A carbonyl reductase mutant and its application in synthesizing 11β-hydroxysteroid compounds

Abstract

The present invention discloses a carbonyl reductase mutant and its application in synthesizing 11β-hydroxysteroid compounds. Carbonyl reductase derived from Homo sapiens is modified by semi-rational design methods to provide a carbonyl reductase mutant. The mutant can efficiently catalyze the synthesis of 11β-hydroxysteroid compounds prednisolone, hydrocortisone and methylprednisolone using prednisone, cortisone and methylprednisolone as substrates.

Description

(1) Technical field

[0001] The invention belongs to the fields of genetic engineering and enzyme engineering, and relates to a carbonyl reductase mutant and an application thereof in synthesizing 11β-hydroxysteroid compounds. (2) Background technology

[0002] 11β-hydroxysteroids are important synthetic precursors for adrenal corticosteroids such as prednisolone, hydrocortisone, and methylprednisolone. They are used to treat diseases caused by adrenal insufficiency, rheumatoid arthritis, rheumatic fever, gout, bronchial asthma, and other conditions. They are also used for atopic dermatitis, seborrheic dermatitis, and pruritus. They are also precursors for budesonide-like drugs such as desonide and budesonide, and have enormous 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 disadvantages such as a long process route, high cost, low total yield and a large number of by-products. Patent CN111518151A discloses a chemical synthesis route of hydrocortisone using 17α-hydroxypregnane-4,9(11)-diene-3,20-dione-21-acetate as a substrate, which has problems such as a complex reaction process, many impurities and a heavy environmental burden. Patent CN106518944A discloses a chemical synthesis route of methylprednisolone using 4-androstenedione as a substrate, which has disadvantages such as 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 reductases are a class of enzyme catalysts that catalyze bidirectional, reversible redox reactions between alcohols and aldehydes / ketones. They possess advantages such as strict stereoselectivity and regioselectivity and high catalytic activity, making them important applications in the synthesis of pharmaceutical intermediates. For example, Zhang et al. modified a guinea pig-derived carbonyl reductase and co-expressed it with glucose dehydrogenase, achieving highly selective biosynthesis of hydrocortisone (Applied Microbiology and Biotechnology, 2014, 98, 8879-8886). Yu et al. coupled a guinea pig-derived carbonyl reductase with a hydrolase to achieve efficient biosynthesis of prednisolone (CN114875003A). The present invention aims to provide a highly regio- and stereoselective carbonyl reductase, laying the foundation for the efficient preparation of 11β-hydroxysteroid compounds. (3) Summary of the invention

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

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

[0007] The present invention provides a carbonyl reductase mutant, which is obtained by performing single mutation or multiple mutations 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, or SEQ ID NO. 7 that has 11β-4HFR activity still falls within the scope of protection 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 the plasmid to an expression vector, and expresses the plasmid in Escherichia coli. Mutants with enhanced activity are obtained through screening.

[0011] The present invention also relates to a gene encoding the carbonyl reductase mutant, a recombinant vector constructed from the gene encoding the gene, and genetically engineered bacteria transformed with the recombinant vector. The recombinant vector can be constructed by connecting the nucleotide sequence of the 11β-4HFR mutant to various vectors using conventional methods in the art. The base vector used to construct the recombinant vector is not limited, as long as it can maintain replication or autonomous replication in various prokaryotic and / or eukaryotic host cells. The base vector can be any conventional vector in the art, such as various plasmids, phage, or viral vectors, preferably pET-28a(+). The host cell into which the DNA encoding the 11β-4HFR mutant of the present invention is introduced is not limited, as long as a recombinant expression system has been established for it, the recombinant expression vector can stably replicate itself, and the 11β-4HFR mutant gene carried by the present invention can be effectively expressed. Examples include Escherichia coli, Bacillus subtilis, yeast, actinomycetes, Aspergillus, as well as animal cells and higher plant cells. Escherichia coli is preferred, and E. coli BL21(DE3) is more preferred.

[0012] The preparation of the 11β-4HFR mutant of the present invention comprises culturing the genetically engineered bacteria of the present invention to induce the production of the 11β-4HFR mutant protein. The culture medium used to culture the genetically engineered bacteria can be any culture medium known in the art that can grow transformants and produce the 11β-4HFR of the present invention, preferably LB culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, distilled water as the solvent, pH 7.2. The culture method and conditions are not particularly limited, as long as the transformants can grow and produce 11β-4HFR. The following method is preferred: the genetically engineered bacteria of the present invention are inoculated into LB culture medium containing 50 μg / ml kanamycin, cultured at 37°C until the optical density (OD600) reaches 0.5-0.7, and then induced with a final concentration of 0.1-1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to efficiently express the 11β-4HFR mutant protein of the present invention.

[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 application is as follows: using wet bacteria obtained by fermentation culture of an engineered bacterium 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 (GDH) as a coenzyme, adding NAD +A conversion system is formed using a buffer solution of pH 6.0 to 8.5 (preferably pH 8.0, 0.1 M PB buffer) as a reaction medium. 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 an 11β-hydroxysteroid compound.

[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-hydroxy steroid 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] Furthermore, 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 at 37°C, 200 rpm overnight, 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. The cells were induced and cultured at 28°C for 12 h. After the culture was completed, the cells were collected by centrifugation at 8000 rpm at 4°C for 10 min, and washed twice with 0.9% saline to obtain the wet cells.

[0018] The 11β-4HFR mutant provided by the present 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 mutants provided by the present invention exhibit superior enzymatic activity compared to the wild-type. The optimal mutant, N104A / V212F / Y265I, showed a 305% increase in enzymatic activity on the substrate prednisone, a 385% increase in enzymatic activity on the substrate cortisone, and a 241% increase in enzymatic activity on the substrate methylprednisone compared to the wild-type.

[0021] The carbonyl reductase mutants provided by the present invention exhibit superior catalytic performance compared to the wild-type. Compared to the wild-type, the optimal mutant, N104A / V212F / Y265I, achieved a conversion rate of over 90.2% for prednisolone synthesis, an optical purity greater than 99%, and a yield of over 87.7%. The conversion rate for hydrochloride synthesis reached over 99.2%, an optical purity greater than 99%, and a yield of 96.6%. The conversion rate for methylprednisolone synthesis reached over 95.6%, an optical purity greater than 99%, and a yield of over 92.8%. (IV) Description of the accompanying 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 optimal mutant N104A / V212F / Y265I catalyzing the synthesis of hydrocortisone.

[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 with reference to 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, amino acid sequence as shown in SEQ ID No. 1, gene sequence as 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 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 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 mutagenesis was performed on 104 sites in the predicted structural and functional hotspots. Using the plasmid pET28a-11β-4HFR as a DNA template, saturation mutagenesis was performed on the asparagine at position 104. This involved replacing the original codon at position 104 with NNK. Degenerate primers were designed and PCR amplification was performed on the entire plasmid.

[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, plasmid pET28-Bd-Nit 1 μL, Phanta Max DNA polymerase 0.5 μL, 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 a total of 30 cycles; (3) final extension at 72°C for 3 min, and storage at 4°C.

[0041] After the reaction, the PCR products were analyzed by 0.9% agarose gel electrophoresis. The original template was digested with Dpn I, the gel was harvested, and the PCR products were purified and introduced into competent E. coli BL21 (DE3) cells by heat shock. The cells were spread on LB plates containing 50 μg / mL kanamycin and cultured overnight at 30°C. Positive clones were screened to obtain a saturated mutant 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 medium (containing 50 μg / mL of kanamycin), cultured in an incubator at 37°C and 180 rpm for 12 h, and then transferred to a new LB liquid 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., the carbonyl reductase wet cells, and the catalytic synthesis of prednisolone and other 11β-hydroxysteroid compounds was studied.

[0045] (2) Mutant activity detection

[0046] The final concentration of 1mL reaction system is: 6g / L prednisone substrate, 20g / L glucose, 2.0mM NAD + , 20 g / L LDH and 100 g / L carbonyl reductase wet cells, K2HPO4-KH2PO4 buffer (100 mM, pH 8.0). The mixture was incubated at 37°C, 150 rpm, and incubated for 2 h. After completion of the reaction, the mixture was extracted with N,N-dimethylformamide and centrifuged. Water (2 times the volume of N,N-dimethylformamide) was added, and the mixture was vacuum evaporated at 60°C for 3 h. The mixture was then filtered with acetonitrile and analyzed for enzyme activity by high-performance liquid chromatography (HPLC).

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

[0048] The results showed that the mutant strain E. coli BL21(DE3)-pET28a-N104A had the highest relative enzyme activity, increasing by 129% compared to the wild-type strain. Under the same conditions, the reaction was carried out using the engineered strain E. coli BL21(DE3)-11β-4HFR expressing the wild-type carbonyl reductase 11β-4HFR (nucleotide sequence shown in SEQ ID NO. 1), and enzyme activity was measured by HPLC. The results are shown in Table 4.

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

[0050] 1. Screening of mutant N104A / V212F

[0051] Based on the mutant strain N104A with enhanced enzyme activity obtained in Example 1, amino acid sites V212 and Y265 around the catalytic active center were further modified. Whole-plasmid PCR amplification was performed using the N104A plasmid as a mutation template and the V212 site saturation mutagenesis primers (Table 2) as primers.

[0052] Table 2. Design of primers 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, retardation 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, storage at 4°C.

[0056] The amplified PCR product was digested with endonuclease Dpn I at 37°C for 3 hours, inactivated at 65°C for 10 minutes, and transformed into E. coli BL21(DE3). The product was plated on LB plates containing kanamycin (50 μg / mL) and incubated overnight at 37°C. The mutant N104A / V212F with the highest activity was screened using the method of Example 1, showing a 229% increase compared to 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, storage at 4°C.

[0063] The amplified PCR product was digested with endonuclease Dpn I at 37°C for 3 hours, inactivated at 65°C for 10 minutes, and transformed into E. coli BL21(DE3). The product was plated on LB plates containing kanamycin (50 μg / mL) and incubated overnight at 37°C. Screening using the method of Example 1 yielded the mutant with the highest activity, E. coli BL21(DE3)-pET28a-N104A / V212F / Y265I, 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 bacterial suspensions prepared in glycerol tubes according to the methods of Examples 1 and 2 were inoculated into 10 mL of liquid LB medium (containing 50 μg / mL of kanamycin) and cultured overnight at 37°C and 200 rpm. The inoculum was transferred to 100 mL of fresh LB medium (containing 50 μg / mL of kanamycin) at a volume concentration of 2%, and the culture was continued until the OD 600 The pH was set to 0.6, IPTG was added to a final concentration of 0.1 mM, and the cells were induced at 28°C for 12 hours. After incubation, the cells were harvested by centrifugation at 8000 rpm for 10 minutes at 4°C and washed twice with 0.9% saline to obtain wet cells of the recombinant carbonyl reductase-engineered bacteria. Wild-type E. coli BL21(DE3)-pET28a-11β-4HFR wet cells were prepared under the same conditions.

[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 / LGDH 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 Table 5. Figure 1 .

[0070] Analysis showed that the wild-type 11β-4HFR catalyzed the conversion of prednisone to prednisolone with a conversion rate of 44.7%, an ee value of over 99%, and a yield of 41.2%; the mutant N104A catalyzed the conversion of prednisone to prednisolone with a conversion rate of 53.9%, an ee value of over 99%, and a yield of 50.1%; the mutant N104A / V212F catalyzed the conversion of prednisone to prednisolone with a conversion rate of 85.6%, an ee value of over 99%, and a yield of 82.6%; and the optimal mutant N104A / V212F / 265I catalyzed the conversion of prednisone to prednisolone with a conversion rate of 90.5%, an ee value of over 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 the reaction system in 1 mL is: 4 g / L cortisone substrate, 20 g / L glucose, 2.0 mM NAD + , 20g / LGDH 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, extract with N,N-dimethylformamide and centrifuge, add 2 times the volume of N,N-dimethylformamide water, 60°C vacuum rotary evaporation 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, among which the liquid chromatography of mutant N104A / V212F / Y265I is shown in Table 6. Figure 2 .

[0075] Liquid chromatography detection conditions: Agilent 1260 Infinity II, equipped with a Fully Porous Silica C18 chiral column (5 μm × 250 mm × 4.6 mm), detection wavelength 265 nm, flow rate 0.8 mL / min, injection volume 2 μL, column temperature 30°C.

[0076] The results showed that the wild-type 11β-4HFR catalyzed the conversion of cortisone to hydrocortisone with a conversion rate of 54.4%, an ee value of over 99%, and a yield of 51.8%; the mutant N104A catalyzed the conversion of cortisone to hydrocortisone with a conversion rate of 65.5%, an ee value of over 99%, and a yield of 62.3%; the mutant N104A / V212F catalyzed the conversion of cortisone to hydrocortisone with a conversion rate of 85.4%, an ee value of over 99%, and a yield of 82.7%; and the optimal mutant N104A / V212F / 265I catalyzed the conversion of cortisone to hydrocortisone with a conversion rate of 99.2%, an ee value of over 99%, and a yield of 96.6%.

[0077] Table 6. Conversion rate and yield of cortisone catalyzed by wild type and mutants

[0078]

[0079] Example 6: Use of recombinant Escherichia coli containing carbonyl reductase to prepare 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). Place at 37 ° C, 150rpm constant temperature shaker conditions for 24h. After the reaction is completed, extract with N, N-dimethylformamide and centrifuge, add 2 times the volume of N, N-dimethylformamide water, 60 ° C vacuum rotary evaporation for 3h until no liquid flows out, then dissolve in acetonitrile and filter, high performance liquid chromatography (HPLC) analysis to determine its enzyme activity, conversion rate and yield, the results are shown in Table 7, wherein the liquid chromatography of mutant N104A / V212F / Y265I is shown in Table 7. Figure 3 .

[0082] Liquid chromatography detection conditions: Agilent 1260 Infinity II, equipped with a Fully Porous Silica C18 chiral column (5 μm × 250 mm × 4.6 mm), detection wavelength 265 nm, flow rate 0.8 mL / min, injection volume 2 μL, column temperature 30°C.

[0083] Through detection and analysis, it was found that the wild-type 11β-4HFR catalyzed the conversion of methylprednisone to 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 conversion of methylprednisone to 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 conversion of methylprednisone to 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 conversion of methylprednisone to 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 methylprednisolone catalyzed by wild type and mutants

[0085]

[0086] The present invention is not limited to the above description. The present invention can be modified in various ways within the scope of the claims, and these modifications are all within the scope of the present invention.

Claims

1. A carbonyl reductase mutant, 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.

2. A genetically engineered bacterium containing a gene encoding the carbonyl reductase mutant according to claim 1.

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

4. The use according to claim 3, characterized in that The 11β-carbonyl steroid compounds include prednisone, cortisone, and methylprednisone.

5. The use according to claim 4, characterized in that The application method is as follows: 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 substrates, 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. 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 liquid is separated and purified to obtain 11β-hydroxysteroid compounds.

6. The use according to claim 5, 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.

7. The use according to claim 5, 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.

8. The use according to claim 5, characterized in that The wet cell was prepared as follows: the engineered bacteria containing the carbonyl reductase mutant encoding gene were inoculated into liquid LB medium containing 50 μg / mL kanamycin, cultured at 37°C, 200 rpm overnight, transferred to fresh LB medium containing 50 μg / mL kanamycin at a volume concentration of 2%, and continued to culture until the 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 h. After the culture was completed, the cells were collected by centrifugation at 8000 rpm at 4°C for 10 min, and washed twice with 0.9% saline to obtain the wet cells.

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