Combinatorial Site-Directed Mutagenesis of the C-Terminus of 7α-Hydroxysteroid Dehydrogenase and Efficient Synthesis of Ursodeoxycholic Acid Intermediate

Abstract

The present invention discloses the combined site-directed mutagenesis of the C-terminus of 7α-hydroxysteroid dehydrogenase and the efficient synthesis of ursodeoxycholic acid intermediate, belonging to the fields of genetic engineering and biocatalysis. By truncating the N-terminal amino acids of 7α-hydroxysteroid dehydrogenase and performing site-directed mutagenesis of specific amino acids in the binding pocket, mutants with significantly improved thermal stability and catalytic efficiency are obtained. The enzyme activity of the best combined mutant ΔN53 / M196I / I258M / K262T is increased by about 323 times, and the k cat / K m value is increased by 267.04 times compared with the wild type, and the T m value is increased by 21.06 °C. When the wild type catalyzes 50 mM substrate chenodeoxycholic acid, the reaction takes 48 h, and the yield of the synthetic product 7-oxolithocholic acid is 84.4%. While when the mutant catalyzes 50 mM substrate chenodeoxycholic acid, the reaction only takes 4 h, and the yield of the synthetic product 7-oxolithocholic acid is as high as 97.1%.

Description

Technical Field

[0001] The present invention relates to the terminal modification combined site mutation of 7α-hydroxysteroid dehydrogenase and the efficient synthesis of ursodeoxycholic acid intermediates, belonging to the technical fields of genetic engineering and biocatalysis. Background Art

[0002] Chenodeoxycholic acid (also known as 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 (also known as 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 (also known as 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 dyspepsia, and prevent COVID-19, etc.

[0003] 7α-Hydroxysteroid dehydrogenase (7α-HSDH, EC 1.1.1.159) is one of the main enzymes used in the production of 7-oxolithocholic acid. 7α-HSDH belongs to the short-chain dehydrogenase / reductase family. The natural 7α-HSDH enzyme has low efficiency in the catalytic synthesis of 7-oxolithocholic acid production, and the thermal stability of the enzyme is poor. In recent years, computer-aided protein design strategies have made great contributions to improving protein catalytic efficiency, stereo / regioselectivity, and stability. For example, the synthesis efficiency of imine reductase for (R)-3-benzylamino-1-Boc-piperidine was improved by structure-guided site-directed mutagenesis, and the catalytic efficiency was increased by 4193 times compared with the wild type, and the T m increased by 16.2 °C, and the optical purity of the product increased from 78% to 99%. Based on the active site replacement of structure-based site-directed mutagenesis, in order to increase the process stability of CYP102A1, Eiben et al. exchanged the unstable reductase domain of CYP102A1 with the stable CYP102A3 domain, and obtained a chimeric fusion protein with enhanced stability, with a half-life of 100 minutes at 50 °C, which was more than 10 times higher than that of the wild type CYP102A1. Proteins with good thermal stability usually have higher rigidity, while flexible regions, such as flexible residues, the N-terminal and C-terminal loops of the protein chain, have few contacts with other amino acids. Therefore, truncating, replacing flexible loops, or introducing point mutations to enhance the rigidity of flexible residues is an effective method to improve stability. Based on structure alignment, Savino et al. truncated the C-terminal fragment of C. absonum 7β-HSDH in segments, and the activity decreased by 1000 times or even inactivated. Lou et al. proposed that the loop structure (residues 194–211) in C. absonum 7α-HSDH is closely related to the thermal stability of the enzyme.

[0004] The inventors screened a 7α-HSDH from Brucella melitensis, which can use chenodeoxycholic acid as a substrate and NAD + as a coenzyme to stably catalyze the synthesis of 7-oxolithocholic acid under alkaline conditions. However, the wild-type enzyme has low catalytic stability and efficiency, resulting in high time and economic costs in the biological preparation process and hindering its industrialization process. The present invention uses a computer-aided protein design strategy to study the effects of terminal modification on protein expression, catalytic efficiency, and thermal stability. First, the three-dimensional structure of 7α-HSDH is obtained based on homology modeling, and molecular docking and kinetic simulations are combined with prior information on protein function to analyze the flexible changes at the protein termini. Based on sequence and structural features, the disordered region of loop N is truncated in segments, and site-directed combinatorial mutations are combined with single-point mutations to investigate the effects of the mutants on enzyme expression, catalytic efficiency, and thermal stability. Mutants with significantly improved enzyme catalytic efficiency and thermal stability are obtained, which has important research significance for the industrial preparation of 7-oxolithocholic acid. Summary of the Invention

[0005] The inventors' team found that 7α-HSDH from B. melitensis can use chenodeoxycholic acid as a substrate to catalyze the synthesis of 7-oxolithocholic acid, but the wild-type enzyme has low stability and catalytic efficiency, resulting in a long catalytic cycle and low conversion efficiency in the biological preparation process.

[0006] After codon optimization of the 7α-HSDH gene from Brucella (B. melitensis) according to the codon preference of Escherichia coli, the gene was chemically synthesized. Then, by combining truncation of different lengths of amino acids at the N-terminus and / or specific amino acid mutations in the enzyme substrate-binding pocket region, 7α-hydroxysteroid dehydrogenase mutants with significantly improved catalytic efficiency and thermal stability were obtained, realizing the efficient synthesis of 7-oxolithocholic acid.

[0007] The 7α-HSDH gene from B. melitensis of the present invention contains 915 bp of bases. The present invention provides a 7α-HSDH gene optimized for 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 304 amino acids, and the amino acid sequence is shown in SEQ ID NO.2.

[0008] The present invention provides a 7α-hydroxysteroid dehydrogenase mutant, which is obtained by truncating the 1st to 53rd amino acids at the N-terminus of the amino acid sequence of 7α-hydroxysteroid dehydrogenase shown in SEQ ID NO.2; or, the 7α-hydroxysteroid dehydrogenase mutant is obtained by truncating the 1st to 53rd amino acids at the N-terminus of the amino acid sequence of 7α-hydroxysteroid dehydrogenase shown in SEQ ID NO.2, and simultaneously mutating one or more of the 196th, 258th, and 262nd amino acids.

[0009] In one embodiment of the present invention, the 7α-hydroxysteroid dehydrogenase mutant is any one of the following (a) to (f):

[0010] (a) Obtained by truncating the 1st to 53rd amino acids at the N-terminus of the amino acid sequence of 7α-hydroxysteroid dehydrogenase shown in SEQ ID NO.2, named: ΔN53;

[0011] (b) Obtained by truncating the 1st to 53rd amino acids at the N-terminus of the amino acid sequence of 7α-hydroxysteroid dehydrogenase shown in SEQ ID NO.2, and simultaneously mutating the 196th methionine to isoleucine, named: ΔN53 / M196I;

[0012] (c) Obtained by truncating the 1st to 53rd amino acids at the N-terminus of the amino acid sequence of 7α-hydroxysteroid dehydrogenase shown in SEQ ID NO.2, and simultaneously mutating the 258th isoleucine to methionine, named: ΔN53 / I258M;

[0013] (d) Obtained by truncating the 1st to 53rd amino acids at the N-terminus of the amino acid sequence of 7α-hydroxysteroid dehydrogenase shown in SEQ ID NO.2, and simultaneously mutating the 262nd lysine to threonine, named: ΔN53 / K262T;

[0014] (e) Obtained by truncating the 1st to 53rd amino acids at the N-terminus of the amino acid sequence of 7α-hydroxysteroid dehydrogenase shown in SEQ ID NO.2, and simultaneously mutating the 258th isoleucine to methionine and the 262nd lysine to threonine, named: ΔN53 / I258M / K262T;

[0015] (f) It was obtained by truncating the 1st to 53rd amino acids at the N-terminus of the 7α-hydroxysteroid dehydrogenase amino acid sequence shown in SEQ ID NO.2, and simultaneously mutating the 196th methionine to isoleucine, the 258th isoleucine to methionine, and the 262nd lysine to threonine, and is named: ΔN53 / M196I / I258M / K262T.

[0016] The present invention also provides a gene encoding the mutant.

[0017] The present invention also provides a recombinant vector carrying the above gene.

[0018] In one embodiment of the present invention, the recombinant vector uses pET-21a or pRSFDuet-1 as the expression vector.

[0019] The present invention also provides a recombinant cell expressing the above mutant, or carrying the above gene, or carrying the above recombinant vector.

[0020] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as the expression host.

[0021] The present invention also provides a recombinant expression transformant comprising the coding gene of the 7α-HSDH mutant or its recombinant expression vector.

[0022] In one embodiment of the present invention, the recombinant expression transformant can be prepared by transforming the above recombinant expression vector into the corresponding host cell by conventional techniques in the art.

[0023] The host cell is a conventional host cell in the art, as long as it can satisfy that the recombinant expression vector can stably self-replicate, and the 7α-HSDH gene encoded by it can be effectively expressed.

[0024] The present invention also provides a catalyst for catalyzing the synthesis of 7-oxolithocholic acid from chenodeoxycholic acid, and the catalyst is the above 7α-hydroxysteroid dehydrogenase mutant, or an enzyme solution or its lyophilized powder containing the 7α-hydroxysteroid dehydrogenase mutant prepared by using the above recombinant cell.

[0025] 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-HSDH 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 protein expression.

[0026] 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.

[0027] In one embodiment of the present invention, the reaction system further contains a buffer salt solution with a pH of 8.0 to 9.5.

[0028] In one embodiment of the present invention, the buffer salt solution can be any conventional buffer in the art as long as its pH range is 8.0 to 9.5, such as sodium phosphate, potassium phosphate, Tris-HCl buffer, sodium carbonate buffer.

[0029] In one embodiment of the present invention, the buffer salt solution is a sodium carbonate-sodium bicarbonate buffer with a pH of 9.5.

[0030] In one embodiment of the present invention, the concentration of the buffer solution can be 0.05 to 0.2 M.

[0031] In one embodiment of the present invention, the concentration of the substrate chenodeoxycholic acid is 2 to 50 mM.

[0032] In one embodiment of the present invention, the reaction temperature is 20 to 40 °C.

[0033] In one embodiment of the present invention, the reaction temperature is 30 °C.

[0034] The present invention also provides a method for synthesizing 7-oxolithocholic acid. The method is to add the recombinant bacterium containing the mutant and the recombinant bacterium expressing the ethanol dehydrogenase mutant to a reaction system containing a buffer (pH 9.5), chenodeoxycholic acid, NAD + , and acetaldehyde solution for reaction to prepare 7-oxolithocholic acid.

[0035] In one embodiment of the present invention, the reaction system comprises 0.1M Na2CO3-NaHCO3 buffer (pH 9.5), 50mM chenodeoxycholic acid, 0.5mM NAD + , 5mL acetaldehyde solution, 10g·L -1 of E.coli / pET-21a-ΔN53 / M196I / I258M / K262T recombinant bacteria, 10g·L -1 of E.coli / pET-21a-ADH recombinant bacteria, and stir the reaction at 30℃ and 800r·min -1 for 48 hours.

[0036] The present invention also provides the use of the above mutant, or the above gene, or the above recombinant vector, or the above recombinant cell in the preparation of 7-oxolithocholic acid or a product containing 7-oxolithocholic acid.

[0037] Beneficial effects

[0038] (1) In the present invention, 7α-HSDH with the amino acid sequence shown in SEQ ID NO.2 in the sequence listing is used as the parent, and the complex structure is obtained by homology modeling and molecular docking. In order to study the influence of the loop region on protein folding and thermal stability, 50ns MD simulation is applied to analyze the flexibility changes of 7α-HSDH at the N-terminus and C-terminus. Through homologous sequence and structure alignment, N-terminal segment truncation and C-terminal site-directed mutagenesis are carried out, and significant changes in the catalytic efficiency and thermal stability of 7α-HSDH are successfully achieved. On this basis, through enzyme activity determination, circular dichroism detection, and high-pressure liquid 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.

[0039] (2) In the present invention, the 7α-HSDH gene is constructed in plasmid pET-21a and expressed in Escherichia coli BL21(DE3). The crude enzyme solution is purified by His-Trap affinity chromatography column to obtain pure enzyme. As the truncation length increases, the protein expression level gradually increases. By optimizing the enzyme activity determination conditions, the specific enzyme activity is measured at 30℃ in Na2CO3-NaHCO3 buffer at pH 9.5. Through enzyme activity determination, for the combined mutant ΔN53 / M196I / I258M / K262T, the enzyme activity is increased by about 323 times, and the T m value is increased by 21℃.

[0040] (3) The 7α-HSDH of the present invention is a hydroxysteroid dehydrogenase, which provides a new way for the asymmetric transformation reaction to synthesize the ursodeoxycholic acid intermediate 7-oxolithocholic acid, solves the problem that the resources of chenodeoxycholic acid metabolites cannot be effectively utilized, helps to reduce the cost of preparing and producing ursodeoxycholic acid, and lays a solid research foundation for realizing industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : SDS-PAGE analysis of N-terminal truncated proteins; M: protein molecular weight standard, CK: supernatant of E. coli cell lysate; Lanes 1-4: expression of WT, ΔN16, ΔN29, and ΔN53 proteins in cells respectively; Lanes 5-8: purified protein products of WT, ΔN16, ΔN29, and ΔN53 respectively.

[0042] Figure 2 : SDS-PAGE analysis of combined mutations; M: protein molecular weight standard; Lanes 1-6: purified protein products of WT, ΔN53 / M196I, ΔN53 / I258M, ΔN53 / K262T, ΔN53 / I258M / K262T, and ΔN53 / M196I / I258M / K262T respectively.

[0043] Figure 3 : Time curve of the catalytic synthesis of 7-oxolithocholic acid by Bm7α-HSDH and the mutant ΔN53 / M196I / I258M / K262T. DETAILED DESCRIPTION OF THE INVENTION

[0044] The chenodeoxycholic acid involved in the following examples was purchased from: J&K Scientific Ltd.

[0045] The culture media involved in the following examples are as follows:

[0046] LB medium: peptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0. Ampicillin (100 μg·mL -1 ) was added before use when needed, and 1.5% agar powder was added to the solid medium.

[0047] The detection methods involved in the following examples are as follows:

[0048] Determination of 7α-HSDH enzyme activity:

[0049] 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: reaction volume 200 μL, and Na2CO3-NaHCO3 (pH 9.5) with a final concentration of 100 mM and NAD with a concentration of 5 mM were added respectively +Incubate with 5 mM CDCA in a 30 °C metal bath for 3 min, add an appropriate amount of pure enzyme (10–200 μM), and scan the change in absorbance at 340 nm using a Bio-Tek Cytation 5 cell imaging multi-functional detection system.

[0050] 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.

[0051] The calculation formula for enzyme activity is: Enzyme activity (U) = EW × V × 10 3 / (6220 × 0.5);

[0052] The calculation formula for specific activity: Specific activity (U·mg -1 ) = Enzyme activity (U) / Protein amount (mg);

[0053] 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).

[0054] Determination of kinetic parameters:

[0055] According to the Michaelis-Menten equation v = V max ×[S] / K m +[S], calculate the K m value of the enzyme for different substrates.

[0056] 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 .

[0057] When the substrate concentration is saturated, V max = k cat / [E], calculate the K cat value.

[0058] Among them, V max : Maximum reaction rate; Et: Concentration of enzyme sites.

[0059] Determination of kinetic parameters:

[0060] The total reaction volume is 200 μL, add 0.1 M Na2CO3-NaHCO3 buffer (pH 9.5), 5.0 mM NAD +, incubate at 30 °C for 2 min, add an appropriate amount of pure enzyme solution and measure the specific enzyme activity at different substrate concentrations (0.1 mM to 5.0 mM). The fitting of the enzyme kinetic curve was achieved by GraphPad software. Using the substrate concentration and specific enzyme activity as the abscissa and ordinate respectively, the Michaelis-Menten equation model was selected for non-linear fitting to obtain the V max value and K m value. The k cat value and k cat / K m value were obtained through data conversion (Table 3).

[0061] CD determination

[0062] Circular dichroism (CD) measurements were performed using a Jasco J720 spectropolarimeter (Jasco, Inc., Easton, MD). Wavelength scan data were collected from 190 to 250 nm in phosphate buffer (pH 8.0) using the following instrument settings (average of 30 scans): response, 1 s; sensitivity of 100 mm; speed of 50 nm min -1 . Scans were repeated every 3 °C between 20 °C and 80 °C. In 50 mM Na / K phosphate buffer (pH 8.0), the protein concentration was approximately 0.01 M, and the decrease in the CD signal with increasing temperature was recorded at 220 nm. The T m values of different proteins were calculated.

[0063] Example 1: Codon optimization and synthesis of the 7α-HSDH gene

[0064] The specific steps are as follows:

[0065] (1) The 7α-HSDH gene from B. melitensis (NCBI accession number: MW202238) contains 915 bp of bases. The 7α-HSDH gene was codon-optimized with reference to the codon preference of Escherichia coli, and the optimized gene sequence is shown in SEQ ID NO: 1.

[0066] (2) Six histidine-tag genes were introduced at the C-terminus of the gene, and then BamH I and Xho I restriction enzyme cleavage site sequences were added to both ends. Finally, it was ligated with pET-21a to obtain the recombinant plasmid pET-21a-7α-HSDH.

[0067] Example 2: Construction of the 7α-HSDH mutant gene and recombinant Escherichia coli expression system

[0068] (1) Construction of the 7α-HSDH mutant

[0069] The full plasmid PCR technique was used to design truncations and / or site-directed mutations for 7α-HSDH. Using the recombinant plasmid pET-21a-7α-HSDH as a template, primer sequences were designed to obtain the following mutants:

[0070] ΔN16 with 16 amino acids truncated at the N-terminal end, ΔN29 with 29 amino acids truncated at the N-terminal end, ΔN53 with 53 amino acids truncated at the N-terminal end, ΔN61 with 61 amino acids truncated at the N-terminal end, and mutants: ΔN53 with 53 amino acids truncated at the N-terminal end and the 196th position mutated to isoleucine (ΔN53 / M196I), ΔN53 with 53 amino acids truncated at the N-terminal end and the 258th position mutated to methionine (ΔN53 / I258M), ΔN53 with 53 amino acids truncated at the N-terminal end and the 262nd position mutated to threonine (ΔN53 / K262T), ΔN53 with 53 amino acids truncated at the N-terminal end and the 258th position mutated to methionine and the 262nd position mutated to threonine (ΔN53 / I258M / K262T), ΔN53 with 53 amino acids truncated at the N-terminal end and the 196th position mutated to isoleucine, the 258th position mutated to methionine, and the 262nd position mutated to threonine (ΔN53 / M196I / I258M / K262T);

[0071] The primer design is shown in Table 1.

[0072] Table 1: Primer design table for constructing mutants

[0073]

[0074] Note: The underlined part indicates the mutation site.

[0075] PCR amplification system: 25.0 μl of DNA polymerase, 1.0 μl of upstream primer (10 pmol·μl -1 )), 1.0 μl of downstream primer (10 pmol·μl -1 ), 1.0 μl of template, 22.0 μl of ddH2O;

[0076] PCR amplification conditions: Pre-denaturation: 98°C for 30 s; Denaturation: 98°C for 10 s; Annealing: 55°C for 15 s; Extension: 72°C for 60 s; Post-extension: 72°C for 10 min; Storage: 4°C.

[0077] After the PCR amplification was completed, the amplification product was digested with Dpn I at 37°C for 2 h to remove the template plasmid. The digested product was transformed into E. coli BL21(DE3) competent cells, spread on plates, single colonies were picked for tube culture, and the mutant sequences were verified by sequencing.

[0078] (2) Transformation of the recombinant plasmid into Escherichia coli E. coli BL21(DE3):

[0079] 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 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, and incubate at 37 °C with shaking at 100 rpm for 1 h. After incubation, centrifuge the bacterial suspension at 3,000×g for 2 min, discard 700 μL of the supernatant, and spread the remaining bacterial suspension evenly on an LB plate containing 50 μg·mL -1 ampicillin, and incubate inverted at 37 °C overnight.

[0080] (3) Selection of positive clones:

[0081] Pick 4 clones and transfer them into 5 mL of LB medium containing 100 μg·mL -1 ampicillin, incubate at 37 °C for 8 h, and extract the plasmid using the Mini-Plasmid Rapid Isolation Kit (Nanjing Novoprotein Science & Technology Co., Ltd.).

[0082] Use the following reaction system for enzyme digestion verification: 2 μL of 10×Buffer H, 5 μL of plasmid DNA, 0.5 μL of BamH I, 0.5 μL of Xho I, and ddH2O to make up the system to 20 μL.

[0083] Respectively obtain positive clones: E. coli / pET-21a-ΔN16, E. coli / pET-21a-ΔN53, E. coli / pET-21a-ΔN29, E. coli / pET-21a-ΔN61, E. coli / pET-21a-ΔN53 / M196I, E. coli / pET-21a-ΔN53 / I258M, E. coli / pET-21a-ΔN53 / K262T, E. coli / pET-21a-ΔN53 / I258M / K262T, E. coli / pET-21a-ΔN53 / M196I / I258M / K262T.

[0084] Meanwhile, prepare a recombinant bacterium containing the wild-type enzyme: E. coli / pET-21a-7α-HSDH according to the above method.

[0085] Example 3: Induced expression culture of recombinant bacteria

[0086] The specific steps are as follows:

[0087] (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.

[0088] (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.

[0089] (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.

[0090] Example 4: Purification of Recombinant Protein

[0091] Purify the recombinant protein using a His-Trap HP affinity column:

[0092] (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 of 3 s, working time of 10 min). Centrifuge at 12,000 rpm for 30 min at 4 °C to collect the supernatant as the crude enzyme solution.

[0093] (2) Purify the above crude enzyme solution using His-Trap affinity chromatography produced by Cytiva. After ultrafiltration and desalting of the pure enzyme solution, it is used for enzyme activity determination.

[0094] Pure enzyme solutions containing wild-type enzyme WT, ΔN16, ΔN29, ΔN53, ΔN61, ΔN53 / M196I, ΔN53 / I258M, ΔN53 / K262T, ΔN53 / I258M / K262T, and ΔN53 / M196I / I258M / K262T are respectively prepared, and the SDS-PAGE analysis diagrams of the proteins are as Figures 1-2 shown.

[0095] The results showed that the purified wild type exhibited three main bands, ΔN16 showed two close bands, and ΔN29, ΔN53, and the truncated combined mutants all showed a single band( Figure 1 and Figure 2 ). It indicates that the disordered N-terminal structure of the full-length protein may interfere with the correct folding of Bm7α-HSDH, thus affecting protein expression

[0096] Example 5: Specific enzyme activity determination

[0097] 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.

[0098] Table 2 Comparison of specific enzyme activities between wild type and mutants

[0099]

[0100] Note: -- indicates not detected; Naming method of mutants in the present invention: The mutants are represented by "amino acid at the original amino acid position replaced by amino acid". For example, I258A means that the amino acid at position 258 is replaced from isoleucine Ile of the parental 7α-HSDH to alanine Ala, and the number of the position corresponds to the corresponding site of the amino acid sequence of the parental 7α-HSDH.

[0101] The results showed that, as shown in Table 2, among the truncated mutants, the specific enzyme activity of the single mutant ΔN53 was significantly increased relative to the wild type, and the specific enzyme activities of other mutants showed different degrees of decrease.

[0102] The dominant truncated mutants were combined with single-point mutants, and finally ΔN53 / M196I, ΔN53 / I258M, ΔN53 / K262T, ΔN53 / I258M / K262T, ΔN53 / M196I / I258M / K262T with significantly increased specific enzyme activities were screened. The results indicate that the combination of truncation of the N-terminal flexible region and mutations at positions 196, 258, and 262 reshaped the substrate binding channel and improved the binding and catalysis of the enzyme to chenodeoxycholic acid.

[0103] Example 6: Determination of kinetic parameters

[0104] Determination of kinetic parameters: The total reaction volume was 200 μL, 0.1 M Na2CO3-NaHCO3 buffer (pH 9.5) was added, 5.0 mM NAD + , 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 to 5.0 mM). The k cat , K m , k cat / K of the 7α-HSDH wild type and mutants were calculatedm (Table 3).

[0105] Table 3 Kinetic parameters of wild type and mutants

[0106]

[0107] a n.d. represents no activity detected;

[0108] As shown in Table 3, compared with the wild type, the k cat / K m values of ΔN53, ΔN53 / M196I, ΔN53 / I258M, ΔN53 / K262T, ΔN53 / I258M / K262T and ΔN53 / M196I / I258M / K262T for the substrate showed a significant increase, significantly improving the catalytic efficiency of chenodeoxycholic acid for synthesizing 7-oxolithocholic acid.

[0109] Example 7: CD determination

[0110] By characterizing the thermal stability of the mutant enzyme and wild-type enzyme obtained in Example 4, mutants with improved thermal stability and catalytic efficiency were obtained, which are excellent enzymes for industrial transformation and synthesis of 7-oxolithocholic acid.

[0111] Circular dichroism (CD) measurements were performed using a Jasco J720 spectropolarimeter (Jasco, Inc., Easton, MD). The T m values of different proteins were calculated (Table 4).

[0112] Table 4: Thermal stability analysis of wild type and dominant mutants of 7α-HSDH

[0113]

[0114] As shown in the table, the T m values of ΔN53, ΔN53 / M196I, ΔN53 / I258M, ΔN53 / K262T, ΔN53 / I258M / K262T and ΔN53 / M196I / I258M / K262T were all improved compared with the wild type. Combining with Example 6, ΔN53 / M196I / I258M / K262T showed the greatest improvement in both thermal stability and catalytic efficiency.

[0115] Example 8: Catalytic synthesis of 7-oxolithocholic acid by 7α-HSDH

[0116] The specific steps are as follows:

[0117] The whole-cell reaction system was carried out in a 500 mL bioreactor. Ethanol dehydrogenase from Saccharomyces cerevisiae (S.cerevisiae ADH) was introduced into the reaction to construct a coenzyme recycling system.

[0118] (1) Construction of recombinant strain:

[0119] Ethanol dehydrogenase from Saccharomyces cerevisiae (UniProtKB entry P00330) was selected, codon-optimized and chemically synthesized by Ascentage Biotechnology Co., Ltd. (Suzhou, China), cloned into the expression plasmid pET21a, and transformed into E. coli BL21(DE3) competent cells to obtain the recombinant strain E. coli / pET-21a-ADH.

[0120] The amino acid sequence of the ethanol dehydrogenase is shown in SEQ ID NO.4, and the nucleotide sequence encoding the ethanol dehydrogenase is shown in SEQ ID NO.3.

[0121] (2) The reaction system included 0.1 M Na2CO3-NaHCO3 buffer (pH 9.5), 50 mM chenodeoxycholic acid, 0.5 mM NAD + , 5 mL acetaldehyde solution, 10 g·L -1 of E. coli / pET-21a-ΔN53 / M196I / I258M / K262T recombinant bacteria, 10 g·L -1 of E. coli / pET-21a-ADH recombinant bacteria. The reaction was stirred at 30 °C and 800 r·min -1 for 48 hours;

[0122] The addition of NaOH solution (1.0 M) was controlled by an automatic potentiometric titrator to maintain the pH of the reaction solution at 9.5. Samples were taken intermittently within 12 hours to detect the reaction conversion rate. The reaction was terminated by adjusting the pH to 10–11 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 suction filtration, the solvent was removed, and dried to a constant weight, and detected by HPLC. The liquid phase column was a Waters XBridge C18 reverse chromatographic column (5 μm, 4.6×250 mm), the detection wavelength was 195 nm, the flow rate was 1 mL min -1 , the column temperature was 30 °C, and the mobile phase ratio (v:v): acetonitrile: 0.1% phosphoric acid aqueous solution = 60:40.

[0123] Meanwhile, the recombinant bacteria E. coli / pET-21a-7α-HSDH containing the wild type was used as a control, and the reaction was carried out according to the above steps.

[0124] The results showed that:

[0125] By measuring the catalytic synthesis of 7-oxolithocholic acid by the wild-type enzyme 7α-HSDH and the combined mutant ΔN53 / M196I / I258M / K262T, it was found that in a 100 mL whole-cell reaction system ( Figure 3 ).

[0126] Using a bioreactor to catalyze 20 mM substrate chenodeoxycholic acid respectively, the yield of the product 7-oxolithocholic acid synthesized by the wild-type strain reached 99.0% after 48 h, and the combined mutant strain reached the maximum yield of 99.7% at 4 h.

[0127] Using a bioreactor to catalyze 50 mM substrate chenodeoxycholic acid respectively, the yield of the product 7-oxolithocholic acid synthesized by the wild-type strain was 84.4% after 48 h, and the combined mutant strain reached the maximum yield of 97.1% at 4 h.

[0128] Therefore, the best combined mutation ΔN53 / M196I / I258M / K262T significantly improved the stability of the enzyme and enhanced its catalytic function, shortening the conversion time from 48 h to 4 h.

[0129] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications 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. 7α-Hydroxysteroid dehydrogenase mutant, characterized in that, Any one of the following (a) to (f): (a) Obtained by truncating the amino acids at positions 1 to 53 at the N-terminus of 7α-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 2; (b) On the basis of (a), simultaneously mutating the methionine at position 196 to isoleucine; (c) On the basis of (a), simultaneously mutating the isoleucine at position 258 to methionine; (d) On the basis of (a), simultaneously mutating the lysine at position 262 to threonine; (e) On the basis of (a), simultaneously mutating the isoleucine at position 258 to methionine and mutating the lysine at position 262 to threonine; (f) On the basis of (a), simultaneously mutating the methionine at position 196 to isoleucine, mutating the isoleucine at position 258 to methionine, and mutating the lysine at position 262 to threonine.

2. A gene encoding the 7α-hydroxysteroid dehydrogenase mutant according to claim 1.

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

4. The recombinant vector according to claim 3, wherein The recombinant vector uses pET-21a or pET-28a as the expression vector.

5. A recombinant cell expressing the 7α-hydroxysteroid dehydrogenase 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 containing the 7α-hydroxysteroid dehydrogenase mutant or its lyophilized powder 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 7α-hydroxysteroid dehydrogenase 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 sodium carbonate-sodium bicarbonate buffer with a pH of 9.5; the concentration of the substrate chenodeoxycholic acid is 1 to 50 mM, and the reaction temperature is 20 to 40 °C.

10. Use of the 7α-hydroxysteroid dehydrogenase 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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