7α-Hydroxysteroid dehydrogenase mutants and their applications in the synthesis of 7-oxolithocholic acid

Abstract

The present invention discloses a 7α-hydroxysteroid dehydrogenase mutant and its application in the synthesis of 7-oxolithocholic acid, belonging to the fields of genetic engineering and biocatalysis technology. By performing site-directed mutagenesis on specific amino acids in the pocket of Brucella melitensis 7α-hydroxysteroid dehydrogenase (Bm7α-HSDH) and combining some advantageous mutations, the present invention obtains mutants with significantly improved thermal stability and catalytic efficiency. In the reaction of catalyzing the synthesis of 7-oxolithocholic acid from the substrate chenodeoxycholic acid, the enzyme activity of the mutant is increased by up to about 45 times at most, the catalytic efficiency is increased by about 122 times, and the T m value can be increased by about 12 °C at most, providing high-quality biocatalysts and precursor compounds for the biocatalytic synthesis of ursodeoxycholic acid and laying a solid research foundation for its industrial production.

Description

Technical Field

[0001] The present invention relates to a 7α-hydroxysteroid dehydrogenase mutant and its application in the synthesis of 7-oxolithocholic acid, belonging to the technical fields of genetic engineering and biocatalysis. Background Art

[0002] Chenodeoxycholic acid (3α,7α-dihydroxy-5-β-cholanic acid) is a steroid containing two hydroxyl groups, and its main function is to reduce the saturation of cholesterol in bile. 7-Oxolithocholic acid (3α-hydroxy-7-oxo-5β-cholanic acid) is the reduction product of chenodeoxycholic acid and serves as an intermediate in the synthesis of ursodeoxycholic acid (3α,7β-dihydroxy-5β-cholestane-24-oic acid). Ursodeoxycholic acid and chenodeoxycholic acid are stereoisomers of each other, and their main functions are to treat gallstones, fatty liver, biliary indigestion, and prevent COVID-19, etc.

[0003] 7α-Hydroxysteroid dehydrogenase (7α-HSDH, EC 1.1.1.159) is one of the main enzymes used for the production of 7-oxolithocholic acid. 7α-Hydroxysteroid dehydrogenase belongs to the short-chain dehydrogenase / reductase family and contains a Rossmann-folded NAD(P)H / NAD(P) + binding domain and an active catalytic triad composed of Ser-Tyr-Lys. Currently, 10 different 7α-HSDH genes from various sources have been cloned and functionally verified. The amino acid sequence homology of these 7α-HSDHs is 17.57%–73.93%, showing different catalytic efficiencies and thermal stabilities in the production of 7-oxolithocholic acid. However, the natural 7α-HSDH enzyme has poor thermal stability and low efficiency in the production of 7-oxolithocholic acid. It is reported that the yield of 7-oxolithocholic acid by 7α-HSDH from Xanthomonas maltophilia is only 80% after 24 h of reaction. The residual activity of 7α-HSDH from Clostridium absonum is less than 50% after incubation at 37 °C for 2 h.

[0004] The inventors screened a 7α-HSDH from Brucella melitensis, which can catalyze the synthesis of 7-oxolithocholic acid using chenodeoxycholic acid as a substrate. However, the wild-type enzyme has low stability and catalytic efficiency, resulting in high time and economic costs in the biological preparation process. Therefore, improving the thermal stability and catalytic efficiency of 7α-HSDH can lay a solid research foundation for the industrial production of 7-oxolithocholic acid, the intermediate for the enzymatic synthesis of ursodeoxycholic acid. Summary of the Invention

[0005] The inventor team found that 7α-HSDH from B. melitensis can catalyze the synthesis of 7-oxolithocholic acid using chenodeoxycholic acid as a substrate. However, the wild-type enzyme has low stability and catalytic efficiency, resulting in high time and economic costs in the biological preparation process.

[0006] In this invention, the 7α-HSDH gene derived from B. melitensis was codon-optimized according to the codon preference of Escherichia coli and its gene was artificially synthesized, named Bm7α-HSDH. Then, by mutating specific amino acids, a 7α-HSDH mutant with improved catalytic efficiency and thermal stability was obtained, which can be used to achieve the efficient synthesis of 7-oxolithocholic acid.

[0007] The 7α-HSDH gene derived from B. melitensis in this invention contains 915 bp bases. This invention provides the gene 7α-HSDH optimized according to the codon preference of Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.1. The 7α-HSDH encoded by the gene shown in SEQ ID NO.1 contains 305 amino acids, and the amino acid sequence is shown in SEQ ID NO.2.

[0008] This invention provides a 7α-hydroxysteroid dehydrogenase mutant, which is obtained by mutating one or more of the 262nd, 301st, and 258th amino acids of the 7α-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO.2.

[0009] This invention provides a 7α-hydroxysteroid dehydrogenase mutant, which is obtained by mutating the 262nd lysine of the 7α-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO.2 to threonine, named: K262T;

[0010] Or, the 7α-hydroxysteroid dehydrogenase mutant is obtained by mutating the 301st glutamine of the 7α-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO.2 to isoleucine, named: Q301I;

[0011] Or, the 7α-hydroxysteroid dehydrogenase mutant is obtained by mutating the 258th isoleucine of the 7α-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO.2 to methionine, and at the same time mutating the 262nd lysine of the 7α-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO.2 to threonine, named: I258M / K262T.

[0012] The present invention also provides a gene encoding the above-mentioned 7α-hydroxysteroid dehydrogenase mutant, and the gene can be obtained by cloning or artificial total sequence synthesis.

[0013] The present invention also provides a recombinant vector carrying the above-mentioned gene.

[0014] In one embodiment of the present invention, the recombinant expression vector can be constructed by ligating the coding gene to various commercially available empty vector by conventional methods in the art.

[0015] In one embodiment of the present invention, the recombinant vector uses pET-21a, pET-21a or pRSFDuet-1 as the expression vector.

[0016] The present invention also provides a recombinant cell expressing the above-mentioned mutant, or carrying the above-mentioned gene, or carrying the above-mentioned recombinant vector.

[0017] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as the expression host.

[0018] In one embodiment of the present invention, the recombinant cell uses Escherichia coli as the expression host.

[0019] The present invention also provides a recombinant expression transformant containing the coding gene of the 7α-HSDH mutant or its recombinant expression vector.

[0020] In one embodiment of the present invention, the recombinant expression transformant can be prepared by transforming the above-mentioned recombinant expression vector into the corresponding host cell by conventional techniques in the art.

[0021] In one embodiment of the present invention, the host cell is a conventional host cell in the art, as long as it can satisfy that the recombinant expression vector can stably replicate itself, and the 7α-HSDH gene encoded by it can be effectively expressed.

[0022] The present invention also provides a catalyst for catalyzing the synthesis of 7-oxolithocholic acid from chenodeoxycholic acid, and the catalyst is the above-mentioned 7α-hydroxysteroid dehydrogenase mutant, or an enzyme solution or its lyophilized powder containing the 7α-hydroxysteroid dehydrogenase mutant prepared by using the above-mentioned recombinant cell.

[0023] In one embodiment of the present invention, the catalyst is any one of the following forms: transformed somatic cells containing the 7α-hydroxysteroid dehydrogenase mutant obtained by culturing the recombinant expression transformant of the present invention; or, crude enzyme solution containing the 7α-hydroxysteroid dehydrogenase mutant obtained by culturing the recombinant expression transformant of the present invention; or, crude enzyme powder obtained by drying the crude enzyme solution containing the 7α-hydroxysteroid dehydrogenase mutant obtained by culturing the recombinant expression transformant of the present invention. Among them, the culturing methods and conditions of the recombinant expression transformant are conventional methods and conditions, and different optimization conditions are adopted for different host systems to achieve high-efficiency expression of the protein.

[0024] The present invention also provides a method for synthesizing 7-oxolithocholic acid. The method is to add the above mutant, or the mutant prepared by using the above recombinant cells, to a reaction system containing the substrate chenodeoxycholic acid and coenzyme NAD + to catalytically prepare 7-oxolithocholic acid;

[0025] In one embodiment of the present invention, the reaction system further contains a buffer salt solution with a pH of 8.0 - 9.5.

[0026] In one embodiment of the present invention, the buffer salt solution can be any conventional buffer solution in the art, as long as its pH range is 8.0 - 9.5, such as sodium phosphate, potassium phosphate, Tris-HCl buffer solution, sodium carbonate buffer solution, and preferably sodium carbonate-sodium bicarbonate buffer solution with a pH of 9.5.

[0027] In one embodiment of the present invention, the concentration of the buffer solution is 0.05 - 0.2 M.

[0028] In one embodiment of the present invention, the concentration of the substrate chenodeoxycholic acid is 2 - 50 mM.

[0029] In one embodiment of the present invention, the reaction temperature in the reaction system is 20 - 40 °C.

[0030] In one embodiment of the present invention, the reaction temperature is 30 °C.

[0031] The above reaction conditions are: carried out in a buffer salt solution with a pH of 8.0 - 9.5, the concentration of the substrate chenodeoxycholic acid is 2 - 50 mM, and the temperature is 20 - 40 °C.

[0032] The present invention also provides the application of the above mutant, or the above gene, or the above recombinant vector, or the above recombinant cells in the preparation of 7-oxolithocholic acid or products containing 7-oxolithocholic acid.

[0033] Beneficial effects

[0034] (1) The present invention uses 7α-HSDH with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing as the parent. By homologous sequence and structure alignment, the key amino acids near the substrate binding site are found. The alanine scanning strategy is adopted to determine the candidate sites for mutagenesis. By using the site-directed mutagenesis method, a significant change in the catalytic efficiency and thermal stability of 7α-HSDH is successfully achieved. On this basis, through enzyme activity determination, CD detection, and liquid phase detection, 7α-HSDH mutants with improved catalytic efficiency are obtained. Compared with the wild type, the preferred mutants can not only efficiently catalyze chenodeoxycholic acid, but also have improved thermal stability.

[0035] (2) The present invention constructs the 7α-HSDH gene in plasmid pET-21a and expresses it in Escherichia coli BL21(DE3). The crude enzyme solution is purified by His-Trap affinity chromatography column to obtain pure enzyme. By optimizing the enzyme activity determination conditions, the specific enzyme activity is measured at 30 °C in a Na 2 CO 3 -NaHCO 3 buffer at pH 9.5. Through enzyme activity determination, for the combined mutant I258M / K262T, the enzyme activity is increased by about 45 times, and the T m value is increased by about 12 °C.

[0036] (3) The 7α-HSDH of the present invention provides a new way for the asymmetric transformation reaction of hydroxy steroid dehydrogenase combined with coenzyme recycling to synthesize ursodeoxycholic acid, solves the problem of insufficient raw materials for the synthesis of ursodeoxycholic acid, and provides excellent strains and enzymes for the synthesis of ursodeoxycholic acid intermediates in industry, which helps to greatly reduce its production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : SDS-PAGE analysis of wild type, single-site dominant mutants and their combined mutants. M: Protein molecular weight standard, CK: Supernatant of Escherichia coli cell lysate; Lane 1 shows the expression of WT in cells; Lanes 2-5: Protein purification products of WT, K262T, Q301I, and I258M / K262T, respectively. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] Chenodeoxycholic acid involved in the following examples was purchased from: J&K Scientific Ltd.

[0039] The following media are involved in the following examples:

[0040] LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0. Ampicillin (100 μg·mL -1) Add 1.5% agar powder to the solid medium.

[0041] The detection methods involved in the following examples are as follows:

[0042] Determination of 7α-HSDH enzyme activity:

[0043] By measuring the change rate of the absorbance of NADH at 340 nm, the enzyme activity of 7α-HSDH oxidizing CDCA was measured at 30 °C. Enzyme activity determination standard: The reaction volume was 200 μL, and Na with a final concentration of 100 mM was added respectively. 2 CO 3 -NaHCO 3 (pH 9.5), 5 mM NAD + and 5 mM CDCA were incubated in a 30 °C metal bath for 3 min, an appropriate amount of pure enzyme (10–200 μM) was added, and the change in absorbance at 340 nm was scanned using a Bio-Tek Cytation 5 cell imaging multi-functional detection system.

[0044] The enzyme activity is defined as: Under the above conditions, the amount of enzyme that catalyzes the formation of 1 μmol of NADH per minute is defined as one unit U.

[0045] The calculation formula for enzyme activity is: Enzyme activity (U) = EW × V × 10 3 / (6220 × 0.5)

[0046] The calculation formula for specific activity: Specific activity (U·mg -1 ) = Enzyme activity (U) / Protein amount (mg)

[0047] Among them, EW: The change in absorbance at 340 nm within 1 min; V: The volume of the reaction solution (mL); 6220: Molar extinction coefficient L·mol -1 ·cm -1 ); 0.5: Optical path distance (cm).

[0048] Determination of kinetic parameters:

[0049] According to the Michaelis equation v = V max ×[S] / K m +[S], the K value of the enzyme for different substrates was calculated. m value.

[0050] Among them, v: Reaction rate (U·mg -1 ); V max : Maximum reaction rate (U·mg -1 ); [S]: Substrate concentration (mM); K m : Substrate concentration when the reaction rate v reaches half of 1 / 2V max value.

[0051] When the substrate concentration is saturated, k cat = V max / Et, and the K cat value is calculated.

[0052] Among them, V max : maximum reaction rate; Et: concentration of enzyme sites.

[0053] Determination of kinetic parameters:

[0054] The total reaction volume is 200 μL. Add 0.1 M Na 2 CO 3 -NaHCO 3 buffer (pH 9.5), 5.0 mM NAD + , incubate at 30 °C for 2 min, and add an appropriate amount of pure enzyme solution to measure the specific enzyme activity at different substrate concentrations (0.1 mM to 5.0 mM). The fitting of the enzyme kinetic curve is achieved by GraphPad software. Using the substrate concentration and specific enzyme activity as the abscissa and ordinate respectively, select the Michaelis-Menten equation model for non-linear fitting to obtain the V max value and the K m value, and obtain the k cat value and the k cat / K m value through data conversion (Table 3).

[0055] CD determination

[0056] Use a Jasco J720 spectropolarimeter to perform circular dichroism measurements. Collect wavelength scan data from 190 to 250 nm in phosphate buffer (pH 8.0) using the following instrument settings (average 30 scans): response, 1 s; sensitivity is 100 mm; speed is 50 nm min -1 . Repeat the scan every 3 °C between 20 °C and 80 °C. In 50 mM Na / K phosphate buffer (pH 8.0), the protein concentration is approximately 0.01 M, and the decrease of the circular dichroism signal with increasing temperature is recorded at 220 nm, and the T m value of different proteins is calculated.

[0057] Example 1: Codon optimization and synthesis of 7α-HSDH gene

[0058] The specific steps are as follows:

[0059] (1) The 7α-HSDH gene from B. melitensis contains 915 bp of bases (NCBI accession number: MW202238). The 7α-HSDH gene was codon optimized with reference to the codon preference of E. coli. The optimized gene sequence is shown in SEQ ID NO:1.

[0060] (2) A six-histidine tag gene was introduced at the C-terminus of the gene, and then BamH I and Xho I restriction enzyme cutting site sequences were added at both ends, respectively, and finally connected with pET-21a to obtain the recombinant plasmid pET-21a-7α-HSDH.

[0061] Example 2: Construction of 7α-HSDH mutant gene and recombinant E. coli expression system

[0062] (1) Construction of 7α-HSDH mutant

[0063] Design of 7α-HSDH by whole plasmid PCR

[0064] For site-directed mutagenesis, the recombinant plasmid pET-21a-7α-HSDH was used as a template, and primer sequences were designed (Table 1 ), and the following mutations were performed: K262C, K262F, K262G, K262T, K262V, K262Y, Q301F, Q301I, Q301L, Q301M, Q301N, and I258M / K262T.

[0065] Table 1: Primer design table for constructing mutants

[0066]

[0067] pmol·μl -1 )1.0μl, template 1.0μl, ddHO 2 O 22.0 μl;

[0068] PCR amplification conditions: pre-deformation: 98℃30s; denaturation: 98℃10s; annealing: 55℃15s; extension: 72℃

[0069] 60s; post-extension: 72℃10min; storage: 4℃.

[0070] After PCR amplification, the amplified product was digested with Dpn I at 37°C for 2 h to remove the template plasmid, and the digested product was transformed into E. coli BL21 (DE3) competent cells, plated, and single colonies were picked for test tube culture, and the mutant sequence was verified by sequencing.

[0071] (2) Transformation of recombinant plasmid into E. coli BL21 (DE3):

[0072] Add 10 μL of the PCR product obtained in step (1) to 100 μL of E. coli BL21(DE3) competent cell suspension in each tube, gently mix, and let stand in an ice bath for 30 min. Transfer to a 42 °C water bath and perform heat shock for 90 s. Quickly transfer to an ice bath and cool for 3 min. Add 700 μL of LB liquid medium to each tube, incubate in a shaker at 37 °C and 100 rpm for 1 h. After incubation, centrifuge the bacterial solution at 3,000×g for 2 min, discard 700 μL of the supernatant, and spread the remaining bacterial solution evenly on an LB plate containing 50 μg·mL -1 ampicillin and incubate upside down at 37 °C overnight.

[0073] (3) Selection of positive clones:

[0074] Pick 4 clones and transfer them into 5 mL of LB medium containing 100 μg·mL -1 ampicillin, culture at 37 °C for 8 h, and extract the plasmid using the Mini-Plasmid Rapid Isolation Kit (Nanjing Novoprotein Scientific Co., Ltd.).

[0075] Use the following reaction system for restriction enzyme verification: 10×Buffer 2 μL, plasmid DNA 5 μL, BamH I 0.5 μL, Xho I 0.5 μL, ddH 2 O make up the system to 20 μL.

[0076] Respectively obtain positive clones: E. coli / pET-21a-K262C, E. coli / pET-21a-K262F, E. coli / pET-21a-K262G, E. coli / pET-21a-K262T, E. coli / pET-21a-K262V, E. coli / pET-21a-K262Y, E. coli / pET-21a-Q301F, E. coli / pET-21a-Q301I, E. coli / pET-21a-Q301L, E. coli / pET-21a-Q301M, E. coli / pET-21a-Q301N, E. coli / pET-21a-I258M / K262T.

[0077] Meanwhile, prepare the recombinant bacterium containing the wild-type enzyme: E. coli / pET-21a-7α-HSDH according to the above method.

[0078] Example 3: Induced expression culture of recombinant bacteria

[0079] The specific steps are as follows:

[0080] (1) Pick positive clones containing mutants and single colonies containing wild-type enzymes prepared in Example 2 and inoculate them into 10 mL of LB liquid medium containing 100 μg·mL -1 ampicillin, and culture them overnight at 37 °C with shaking at 200 rpm.

[0081] (2) Transfer 10 mL of the culture solution obtained in step (1) to 1 L of LB liquid medium containing 100 μg·mL -1 ampicillin, and culture it at 37 °C with shaking at 200 rpm until the OD 600 is approximately 0.6 - 0.8 to obtain a culture; add isopropyl-β-D-thiogalactoside with a final concentration of 0.1 mM to the culture and perform induction culture at 25 °C for 12 h.

[0082] (3) After the induction culture is completed, centrifuge the culture solution at 6,000×g for 10 min to collect recombinant Escherichia coli cells, wash them three times with physiological saline, and then collect them.

[0083] Example 4: Purification of Recombinant Protein

[0084] Purify the recombinant protein using a His-Trap HP affinity column:

[0085] (1) Weigh 2 g of the wet bacterial cells collected in Example 3 respectively, add an appropriate amount of 20 mM Tris-HCl (pH 8.0) buffer to suspend the cells, and perform ultrasonic disruption in an ice bath (working for 2 s, interval for 3 s, working time for 10 min). Centrifuge at 12,000×g for 30 min at 4 °C to collect the supernatant as the crude enzyme solution.

[0086] (2) Purify the above crude enzyme solution using a His-Trap affinity chromatography produced by Cytiva, and analyze the purification products of wild-type and its dominant mutant proteins by SDS-PAGE ( Figure 1 ). The expression bands of wild-type in the cells and the bands after purification are both shown as non-single bands, which may be due to the flexible ends of the full-length protein affecting the protein expression. The pure enzyme solution is ultrafiltered and desalted for enzyme activity determination.

[0087] Prepare pure enzyme solutions containing wild-type enzyme WT, pure enzyme solutions containing K262C, pure enzyme solutions containing K262F, pure enzyme solutions containing K262G, pure enzyme solutions containing K262T, pure enzyme solutions containing K262V, pure enzyme solutions containing K262Y, pure enzyme solutions containing Q301F, pure enzyme solutions containing Q301I, pure enzyme solutions containing Q301L, pure enzyme solutions containing Q301M, pure enzyme solutions containing Q301N, and pure enzyme solutions containing I258M / K262T respectively.

[0088] Example 5: Specific Enzyme Activity Assay

[0089] The specific enzyme activities of the wild-type enzyme and mutant enzymes obtained in Example 4 against chenodeoxycholic acid were detected respectively, and the results are shown in the following table.

[0090] Table 2: Comparison of Specific Enzyme Activities between Wild-Type and Mutant

[0091]

[0092] Note: n.d. a Indicates that no activity was detected; Nomenclature of mutants in the present invention: The mutant is represented by "amino acid at the original amino acid position replaced by amino acid". For example, K262C means that the amino acid at position 262 is replaced by cysteine Cys from lysine Lys of the parental 7α-HSDH, and the numbering of the position corresponds to the corresponding site of the amino acid sequence of the parental 7α-HSDH.

[0093] As shown in Table 2, the specific enzyme activities of the wild-type and mutants catalyzing chenodeoxycholic acid were measured respectively. The results showed that the specific enzyme activities of the single mutants K262C, K262G, K262T, and Q301I were significantly increased relative to the wild-type, while the specific activities of other single mutants showed different degrees of decrease.

[0094] The dominant single mutants were subjected to combined mutagenesis, and finally I258M / K262T with a significantly increased specific enzyme activity was screened. The results showed that the changes in the steric hindrance and polarity of different amino acid side chains at positions 258, 262, and 301 played an important role in the catalytic activity of catalyzing chenodeoxycholic acid.

[0095] Example 6: Determination of Kinetic Parameters

[0096] Determination of kinetic parameters: The total reaction volume was 200 μL, 0.1 M Na 2 CO 3 -NaHCO 3 buffer (pH 9.5), 5.0 mM NAD + was added, incubated at 30 °C for 2 min, and an appropriate amount of pure enzyme solution was added to measure the specific enzyme activity at different substrates (0.1 mM - 5.0 mM). The k cat , K m , k cat / K m of the wild-type and mutants of 7α-HSDH were calculated. (Table 3).

[0097] Table 3: Kinetic Parameters of Wild-Type and Mutant

[0098]

[0099] As shown in Table 3, the k cat / K m values of K262G, K262T, Q301I and I258M / K262T mutants for catalyzing the substrate chenodeoxycholic acid showed a significant increase, which was beneficial to improving the catalytic efficiency of the enzyme for catalyzing the substrate chenodeoxycholic acid to synthesize 7-oxolithocholic acid.

[0100] Example 7: CD determination

[0101] By characterizing the thermal stability of the mutants obtained in Example 4, mutants with improved thermal stability and catalytic efficiency were obtained, which were used as excellent enzymes for industrial transformation to synthesize 7-oxolithocholic acid. Circular dichroism measurements were performed using a Jasco J720 spectropolarimeter to calculate the T m values of different proteins (Table 4).

[0102] Table 4: Thermal stability analysis of 7α-HSDH wild type and dominant mutants

[0103]

[0104] As shown in Table 4, compared with the wild type, the T m values of K262T, Q301I and I258M / K262T all showed significant improvement. Combining with Example 6, K262T and I258M / K262T simultaneously showed improved thermal stability and catalytic efficiency.

[0105] Example 8: Catalytic synthesis of 7-oxolithocholic acid by 7α-HSDH

[0106] The specific steps are as follows:

[0107] The pure enzyme-catalyzed reaction system was carried out in a 50 mL centrifuge tube. The 10 mL reaction mixture consisted of 0.1 M Na 2 CO 3 -NaHCO 3 buffer (pH 9.5), 2 mM NAD + , 2 mM chenodeoxycholic acid and 20 μg pure enzyme. The reaction was carried out with shaking at 30 °C and 200 r·min -1 for 12 hours, and the product yield was detected by sampling. The reaction was terminated by adjusting the pH to 10.0 - 11.0 with 2 M NaOH, and extracted three times with an equal volume of acetonitrile. The extraction solutions were mixed, then rotary evaporated and concentrated until crystals precipitated. After filtration, the solvent was removed, dried to constant weight, and detected by HPLC. The reaction conversion rate was analyzed by liquid chromatography using a C-18 column (250 mm × 4.6 mm), with the mobile phase of acetonitrile: water (0.1% phosphoric acid) = 60:40, column temperature of 30 °C, flow rate of 1.0 mL min -1 , and detection wavelength of 195 nm. The results are shown in Table 5.

[0108] Table 5: Yields of 7α-HSDH Wild-Type and Mutants

[0109]

[0110] As shown in Table 5, the K262T and I258M / K262T mutants showed significant improvement in yield. For the remaining mutants, due to changes in enzyme activity or thermal stability, their yields showed no obvious improvement compared to the wild-type. This indicates that the improvement in catalytic efficiency and thermal stability of the mutants may play a decisive role in the efficient preparation of 7-oxolithocholic acid from chenodeoxycholic acid.

[0111] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A 7α-hydroxysteroid dehydrogenase mutant, characterized in that, the 7α-hydroxysteroid dehydrogenase mutant is obtained by mutating glutamine at position 301 of the amino acid sequence of 7α-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO.2 to isoleucine.

2. A gene encoding the mutant according to claim 1.

3. A recombinant vector carrying the gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that, the recombinant vector uses pET-21a or pRSFDuet-1 as the expression vector.

5. A recombinant cell expressing the mutant according to claim 1, or carrying the gene according to claim 2, or carrying the recombinant vector according to claim 3 or 4.

6. The recombinant cell according to claim 5, characterized in that, the recombinant cell uses bacteria or fungi as the expression host.

7. A catalyst for catalyzing the synthesis of 7-oxolithocholic acid from chenodeoxycholic acid, characterized in that, the catalyst is the 7α-hydroxysteroid dehydrogenase mutant according to claim 1, or an enzyme solution or its lyophilized powder containing the 7α-hydroxysteroid dehydrogenase mutant prepared using the recombinant cell according to claim 5 or 6.

8. A method for synthesizing 7-oxolithocholic acid, characterized in that, The method is to add the mutant described in claim 1 or the mutant prepared by using the recombinant cell described in claim 5 or 6 to a reaction system containing the substrate chenodeoxycholic acid and the coenzyme NAD + to catalytically prepare 7-oxolithocholic acid.

9. The method according to claim 8, characterized in that, the reaction system further contains a buffer salt solution with a pH of 8.0 to 9.5; the concentration of the substrate chenodeoxycholic acid is 2 to 50 mM, and the reaction temperature is 20 to 40 °C.

10. Use of the mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3 or 4, or the recombinant cell according to claim 5 or 6 in the preparation of 7-oxolithocholic acid or a product containing 7-oxolithocholic acid.

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