Carbonyl reductase mutant and application thereof in synthesis of 11beta-hydroxy steroid compound

Abstract

The invention discloses a carbonyl reductase mutant and application of the carbonyl reductase mutant in synthesis of 11 beta-hydroxy steroid compounds, carbonyl reductase from Homo sapiens is modified by a semi-rational design means, the carbonyl reductase mutant is provided, prednisone, cortisone and methylprednisone are used as substrates, and the carbonyl reductase mutant is synthesized by a one-step method. And the 11 beta-hydroxy steroid compounds such as prednisolone, hydrocortisone and methylprednisolone are efficiently and catalytically synthesized.

Description

(I) Technical field

[0001] The invention belongs to the field of genetic engineering and enzyme engineering, and relates to a carbonyl reductase mutant and an application thereof in synthesizing 11β-hydroxysteroid compounds. (II) Background technology

[0002] 11β-Hydroxysteroids are important synthetic precursors of adrenal cortical hormone drugs such as prednisolone, hydrocortisone and methylprednisolone. They can be used for diseases caused by adrenal insufficiency, rheumatoid arthritis, rheumatic fever, gout, bronchial asthma, etc. They can also be used for allergic dermatitis, seborrheic dermatitis, pruritus, etc. At the same time, they are also precursors of desonide, budesonide and other budesonide drugs, and have huge market potential.

[0003] At present, the synthesis of 11β-hydroxysteroid compounds is mainly based on chemical methods. For example, patent CN101397324A discloses a chemical synthesis route of prednisolone using 17α-hydroxyprogesterone as a substrate, which has the disadvantages of long process route, high cost, low total yield and more by-products. Patent CN111518151A discloses a chemical synthesis route of hydrocortisone using 17α-hydroxypregnant-4,9(11)-diene-3,20-dione-21-acetate as a substrate, which has the problems of complex reaction process, many impurities and heavy environmental burden. Patent CN106518944A discloses a chemical synthesis route of methylprednisolone using 4-androstenedione as a substrate, which has the disadvantages of expensive raw materials, difficult sewage treatment, complex process operation and high production cost. Therefore, the development of a green and efficient new method for the synthesis of 11β-hydroxysteroid compounds is of great significance for their large-scale application.

[0004] Carbonyl reductase is a type of enzyme catalyst that can catalyze bidirectional reversible redox reactions between alcohols and aldehydes / ketones. It has the advantages of strict stereo / regional selectivity and high catalytic activity, and has important applications in the synthesis of pharmaceutical intermediates. For example, Zhang et al. modified the carbonyl reductase from guinea pigs and co-expressed it with glucose dehydrogenase to achieve highly selective biosynthesis of hydrocortisone (Applied Microbiology and Biotechnology, 2014, 98, 8879-8886); Yu et al. coupled the carbonyl reductase from guinea pigs with hydrolases to achieve efficient biosynthesis of prednisolone (CN114875003A). The present invention aims to provide a highly regio- and stereo-selective carbonyl reductase, which lays the foundation for the efficient preparation of 11β-hydroxysteroid compounds. (III) Summary of the invention

[0005] The present invention aims to provide a carbonyl reductase mutant and its application in synthesizing 11β-hydroxysteroid compounds. Carbonyl reductase (11β-4HFR, amino acid sequence as shown in SEQ ID No.1, gene sequence as shown in SEQ ID No.2) derived from Homo sapiens is modified by semi-rational design means to provide a carbonyl reductase mutant. With prednisone, cortisone and methylprednisolone as substrates, the mutant can be efficiently catalyzed to synthesize 11β-hydroxysteroid compounds prednisolone, hydrocortisone and methylprednisolone, thereby solving the problems of low carbonyl reductase activity in the prior art.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides a carbonyl reductase mutant, wherein the mutant is obtained by performing single mutation or multiple mutation on the 104th, 212th or 265th position of the amino acid sequence shown in SEQ ID NO.1.

[0008] Furthermore, the carbonyl reductase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.1 is mutated into one of the following: (1) Asparagine at position 104 is mutated into alanine (N104A, amino acid sequence is SEQ ID NO.3, nucleotide sequence is SEQ ID NO.4). (2) Asparagine at position 104 is mutated into alanine and valine at position 212 is mutated into phenylalanine (N104A / V212F, amino acid sequence is SEQ ID NO.5, nucleotide sequence is SEQ ID NO.6). (3) Asparagine at position 104 is mutated into alanine, valine at position 212 is mutated into phenylalanine and tyrosine at position 265 is mutated into isoleucine (N104A / V212F / Y265I, amino acid sequence is SEQ ID NO.7, nucleotide sequence is SEQ ID NO.8).

[0009] Any deletion, insertion or substitution of one or more amino acids in the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7 which has 11β-4HFR activity still falls within the protection scope of the present invention.

[0010] The invention extracts a plasmid containing wild-type carbonyl reductase 11β-4HFR from Escherichia coli, mutates the 11β-4HFR gene using a gene site saturation mutagenesis method, connects it to an expression vector, and expresses it in Escherichia coli, and obtains mutants with improved activity through screening.

[0011] The present invention also relates to the gene encoding the carbonyl reductase mutant, the recombinant vector constructed by the encoding gene, and the genetically engineered bacteria transformed by the recombinant vector. The recombinant vector can be constructed by connecting the nucleotide sequence of the 11β-4HFR mutant to various vectors by conventional methods in the art. The basic vector used to construct the recombinant vector of the present invention is not limited, as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and / or eukaryotic cells. The basic vector can be various conventional vectors in the art, such as various plasmids, phages or viral vectors, etc., preferably pET-28a (+). There is no limitation on the host cell into which the DNA encoding the 11β-4HFR mutant of the present invention is introduced, as long as a recombinant expression system has been established for it, and the recombinant expression vector can stably self-replicate and the 11β-4HFR mutant gene of the present invention carried can be effectively expressed. For example, Escherichia coli, Bacillus subtilis, yeast, actinomycetes, Aspergillus, as well as animal cells and higher plant cells. The present invention preferably uses Escherichia coli, and more preferably Escherichia coli E.coli BL21 (DE3).

[0012] The preparation of the 11β-4HFR mutant of the present invention includes culturing the genetically engineered bacteria of the present invention to induce the 11β-4HFR mutant protein. The culture medium used for culturing the genetically engineered bacteria can be a culture medium in the art that can make the transformant grow and produce the 11β-4HFR of the present invention, preferably LB culture medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, solvent is distilled water, pH 7.2. There are no special restrictions on the culture method and culture conditions, as long as the transformant can grow and produce 11β-4HFR. The following method is preferred: the genetically engineered bacteria of the present invention are inoculated into an LB culture medium containing 50μg / ml kanamycin, and cultured at 37°C until the optical density OD600 reaches 0.5-0.7, and under the induction of a final concentration of 0.1-1.0mM isopropyl-β-D-thiogalactopyranoside (IPTG), the 11β-4HFR mutant protein of the present invention can be efficiently expressed.

[0013] The present invention also relates to the use of the carbonyl reductase mutant in catalyzing the synthesis of 11β-hydroxysteroid compounds from 11β-carbonyl steroid compounds. The method of the use is: using wet bacteria obtained by fermentation culture of engineered bacteria containing carbonyl reductase mutant encoding genes as catalysts, using 11β-carbonyl steroid compounds as substrates, using glucose as cosubstrate, using glucose dehydrogenase (GDH) as coenzyme, adding NAD +A conversion system is formed with a buffer solution of pH 6.0 to 8.5 (preferably pH 8.0, 0.1 M PB buffer) as a reaction medium, and the conversion reaction is carried out at a temperature of 30 to 40° C. (preferably 37° C.) and a rotation speed of 100 to 200 rpm (preferably 150 rpm). After the reaction is completed, the reaction solution is separated and purified to obtain 11β-hydroxysteroid compounds.

[0014] Furthermore, the 11β-carbonyl steroid compound has a carbonyl group at position 11 of the steroid nucleus, such as prednisone, cortisone, and methylprednisone; the 11β-hydroxysteroid compound has a hydroxyl group at position 11 of the steroid nucleus, such as prednisolone, hydrocortisone, and methylprednisolone.

[0015] Furthermore, in the transformation system, the substrate is added to a final concentration of 1 to 10 g / L (preferably 2 to 6 g / L), glucose is added to a final concentration of 5 to 50 g / L (preferably 20 g / L), glucose dehydrogenase is added to a final concentration of 5 to 50 g / L (preferably 20 g / L), the wet cell dosage is 50 to 150 g / L (preferably 100 g / L), NAD + Add to a final concentration of 1-5 mM (preferably 2 mM).

[0016] Furthermore, when the 11β-carbonyl steroid compound is prednisone, the final concentration is 5-7 g / L; when the 11β-carbonyl steroid compound is cortisone, the final concentration is 3-5 g / L; when the 11β-carbonyl steroid compound is methylprednisone, the final concentration is 2-4 g / L.

[0017] Further, the wet cells were prepared as follows: the engineered bacteria containing the carbonyl reductase mutant encoding gene were inoculated into a liquid LB medium containing 50 μg / mL kanamycin, cultured overnight at 37°C and 200 rpm, transferred to a fresh LB medium containing 50 μg / mL kanamycin at a volume concentration of 2%, and continued to be cultured until the OD 600 The concentration of IPTG was 0.6, and IPTG was added at a final concentration of 0.1 mM, and the cells were induced and cultured at 28°C for 12 hours. After the culture was completed, the cells were collected by centrifugation at 8000 rpm for 10 minutes at 4°C, and washed twice with 0.9% saline to obtain the wet cells.

[0018] The 11β-4HFR mutant provided by the invention can catalyze the synthesis of prednisolone and other 11β-carbonyl steroid compounds in the form of free enzyme, immobilized enzyme and recombinant free cells.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0020] The carbonyl reductase mutant provided by the present invention has better enzyme activity than the wild type. Compared with the wild type, the optimal mutant N104A / V212F / Y265I has an enzyme activity of 305% higher for substrate prednisone, 385% higher for substrate cortisone, and 241% higher for substrate methylprednisone.

[0021] The carbonyl reductase mutant provided by the present invention has better catalytic performance than the wild type. Compared with the wild type, the optimal mutant N104A / V212F / Y265I has a conversion rate of more than 90.2% for synthesizing prednisolone, an optical purity of more than 99%, and a yield of more than 87.7%; a conversion rate of more than 99.2% for synthesizing hydrochloric acid, an optical purity of more than 99%, and a yield of 96.6%; a conversion rate of more than 95.6% for synthesizing methylprednisolone, an optical purity of more than 99%, and a yield of more than 92.8%. (IV) Description of the drawings

[0022] Figure 1 , Liquid chromatogram of the synthesis of prednisolone catalyzed by the optimal mutant N104A / V212F / Y265I.

[0023] Figure 2 , Liquid chromatogram of the synthesis of hydrocortisone catalyzed by the optimal mutant N104A / V212F / Y265I.

[0024] Figure 3 , Liquid chromatogram of the synthesis of methylprednisolone catalyzed by the optimal mutant N104A / V212F / Y265I. (V) Specific implementation methods

[0025] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0026] The LB liquid culture medium of the present invention is composed of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and pH 7.0.

[0027] The composition of LB solid medium is: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 18 g / L, pH 7.0.

[0028] Example 1. Construction of a carbonyl reductase mutant library

[0029] 1. Screening of active sites

[0030] The carbonyl reductase from Homo sapiens (11β-4HFR, the amino acid sequence is shown in SEQ ID No. 1, and the gene sequence is shown in SEQ ID No. 2) was fully synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0031] The structure of 11β-4HFR protein (amino acid sequence as shown in SEQ ID NO.1) was modeled using AlphaFold2 (AlphaFold2 TIB Server (biodesign.ac.cn)) website, and the prednisone substrate was used as the center to analyze The points in the range and the sites of the substrate channel were predicted using HotSpot wizard, and the active sites 104, 212, and 265 were screened.

[0032] SEQ ID No.1

[0033] MGGGNEEFRPEMLQGK KV IVTGASKGIGREMAYH LA KMGAHVVVTARSKET LQ KVVSHCLELGAASAHYIAGT ME DMTFAEQFVAQ AG KLMGGLDMLILNHITNTSLNL F HDDI HH VRKSMEVNFLSYVVLTVAALP MLKQSNGSIVVVSSLA GK VA YPLVAAYSASKFALDGFFSSI RKEYSVSRVNVSIT LC VLGL ID TETAMKAVSGIVHMQAAPKEE CALE II KGGAL RQ EEVYYDSSRWTTLLIRNPSRK ILEEL YS TSYNWDRFI NK LEHHH HH H

[0034] 2. Saturation mutagenesis at site 104

[0035] Saturation mutation was performed on 104 sites in the predicted structural hot spots and functional hot spots. Using plasmid pET28a-11β-4HFR as a DNA template, saturation mutation was performed on asparagine at position 104, that is, NNK was used to replace the original codon for the nucleotide corresponding to position 104, degenerate primers were designed, and whole plasmid PCR amplification was performed.

[0036] Table 1. Primer design for 104-site site-directed saturation mutagenesis

[0037]

[0038] Note: N=A / G / C / T, K=G / T, M=A / C

[0039] PCR reaction system (50 μL): 2× phanta Max buffer 25 μL, dNTP mixture (10 mM) 1 μL, 2 μL of each 10 μM mutant primer as shown in Table 1, 1 μL of plasmid pET28-Bd-Nit, 0.5 μL of Phanta Max DNA polymerase, and ddH2O to 50 μL.

[0040] PCR conditions: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 15 s, annealing at 59°C for 5 s, extension at 72°C for 30 s, step (2) for 30 cycles; (3) final extension at 72°C for 3 min, and storage at 4°C.

[0041] After the reaction, the PCR product was analyzed by 0.9% agarose gel electrophoresis, and then the original template was digested with Dpn I, the gel was cut and recovered, the PCR product was purified, and heat-shocked into E. coli BL21 (DE3) competent cells, spread on LB plates containing 50 μg / mL kanamycin, and cultured at 30°C overnight to screen positive clones to obtain a saturated mutation library.

[0042] 3. Screening of mutant N104A

[0043] (1) Cultivation of positive clones

[0044] A single colony from the saturated mutation library in step 2 was picked and inoculated into LB liquid culture medium (containing 50 μg / mL of kanamycin), and cultured in an incubator at 37°C and 180 rpm for 12 h. The culture was transferred to a new LB liquid culture medium (containing 50 μg / mL of kanamycin) at a volume concentration of 1%. After further culture at 37°C and 180 rpm for 2 h, 0.1 mM isopropylthiogalactoside (IPTG) was added, and the culture was continued at 25°C and 180 rpm for 12 h. The wet cells were collected by centrifugation, i.e., carbonyl reductase wet cells, and the catalytic synthesis of prednisolone and other 11β-hydroxysteroid compounds was studied.

[0045] (2) Mutant Viability Detection

[0046] The final concentration of 1mL reaction system is: 6g / L prednisone substrate, 20g / L glucose, 2.0mM NAD + , 20g / L GDH and 100g / L carbonyl reductase wet cells, K2HPO4-KH2PO4 buffer (100mM, pH 8.0). Place at 37°C, 150rpm constant temperature shaker for 2h. After the reaction, extract with N,N-dimethylformamide and centrifuge, add 2 times the volume of N,N-dimethylformamide water, vacuum rotary evaporation at 60°C for 3h, then filter with acetonitrile, and analyze the enzyme activity with high performance liquid chromatography (HPLC).

[0047] Liquid phase detection conditions: Agilent 1260 InfinityⅡ, chromatographic column is Fully Porous Silica C18 chiral column (5μm×250mm×4.6mm), detection wavelength is 265nm, flow rate is 1mL / min, injection volume is 2μL, column temperature is 30℃.

[0048] The results showed that the mutant strain E. coli BL21 (DE3)-pET28a-N104A had the highest relative enzyme activity, which was 129% higher than the wild type. Under the same conditions, the wild type carbonyl reductase 11β-4HFR (nucleotide sequence as shown in SEQ ID NO.1) was used for the reaction, and the enzyme activity was measured by HPLC. The results are shown in Table 4.

[0049] Example 2: Iterative saturation mutation of carbonyl reductase

[0050] 1. Screening of mutant N104A / V212F

[0051] Based on the mutant strain N104A with improved enzyme activity obtained in Example 1, amino acid sites V212 and Y265 around the catalytic active center were further selected for modification. N104A plasmid was used as a mutation template, and V212 site saturation mutation primers (Table 2) were used as primers for whole plasmid PCR amplification.

[0052] Table 2. Primer design for site-directed saturation mutagenesis at 212 sites

[0053]

[0054] Note: N=A / G / C / T, K=G / T, M=A / C

[0055] PCR conditions: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 15 s, annealing at 60°C for 5 s, extension at 72°C for 3.5 min, step (2) for 35 cycles; (3) final extension at 72°C for 5 min, and storage at 4°C.

[0056] The amplified PCR product was digested with endonuclease Dpn I at 37°C for 3h, inactivated at 65°C for 10min, transformed into E. coli BL21 (DE3), plated on LB plates containing kanamycin (50 μg / mL), and cultured overnight at 37°C. The mutant N104A / V212F with the highest activity was screened using the method of Example 1, which was 229% higher than the wild type.

[0057] 2. Screening of mutant N104A / V212F / Y265I

[0058] The pET28a-N104A / V212F plasmid was used as a mutation template, and the Y265 site saturation mutation primer (Table 3) was used as a primer for PCR reaction. The primer sequences are shown in Table 3 below:

[0059] Table 3. Primers designed for site-directed saturation mutagenesis at site 265

[0060]

[0061] Note: N=A / G / C / T, K=G / T, M=A / C

[0062] PCR conditions: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 15 s, annealing at 60°C for 5 s, extension at 72°C for 3.5 min, step (2) for 35 cycles; (3) final extension at 72°C for 5 min, and storage at 4°C.

[0063] The amplified PCR product was digested with endonuclease Dpn I at 37°C for 3h, inactivated at 65°C for 10min, transformed into E. coli BL21 (DE3), spread on LB plates containing kanamycin (50 μg / mL), and cultured overnight at 37°C. Screening by the method of Example 1 obtained the mutant E. coli BL21 (DE3)-pET28a-N104A / V212F / Y265I with the highest activity, which was 305% higher than the wild type (Table 4).

[0064] Table 4 Comparison of carbonyl reductase activity

[0065]

[0066] Example 3: Inducible expression of recombinant carbonyl reductase mutants

[0067] 10 μL of E. coli BL21 (DE3) -pET28a-N104A, E. coli BL21 (DE3) -pET28a-N104A / 212F, and E. coli BL21 (DE3) -pET28a-N104A / 212F / Y265I prepared by the methods of Examples 1 and 2 were inoculated into 10 mL of liquid LB medium (containing 50 μg / mL of kanamycin), cultured overnight at 37°C, 200 rpm, and transferred to 100 mL of fresh LB medium (containing 50 μg / mL of kanamycin) at a volume concentration of 2%, and continued to culture until OD 600 The concentration of IPTG was 0.6, and IPTG was added at a final concentration of 0.1 mM. The cells were induced and cultured at 28°C for 12 hours. After the culture was completed, the cells were collected by centrifugation at 8000 rpm for 10 minutes at 4°C, and washed twice with 0.9% saline to obtain wet bacterial cells of recombinant carbonyl reductase genetically engineered bacteria. Under the same conditions, wild-type E. coli BL21 (DE3)-pET28a-11β-4HFR wet bacterial cells were prepared.

[0068] Example 4: Application of recombinant Escherichia coli containing carbonyl reductase in the preparation of prednisolone

[0069] The final concentration of 1mL reaction system is: 6g / L prednisone substrate, 20g / L glucose, 2.0mM NAD + , 20g / L GDH and 100g / L wet bacteria of carbonyl reductase prepared by the method of Example 3, K2HPO4-KH2PO4 buffer (100mM, pH8.0). Place at 37°C, 150rpm constant temperature shaker to react for 24h. After the reaction is completed, extract with N,N-dimethylformamide and centrifuge, add 2 times the volume of N,N-dimethylformamide water to the precipitate, vacuum evaporate at 60°C for 3h until no liquid flows out, then dissolve in acetonitrile and filter, the filtrate is analyzed by high performance liquid chromatography (HPLC) in Example 1 to determine its conversion rate and yield. The results are shown in Table 5, where the liquid chromatography of mutant N104A / V212F / Y265I is shown in Figure 1 .

[0070] Through analysis, the wild-type 11β-4HFR catalyzes the synthesis of prednisolone from prednisone with a conversion rate of 44.7%, an ee value of more than 99%, and a yield of 41.2%; the mutant N104A catalyzes the synthesis of prednisolone from prednisone with a conversion rate of 53.9%, an ee value of more than 99%, and a yield of 50.1%; the mutant N104A / V212F catalyzes the synthesis of prednisolone from prednisone with a conversion rate of 85.6%, an ee value of more than 99%, and a yield of 82.6%; the optimal mutant N104A / V212F / 265I catalyzes the synthesis of prednisolone from prednisone with a conversion rate of 90.5%, an ee value of more than 99%, and a yield of 87.7%.

[0071] Table 5 Conversion rate and yield of prednisone catalyzed by wild type and mutants

[0072]

[0073] Example 5: Application of recombinant Escherichia coli containing carbonyl reductase in the preparation of hydrocortisone

[0074] The final concentration of 1mL reaction system is: 4g / L cortisone substrate, 20g / L glucose, 2.0mM NAD + , 20g / L GDH and 100g / L wet bacteria of carbonyl reductase prepared by the method of Example 3, K2HPO4-KH2PO4 buffer (100mM, pH8.0). Place at 37°C, 150rpm constant temperature shaker to react for 24h. After the reaction is completed, extract with N,N-dimethylformamide and centrifuge, add 2 times the volume of N,N-dimethylformamide water, vacuum evaporate at 60°C for 3h until there is no liquid, then dissolve in acetonitrile and filter, the filtrate is analyzed by high performance liquid chromatography (HPLC) to determine its enzyme activity, conversion rate and yield. The results are shown in Table 6, where the liquid chromatography of mutant N104A / V212F / Y265I is shown in Figure 2 .

[0075] Liquid phase detection conditions: Agilent 1260 InfinityⅡ, the column is equipped with a Fully Porous Silica C18 chiral column (5μm×250mm×4.6mm), the detection wavelength is 265nm, the flow rate is 0.8mL / min, the injection volume is 2μL, and the column temperature is 30℃.

[0076] Through analysis, it was found that the wild-type 11β-4HFR catalyzed the synthesis of hydrocortisone from cortisone with a conversion rate of 54.4%, an ee value of more than 99%, and a yield of 51.8%; the mutant N104A catalyzed the synthesis of hydrocortisone from cortisone with a conversion rate of 65.5%, an ee value of more than 99%, and a yield of 62.3%; the mutant N104A / V212F catalyzed the synthesis of hydrocortisone from cortisone with a conversion rate of 85.4%, an ee value of more than 99%, and a yield of 82.7%; the optimal mutant N104A / V212F / 265I catalyzed the synthesis of hydrocortisone from cortisone with a conversion rate of 99.2%, an ee value of more than 99%, and a yield of 96.6%.

[0077] Table 6 Wild type and mutant catalytic cortisone conversion rate and yield

[0078]

[0079] Example 6: Application of recombinant Escherichia coli containing carbonyl reductase in the preparation of methylprednisone

[0080] The final concentration of 1mL reaction system is: 2.5g / L methylprednisolone substrate, 20g / L glucose, 2.0mM NAD + , 20 g / L GDH and 100 g / L carbonyl reductase wet cells prepared by the method of Example 3,

[0081] K2HPO4-KH2PO4 buffer (100mM, pH 8.0). The mixture was placed at 37°C and 150rpm constant temperature shaker for 24h. After the reaction, it was extracted with N,N-dimethylformamide and centrifuged. Water twice the volume of N,N-dimethylformamide was added and vacuum evaporated at 60°C for 3h until no liquid flowed out. The mixture was then dissolved in acetonitrile and filtered. The enzyme activity, conversion rate and yield were determined by high performance liquid chromatography (HPLC). The results are shown in Table 7. The liquid chromatography of mutant N104A / V212F / Y265I is shown in Table 7. Figure 3 .

[0082] Liquid phase detection conditions: Agilent 1260 InfinityⅡ, the column is equipped with a Fully Porous Silica C18 chiral column (5μm×250mm×4.6mm), the detection wavelength is 265nm, the flow rate is 0.8mL / min, the injection volume is 2μL, and the column temperature is 30℃.

[0083] Through detection and analysis, it was found that the wild-type 11β-4HFR catalyzed the synthesis of methylprednisone from methylprednisolone with a conversion rate of 60.5%, an ee value of more than 99%, and a yield of 57.2%; the mutant N104A catalyzed the synthesis of methylprednisone from methylprednisolone with a conversion rate of 67.7%, an ee value of more than 99%, and a yield of 62.9%; the mutant N104A / V212F catalyzed the synthesis of methylprednisone from methylprednisolone with a conversion rate of 83.9%, an ee value of more than 99%, and a yield of 79.0%; the optimal mutant N104A / V212F / 265I catalyzed the synthesis of methylprednisone from methylprednisolone with a conversion rate of 95.6%, an ee value of more than 99%, and a yield of 92.8%.

[0084] Table 7 Conversion rate and yield of methylprednisone catalyzed by wild type and mutants

[0085]

[0086] The present invention is not limited by the above textual description. The present invention can make various changes within the scope of the claims, and these changes are all within the scope of the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that: The mutant is obtained by performing single mutation or multiple mutation on the 104th, 212th or 265th position of the amino acid sequence shown in SEQ ID NO.

1.

2. The carbonyl reductase mutant according to claim 1, characterized in that The carbonyl reductase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.1 is mutated into one of the following: (1) asparagine at position 104 is mutated into alanine; (2) asparagine at position 104 is mutated into alanine and valine at position 212 is mutated into phenylalanine; (3) asparagine at position 104 is mutated into alanine, valine at position 212 is mutated into phenylalanine and tyrosine at position 265 is mutated into isoleucine.

3. A genetically engineered bacterium containing a gene encoding the carbonyl reductase mutant.

4. Use of the carbonyl reductase mutant according to claim 1 in catalyzing the synthesis of 11β-hydroxysteroid compounds from 11β-carbonyl steroid compounds.

5. The use according to claim 4, characterized in that The 11β-carbonyl steroid compounds include prednisone, cortisone, and methylprednisone.

6. The use according to claim 5, characterized in that The application method comprises: using wet bacteria obtained by fermentation culture of engineered bacteria containing a carbonyl reductase mutant encoding gene as a catalyst, using 11β-carbonyl steroid compounds as a substrate, using glucose as a cosubstrate, using glucose dehydrogenase as a coenzyme, adding NAD + A conversion system is formed with a buffer solution of pH 6.0 to 8.5 as a reaction medium, and the conversion reaction is carried out at a temperature of 30 to 40° C. and a rotation speed of 100 to 200 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain 11β-hydroxysteroid compounds.

7. The use according to claim 6, characterized in that In the transformation system, the substrate is added to a final concentration of 1 to 10 g / L, glucose is added to a final concentration of 5 to 50 g / L, glucose dehydrogenase is added to a final concentration of 5 to 50 g / L, the wet cell dosage is 50 to 150 g / L, NAD + Add to a final concentration of 1-5 mM.

8. The use according to claim 6, characterized in that When the 11β-carbonyl steroid compound is prednisone, the final concentration is 5-7 g / L; when the 11β-carbonyl steroid compound is cortisone, the final concentration is 3-5 g / L; when the 11β-carbonyl steroid compound is methylprednisone, the final concentration is 2-4 g / L.

9. The use according to claim 6, characterized in that The wet cells were prepared as follows: the engineered bacteria containing the carbonyl reductase mutant encoding gene were inoculated into a liquid LB medium containing 50 μg / mL kanamycin, cultured overnight at 37°C and 200 rpm, transferred to a fresh LB medium containing 50 μg / mL kanamycin at a volume concentration of 2%, and continued to be cultured until the OD 600 The concentration of IPTG was 0.6, and IPTG was added at a final concentration of 0.1 mM, and the cells were induced and cultured at 28°C for 12 hours. After the culture was completed, the cells were collected by centrifugation at 8000 rpm for 10 minutes at 4°C, and washed twice with 0.9% saline to obtain the wet cells.

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