion was started. The conversion temperature was selected to be 30°C, the pH was controlled at 7.5±0.2 during the conversion process using 2M NaOH, the conversion time was 24h, after the pH was unchanged, the filter residue was collected by filtration, and the sample was detected by liquid chromatography. The liquid chromatography detection product purity was 99.1%, after dissolving in dichloromethane-methanol mixed solvent (dichloromethane and methanol, volume ratio 1:1), filtration, the filtrate was first concentrated under reduced pressure to remove dichloromethane, then concentrated to remove methanol, and after water replacement, the remaining methanol was continuously concentrated, and the product crystal was collected by filtration. NMR detection was prednisolone, and the molar yield was 98.2%.

[0042] The reaction formula is as follows: .

[0043] Prednisolone acetate (self-made): 1 H NMR (400 MHz, DMSO- d 6) δ 7.60 (d, J = 10.2 Hz,1H), 6.11 (dd, J = 10.3, 1.9 Hz, 1H), 6.01 (t, J = 1.6 Hz, 1H), 5.82 (s, 1H),4.99 – 4.71 (m, 2H), 2.90 (d, J = 12.1 Hz, 1H), 2.37 (tt, J = 11.4, 6.4 Hz,2H), 2.19 (dd, J = 11.8, 3.8 Hz, 2H), 2.09 (s, 3H), 2.01 (ddd, J = 19.7,10.7, 4.4 Hz, 2H), 1.83 – 1.71 (m, 1H), 1.65 (ddd, J = 14.8, 9.4, 5.5 Hz,1H), 1.40 (dd, J = 12.0, 5.7 Hz, 1H), 1.36 (s, 3H), 1.20 (qd, J = 12.3, 4.0Hz, 1H), 0.50 (s, 3H). 13C NMR (101 MHz, DMSO- d 6) δ 210.1, 205.4, 185.1, 170.0,167.2, 155.1, 127.0, 123.8, 87.7, 68.0, 58.8, 50.8, 49.3, 48.8, 42.0, 35.5,33.7, 33.1, 31.5, 22.8, 20.4, 18.8, 15.0.

[0044] Prednisone: 1 H NMR (400 MHz, DMSO- d 6) δ 7.59 (d, J = 10.2 Hz, 1H), 6.10 (dd, J = 10.3, 2.0 Hz, 1H), 6.01 (t, J = 1.7 Hz, 1H), 4.70 (dd, J = 6.6, 5.4Hz, 1H), 4.44 (dd, J = 19.3, 6.6 Hz, 1H), 4.13 – 4.02 (m, 1H), 2.88 (d, J =12.1 Hz, 1H), 2.57 (ddd, J = 14.6, 11.6, 3.2 Hz, 1H), 2.40 – 2.31 (m, 2H),2.18 (d, J = 11.3 Hz, 1H), 2.08 – 1.94 (m, 3H), 1.76 (qd, J = 8.6, 7.9, 3.0Hz, 1H), 1.62 (ddd, J = 14.9, 9.5, 5.8 Hz, 1H), 1.35 (s, 3H), 1.20 (qd, J =12.4, 4.3 Hz, 1H), 0.50 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ 211.5, 210.3,185.1, 167.2, 155.1, 127.0, 123.8, 87.4, 66.1, 58.8, 50.5, 49.8, 48.8, 41.9,35.6, 33.6, 33.2, 31.5, 22.8, 18.8, 15.5.

[0045] Example 5 The enzyme solution prepared in Example 2 was used for conversion. The conversion tank was filled with 600 ml of conversion system in a 1L conversion tank. First, 50% of the conversion volume of water was added to the system, followed by 10% of the volume of buffer solution. The buffer solution was 0.5M Tris-HCl (pH 7.5) buffer. Then, 10% of the concentration of isopropanol was added as a solubilizing solvent. Next, the conversion substrate compound (prednisolone acetate) was added to a final concentration of 12%. Then, 20% of the volume of the prepared enzyme solution was added to the system, and the volume was adjusted to the final volume with water. The pH was then adjusted to 8.5 with (liquid alkali / hydrochloric acid) and the conversion began. The conversion temperature was selected at 30℃, and the pH was adjusted with 2M NaOH solution. The pH was controlled at 8.5±0.2 during the conversion process, and the conversion time was 48h. After the pH did not change, the filter residue was collected and sampled for liquid chromatography analysis. The purity of the product was 97.4%. The product was dissolved in a dichloromethane and methanol mixed solvent (dichloromethane and methanol, volume ratio 1:1) and then filtered. The filtrate was first concentrated under reduced pressure to remove dichloromethane, and then concentrated to remove methanol. After reducing the volume to a small amount, water was added to replace the dichloromethane, and the remaining methanol was further concentrated. The product crystals were collected by vacuum filtration and identified as prednisolone by NMR analysis, with a molar yield of 96.3%.

[0046] The reaction formula is as follows: .

[0047] Prednisolone acetate (homemade): 1 H NMR (400 MHz, DMSO- d 6) δ 5.81 (s, 1H), 5.64(s, 1H), 4.98 – 4.74 (m, 2H), 2.89 (d, J = 12.1 Hz, 1H), 2.47 – 2.31 (m, 4H), 2.26 (d, J = 13.8 Hz, 1H), 2.20 – 2.11 (m, 3H), 2.09 (s, 3H), 1.99 (s, 1H),1.89 (t, J = 10.3 Hz, 2H), 1.79 (d, J = 9.7 Hz, 1H), 1.66 (ddd, J = 14.6,11.4, 5.1 Hz, 2H), 1.32 (s, 3H), 1.26 – 1.13 (m, 2H), 0.47 (s, 3H). 13 C NMR (101 MHz, DMSO-) d6) δ 210.6, 205.9, 198.8, 170.4, 169.6, 124.2, 88.4, 68.4,61.5, 51.1, 50.1, 49.5, 38.2, 36.4, 34.3, 33.8, 32.4, 32.1, 23.2, 20.8, 17.3,15.3.

[0048] Prednisolone: 1 H NMR (400 MHz, DMSO- d 6) δ 7.31 (d, J = 10.1 Hz, 1H), 6.15 (dd, J = 10.1, 1.8 Hz, 1H), 5.91 (t, J = 1.6 Hz, 1H), 5.18 (s, 1H), 4.73 –4.61 (m, 2H), 4.49 (dd, J = 19.1, 5.9 Hz, 1H), 4.27 (t, J = 3.3 Hz, 1H), 4.07(dd, J = 19.1, 5.9 Hz, 1H), 2.62 – 2.52 (m, 2H), 2.29 (dd, J = 13.1, 4.3 Hz,1H), 2.01 (dd, J = 11.7, 7.7 Hz, 2H), 1.85 (dd, J = 13.6, 3.6 Hz, 1H), 1.71 –1.50 (m, 3H), 1.38 (s, 3H), 1.34 – 1.20 (m, 1H), 1.00 (qd, J = 13.0, 4.5 Hz,1H), 0.88 (dd, J = 11.0, 3.4 Hz, 1H), 0.77 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ212.1, 187.7, 172.8, 158.4, 126.8, 121.5, 88.7, 69.4, 66.6, 55.3, 51.1, 47.3,44.5, 39.1, 34.0, 33.4, 32.0, 31.1, 23.7, 20.5, 16.8.

[0049] Example 6 The enzyme solution prepared in Example 2 was used for conversion. The conversion tank was filled with 600 ml of conversion system in a 1L conversion tank. First, 30% of the conversion volume of water was added to the system, followed by 10% of the volume of buffer solution. The buffer solution was 0.5M Tris-HCl (pH 7.5) buffer. Then, 20% of the concentration of isopropanol was added as a solubilizing solvent. Next, 12% of the conversion substrate compound (16-α-hydroxyprednisolone acetate) was added to the system. Then, 20% of the volume of the prepared enzyme solution was added to the system, and the volume was adjusted to the final volume with water. The pH was then adjusted to 7.5 with liquid alkali / hydrochloric acid, and the conversion began. The conversion temperature was selected at 30℃, and the pH was adjusted with 2M NaOH solution. The pH was controlled at 7.5±0.2 during the conversion process, and the conversion time was 24h. After the pH did not change, the filter residue was collected and sampled for liquid chromatography analysis. The purity of the product was 99.3%. The product was dissolved in a dichloromethane and methanol mixed solvent (dichloromethane and methanol, volume ratio 1:1) and then filtered. The filtrate was first concentrated under reduced pressure to remove dichloromethane, and then concentrated to remove methanol. After reducing the volume to a small amount, water was added to replace the dichloromethane, and the remaining methanol was further concentrated. The product crystals were collected by vacuum filtration and identified by NMR as 16-α-hydroxyprednisolone, with a molar yield of 98.4%.

[0050] The reaction formula is as follows: .

[0051] 16α-Hydroxyprednisolone acetate (homemade): 1 H NMR (400 MHz, DMSO- d 6) δ 7.30 (d, J =10.1 Hz, 1H), 6.15 (dd, J = 10.1, 1.9 Hz, 1H), 5.91 (t, J = 1.5 Hz, 1H), 5.01(d, J = 17.7 Hz, 1H), 4.76 (dd, J = 11.1, 6.6 Hz, 3H), 4.72 – 4.66 (m, 1H), 4.26 (t, J = 3.4 Hz, 1H), 2.33 – 2.23 (m, 1H), 2.09 (s, 3H), 2.00 – 1.86 (m,3H), 1.84 – 1.76 (m, 2H), 1.63 (dd, J = 13.5, 2.5 Hz, 1H), 1.37 (s, 4H), 0.99 (td, J = 13.0, 4.6 Hz, 1H), 0.90 (dd, J = 11.0, 3.5 Hz, 1H), 0.84 (s, 3H). 13 CNMR (101 MHz, DMSO- d6) δ 206.1, 185.6, 170.9, 170.2, 157.1, 127.5, 122.1,88.5, 72.1, 68.7, 68.6, 55.7, 50.3, 47.9, 44.2, 39.5, 34.5, 34.3, 31.7, 31.1, 21.3, 20.9, 17.1.

[0052] 16α-Hydroxyprednisolone: 1 H NMR (400 MHz, DMSO- d 6) δ 7.30 (d, J = 10.1 Hz, 1H), 6.15 (dd, J = 10.1, 1.9 Hz, 1H), 5.90 (t, J = 1.6 Hz, 1H), 5.36 (d, J =5.2 Hz, 1H), 4.81 – 4.72 (m, 1H), 4.67 (d, J = 3.5 Hz, 2H), 4.53 (s, 1H), 4.48 (d, J = 19.3 Hz, 1H), 4.25 (t, J = 3.3 Hz, 1H), 4.07 (d, J = 19.4 Hz,1H), 2.57 – 2.44 (m, 3H), 2.34 – 2.23 (m, 1H), 2.04 – 1.91 (m, 2H), 1.86 (dd,J = 13.5, 3.7 Hz, 1H), 1.82 – 1.70 (m, 2H), 1.49 (dd, J = 13.6, 2.5 Hz, 1H), 1.37 (s, 4H), 0.97 (dd, J = 13.2, 4.7 Hz, 1H), 0.89 (dd, J = 11.1, 3.6 Hz, 1H), 0.84 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ 212.3, 185.6, 170.8, 157.1,127.6, 122.1, 88.2, 71.8, 68.8, 67.1, 55.6, 50.2, 47.5, 44.2, 39.7, 34.6,34.3, 31.8, 31.1, 21.3, 17.6.

[0053] Example 7 The enzyme solution prepared in Example 2 was used for conversion. The conversion tank was filled with 600 ml of conversion system in a 1L conversion tank. First, 50% of the conversion volume of water was added to the system, followed by 10% of the volume of buffer solution. The buffer solution was 0.5M Tris-HCl (pH 7.5) buffer. Then, 10% of the concentration of ethanol was added as a solubilizing solvent. Next, the conversion substrate compound (methylprednisolone acetate) was added to a final concentration of 12%. Then, 20% of the volume of the prepared enzyme solution was added to the system, and the volume was adjusted to the final volume with water. The pH was then adjusted to 7.5 with (liquid alkali / hydrochloric acid) and the conversion began. The conversion temperature was selected at 30℃, and the pH was adjusted with 2M NaOH solution. The pH was controlled at 7.5±0.2 during the conversion process, and the conversion time was 48h. After the pH did not change, the filter residue was collected and sampled for liquid chromatography analysis. The purity of the product was 85.9%. The product was dissolved in a dichloromethane and methanol mixed solvent (dichloromethane and methanol, volume ratio 1:1) and then filtered. The filtrate was first concentrated under reduced pressure to remove dichloromethane, and then concentrated to remove methanol. After reducing the volume to a small amount, water was added to replace the dichloromethane, and the remaining methanol was concentrated further. The NMR analysis showed that the product was methylprednisolone, with a molar yield of 92.1%.

[0054] The reaction formula is as follows: .

[0055] Methylprednisolone acetate (homemade): 1 H NMR (400 MHz, DMSO- d6) δ 7.33 (d, J = 10.1 Hz, 1H, H-1), 6.18 (dd, J = 10.1, 1.8 Hz, 1H, H-2), 5.82 (t, J = 1.7 Hz, 1H, H-4), 5.40 (s, 1H, OH-17), 5.06 (d, J = 17.6 Hz, 1H, H-21), 4.74 (d, J = 11.1 Hz, 1H, H-21), 4.71 (d, J = 2.6 Hz, 1H, OH-11), 4.28 (p, J = 3.3 Hz, 1H, H-11), 3.17 (d, J = 5.2 Hz, 3H), 2.71 – 2.58 (m, 1H), 2.16 – 2.09 (m, 1H), 2.09 (s, 3H), 2.03 (dt, J = 12.5, 4.3 Hz, 1H), 1.92 – 1.83 (m, 1H), 1.62 (ddd, J = 15.9, 10.2, 2.8 Hz, 3H), 1.50 – 1.41 (m, 1H), 1.38 (s, 3H), 1.30 (dd, J = 11.3, 6.1 Hz, 1H), 1.04 (d, J = 6.3 Hz, 3H, CH3-6), 0.85 (dd, J = 11.2, 3.3 Hz, 1H), 0.78 (s, 3H), 0.70 (q, J = 12.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d 6) δ 205.3 (C-20), 185.1 (C-3), 173.5 (C-5), 169.8 (OCCH3), 157.3 (C-1), 126.7 (C-2), 118.8 (C-4), 88.6, 68.3, 67.5, 55.9, 51.0, 48.6, 47.0, 44.0, 42.9, 33.1, 32.4, 30.8, 23.5, 21.3, 20.4, 17.6, 16.5。

[0056] Methylprednisolone: 1 H NMR (400 MHz, DMSO- d6) δ 7.32 (d, J = 10.1 Hz, 1H, H-1), 6.17 (dd, J = 10.1, 1.8 Hz, 1H, H-2), 5.82 (t, J = 1.7 Hz, 1H, H-4), 5.76 (s, 1H, OH-21), 5.49 (s, 1H, OH-17), 4.74 (d, J = 17.0 Hz, 1H, H-21), 4.65 (d, J = 3.2 Hz, 1H, OH-11), 4.38 (d, J = 17.0 Hz, 1H, H-21), 4.33 – 4.22 (m, 1H, H-11), 2.65 (dq, J = 11.4, 5.2 Hz, 1H), 2.56 (ddd, J = 14.6, 11.1, 2.2 Hz, 1H), 2.12 (td, J = 11.0, 4.1 Hz, 1H), 2.04 (dq, J = 12.5, 4.6, 4.2 Hz, 1H), 1.88 (dd, J = 13.6, 3.6 Hz, 1H), 1.70 – 1.52 (m, 3H), 1.51 – 1.40 (m, 1H), 1.38 (s, 3H), 1.31 (dd, J = 11.2, 6.1 Hz, 1H), 1.04 (d, J = 6.3 Hz, 3H, CH3-6), 0.85 (dd, J = 11.2, 3.4 Hz, 1H), 0.78 (s, 3H), 0.71 (q, J = 12.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d 6) δ 203.2 (C-20), 185.1 (C-3), 173.4 (C-5), 157.2 (C-1), 126.7 (C-2), 118.8 (C-4), 89.2, 68.3, 55.8, 54.9, 50.8, 48.8, 46.7, 44.0, 42.8, 33.0, 32.4, 30.8, 23.4, 21.3, 17.6, 17.0。

[0057] Example 8 The enzyme solution prepared in Example 2 was used for conversion. The conversion tank was filled with 600 ml of conversion system in a 1L conversion tank. First, 50% of the conversion volume of water was added to the system, followed by 10% of the volume of buffer solution. The buffer solution was 0.5M Tris-HCl (pH 7.5) buffer. Then, 10% of the concentration of isopropanol was added as a solubilizing solvent. Next, 12% of the conversion substrate compound (dexamethasone epoxy ester) was added to the system. Then, 20% of the volume of the prepared enzyme solution was added to the system, and the volume was adjusted to the final volume with water. The pH was then adjusted to 7.5 with (liquid alkali / hydrochloric acid) and the conversion began. The conversion temperature was selected at 45℃, and the pH was adjusted with 2M NaOH solution. The pH was controlled at 7.5±0.2 during the conversion process, and the conversion time was 16h. After the pH did not change, the filter residue was collected and sampled for liquid chromatography analysis. The purity of the product was 99.4%. The product was dissolved in a dichloromethane and methanol mixed solvent (dichloromethane and methanol, volume ratio 1:1) and then filtered. The filtrate was first concentrated under reduced pressure to remove dichloromethane, and then concentrated to remove methanol. After reducing the volume to a small amount, water was added to replace the dichloromethane, and the remaining methanol was further concentrated. The product crystals were collected by vacuum filtration and identified by NMR as dexamethasone epoxide hydrolysate with a molar yield of 98.4%.

[0058] The reaction formula is as follows: .

[0059] Dexamethasone epoxy ester (homemade): 1H NMR (500 MHz, CDCl3) δ 6.59 (d, J = 10.1Hz, 1H), 6.17 (dd, J = 10.1, 1.8 Hz, 1H), 6.12 (t, J = 1.5 Hz, 1H), 4.84 (dd,J = 180.6, 17.2 Hz, 2H), 3.22 – 3.16 (m, 1H), 3.09 – 2.98 (m, 1H), 2.65 (dddd, J = 15.7, 10.4, 5.4, 1.6 Hz, 1H), 2.47 (dt, J = 15.5, 5.8 Hz, 1H), 2.38 – 2.28 (m, 2H), 2.22 (dt, J = 11.4, 7.6 Hz, 1H), 2.14 (s, 3H), 1.92 –1.77 (m, 3H), 1.60 (dt, J = 12.8, 11.3 Hz, 1H), 1.46-1.39 (m, 1H), 1.42 (s,3H), 1.31 (ddd, J = 13.0, 8.5, 4.9 Hz, 1H), 0.91 (s, 3H), 0.87 (d, J = 7.2Hz, 3H). 13 C NMR (126 MHz, CDCl3) δ 204.7, 186.2, 170.7, 165.4, 152.5, 127.9,125.1, 90.7, 67.7, 66.1, 62.8, 48.1, 47.7, 44.3, 35.5, 34.3, 33.3, 30.5,29.8, 29.2, 23.8, 20.6, 17.4, 14.6.

[0060] Dexamethasone epoxide hydrolysate: 1H NMR (400 MHz, CDCl3) δ 6.59 (d, J = 10.1 Hz, 1H), 6.18 (dd, J = 10.1, 1.8 Hz, 1H), 6.14 (q, J = 1.4 Hz, 1H), 4.41 (dd, J =156.8, 19.9 Hz, 2H), 3.21 (t, J = 2.1 Hz, 1H), 3.15 – 2.98 (m, 2H), 2.66 (dddd, J = 15.7, 10.5, 5.4, 1.7 Hz, 1H), 2.48 (dt, J = 15.4, 5.6 Hz, 1H), 2.40 – 2.17 (m, 3H), 1.82 (td, J = 11.3, 8.4 Hz, 1H), 1.74 – 1.59 (m, 3H), 1.43 (s, 4H), 1.35 (ddd, J = 13.0, 8.4, 4.8 Hz, 1H), 0.92 (s, 3H), 0.89 (d, J= 7.2 Hz, 3H), 0.83 (dt, J = 5.3, 2.5 Hz, 1H). 13 C NMR (101 MHz, CDCl3) δ211.8, 186.1, 165.1, 152.3, 128.1, 125.2, 89.9, 67.7, 66.3, 62.8, 48.3, 48.2,44.2, 35.8, 34.4, 33.4, 30.6, 30.0, 29.3, 23.8, 17.8, 14.7.

[0061] Analysis of the conversion results from the examples shows that the unmutated original enzyme has poor conversion efficiency for the substrate hydrocortisone acetate. Single-point mutations in A160D and V254S improve conversion efficiency, with V254S showing a significant improvement. The enzyme with dual-point mutations exhibits the best conversion efficiency for hydrocortisone acetate, reaching a conversion rate of 99.4% under suitable conditions. Small-volume conversions are less effective than those in 1L tanks, possibly due to differences in equipment and pH control. Increasing the substrate concentration leads to higher substrate residue at the conversion endpoint, as does decreasing the enzyme dosage. The optimal conversion conditions are a substrate concentration of 12% and an enzyme dosage of 20%. Increasing the solvent volume and conversion temperature slightly improves product purity but has minimal impact on conversion. Considering production costs, a 10% isopropanol concentration and conversion at 30°C are preferred. After 24 hours of conversion, product purity can reach over 99%, meeting production requirements.

[0062] This invention enables an enzyme that originally had a poor conversion effect on hydrocortisone acetate to have a better conversion effect through amino acid mutation, thus giving it production application value.

[0063] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0064] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A specific, highly active hydrolytic enzyme, characterized in that, It is obtained by mutating the original hydrolase, the sequence of which is SEQ ID NO.

1. The mutation is as follows: the 160th site is mutated from A to D, and the 254th site is mutated from V to S, or the 160th site is mutated from A to D and the 254th site is mutated from V to S.

2. The specific high-activity hydrolase as described in claim 1, characterized in that, The mutations are as follows: site 160 changes from A to D and site 254 changes from V to S.

3. A gene encoding a specific, highly active hydrolase as described in claim 1 or 2.

4. An engineered bacterium, characterized in that, The engineered bacteria express the specific high-activity hydrolytic enzyme as described in claim 1 or 2, or contain the gene as described in claim 3.

5. The engineered bacteria as described in claim 4, characterized in that, The host microorganism of the engineered bacteria is Escherichia coli.

6. Use of a specific, highly active hydrolytic enzyme as described in claim 1 or 2, or an engineered bacterium as described in claim 4 or 5, in the hydrolysis of steroid hormones.

7. The use as described in claim 6, characterized in that, The substrate for hydrolysis is a steroidal 21-hydroxyacetate compound. The hydrolysis system includes a buffer solution and a co-solvent. The pH of the system is controlled at 7–9, and the hydrolysis temperature is 20–45°C. The reaction route is as follows: ； The positions indicated by the dashed lines represent single or double bonds, and R1 is H, CH3, Cl, or F; R2 is H, F, Cl, Br, OH or has no group, R3 is carbonyl, OH or H, or R2 and R3 are epoxy groups; R4 is H, α-OH, α-CH3 or β-CH3.

8. The use as described in claim 7, characterized in that, The buffer solution is Tris-HCl buffer, and the co-solvent is methanol, ethanol, or acetone. The pH of the system is controlled at 7.3-7.7, and the hydrolysis temperature is 25-35℃.

9. The use as described in claim 8, characterized in that, The Tris-HCl buffer solution has a volume concentration of 5-30%, and the co-solvent has a volume concentration of 2-30%.

10. The use as described in any one of claims 7-9, characterized in that, The amount of substrate added is 5~20 wt%.

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