7beta-hydroxysteroid dehydrogenase mutant and application of 7beta-hydroxysteroid dehydrogenase mutant in synthesis of ursodesoxycholic acid by biological enzyme method

Abstract

The invention provides a 7beta-hydroxysteroid dehydrogenase mutant and application thereof in synthesis of ursodesoxycholic acid by a biological enzyme method, through a genetic engineering technology and computer-aided design, phosphoric acid coordination residues of protein are modified, the 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) mutant with higher activity under an NADH (Nicotinamide Adenine Dinucleotide Hydroxide) system is constructed, and the 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) mutant can be used for synthesizing ursodesoxycholic acid. The modification strategy is suitable for other proteins for modifying cofactor preference and improving enzyme activity, and has remarkable industrial value when being used for efficiently catalyzing CDCA to be converted into UDCA through a one-pot one-step method.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme modification, and particularly relates to a 7β-hydroxysteroid dehydrogenase mutant and its application in the bioenzymatic synthesis of ursodeoxycholic acid. Background Art

[0002] Ursodeoxycholic acid (UDCA) is an endogenous bile acid derivative that exerts its therapeutic effects by regulating bile acid metabolism and improving bile composition. It has low toxicity and a high safety profile and is commonly used to prevent and treat diseases caused by high cholesterol, such as atherosclerosis, coronary heart disease, cholecystitis, and cholelithiasis. Therefore, UDCA plays a key role in lipid-lowering and choleretic treatments, and has broad market prospects.

[0003] Currently, ursodeoxycholic acid is primarily synthesized industrially using chemical methods, using chenodeoxycholic acid or cholic acid as raw materials through multiple oxidation and reduction reactions. However, this method is characterized by difficult chirality control, harsh reaction conditions, high energy consumption, low yield, and severe environmental pollution, making large-scale production unfeasible. In contrast, the enzymatic method offers advantages such as environmental friendliness, short cycle times, continuous production, and mild reaction conditions.

[0004] The bioenzymatic synthesis of ursodeoxycholic acid is based on chenodeoxycholic acid (CDCA) as a substrate and is catalyzed by two enzyme cascades: 1) CDCA binds to the oxidized coenzyme NAD(P) under the action of 7α-hydroxysteroid dehydrogenase (7α-HSDH) + Produces 7-ketolithocholic acid (7K-LCA); 2) 7K-LCA is reduced to UDCA under the action of 7β-HSDH combined with reduced coenzyme NAD(P)H.

[0005] Research on 7α-HSDH is relatively mature, with good activity and stability, making it a non-limiting factor in the one-step enzymatic synthesis of UDCA. However, there are few reports on 7β-HSDH, and the natural enzyme has low catalytic efficiency, requiring improvements in its activity and stability in the NAD(P)H system.

[0006] Therefore, it is very necessary to develop 7β-HSDH mutants that can efficiently catalyze the conversion of 7K-LCA to ursodeoxycholic acid. Summary of the Invention

[0007] The purpose of the present invention is to propose a 7β-hydroxysteroid dehydrogenase mutant suitable for carrying out a 7-position carbonyl reduction reaction using lithocholic acid-related compounds containing a 7-position carbonyl structure as substrates, so as to improve the efficiency of the 7-position carbonyl reduction reaction and promote the industrialization process of efficiently catalyzing the conversion of 7K-LCA to ursodeoxycholic acid.

[0008] The first aspect of the present invention is to provide a 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant having an amino acid sequence as shown in SEQ ID NO: 5.

[0009] The second aspect of the present invention is to provide a gene encoding the 7β-hydroxysteroid dehydrogenase mutant described in the first aspect.

[0010] The third aspect of the present invention is to provide an expression vector containing the gene described in the second aspect.

[0011] The fourth aspect of the present invention is to provide a strain containing the expression vector described in the third aspect.

[0012] The fifth aspect of the present invention is to propose the use of 7β-hydroxysteroid dehydrogenase in the reduction reaction of 7-carbonyl-lithocholic acid, and the amino acid sequence of the enzyme mutant is shown in SEQ ID NO:5.

[0013] It can be understood that the reduction reaction is applicable to lithocholic acid-related compounds containing a 7-carbonyl structure as a substrate for a 7-carbonyl reduction reaction to obtain a 7β-hydroxyl group. Taking 7-ketolithocholic acid (7K-LCA) as an example, ursodeoxycholic acid is generated under the catalysis of the above-mentioned 7β-hydroxysteroid dehydrogenase.

[0014] The sixth aspect of the present invention is to provide a method for preparing ursodeoxycholic acid, which is obtained by catalytic reduction of 7-ketolithocholic acid (7K-LCA) as a substrate using the 7β-hydroxysteroid dehydrogenase mutant described in the first aspect.

[0015] Furthermore, 7-ketolithocholic acid (7K-LCA) can be a commercially available intermediate or can be prepared in-house. The preparation of 7-ketolithocholic acid can be carried out by catalyzing chenodeoxycholic acid with 7α-hydroxysteroid dehydrogenase (7α-HSDH); more preferably, chenodeoxycholic acid can be directly used as a substrate, and 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) can be added in a one-pot method to directly catalyze the reaction to obtain ursodeoxycholic acid.

[0016] Furthermore, the amino acid sequence of the 7α-hydroxysteroid dehydrogenase is shown in SEQ ID NO: 1, which is derived from 根瘤菌目细菌 .

[0017] Furthermore, the catalytic reduction process is carried out under the synergistic action of the NAD(P) / NAD(P)H coenzyme cycle system; specifically, in the process of preparing ursodeoxycholic acid in one pot using chenodeoxycholic acid as a substrate, isopropanol and NAD + As a coenzyme cycle.

[0018] Furthermore, in the one-pot process for preparing ursodeoxycholic acid, under the conditions of a reaction temperature of 25-45°C, a pH value of 6.5-8.5, and a concentration of the substrate chenodeoxycholic acid of 1-60 g / L, the conversion rate is above 90%.

[0019] Preferably, a one-pot one-step overnight reaction is carried out at 30° C. and pH 8.0 in PBS buffer, and the conversion rate of 82.6 U / mL of 7α-hydroxysteroid dehydrogenase crude enzyme solution and 47.2 U / mL of 7β-hydroxysteroid dehydrogenase mutant crude enzyme solution to 40 g / L substrate CDCA is greater than 99%.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses genetic engineering technology and computer-aided design to modify the phosphate coordination residues of the protein and construct a 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant with higher activity under the NADH system. The mutant is suitable for the 7-position carbonyl reduction reaction using lithocholic acid-related compounds containing a 7-position carbonyl structure as substrates, and is used to efficiently catalyze the conversion of 7K-LCA to UDCA, which has significant industrial value.

[0021] The present invention uses a 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant in combination with 7α-hydroxysteroid dehydrogenase (7α-HSDH) to enzymatically catalyze the production of ursodeoxycholic acid in a one-pot, one-step manner using chenodeoxycholic acid as a substrate. The reaction conditions are mild, the reaction speed is fast, the efficiency is high, the product is environmentally friendly, and the cost is low, making it suitable for industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Schematic diagram of the reaction process for preparing ursodeoxycholic acid in Example 5 of the present invention. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0025] In the present invention, the term "An" means that the amino acid A at position n is changed into multiple amino acids, i.e., a single-point saturation mutation; "AnB" means that the amino acid A at position n is changed into amino acid B, such as Q108V means that the amino acid Q at position 108 is mutated into amino acid V, and so on.

[0026] Example 1 Rb 7α、 Co 7β complete gene synthesis

[0027] Obtained from NCBI database 根瘤菌目细菌 7α-Hydroxysteroid dehydrogenase 7α-HSDH (GenBank No.: MBN8535232.1) 放线杆菌纲 7β-Hydroxysteroid Dehydrogenase 7β-HSDH (GenBank No. WP_331880262.1) from Rb 7α、 Co 7β connected to the Nde I and Xho I double-digested pET26b expression vector to obtain pET26b- Rb 7α, pET26b- Co 7β recombinant expression vector. Rb The amino acid sequence of 7α is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2; Co The amino acid sequence of 7β is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 4.

[0028] The above recombinant expression vector is transformed into a suitable microbial host. The host microorganism is any of the conventional host microorganisms in the art, as long as the above recombinant expression vector can stably replicate and the genes of 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase can be effectively expressed. In this embodiment, the above recombinant expression plasmid is introduced into the host cell by electroporation. 大肠杆菌 In the BL21 (DE3) competent state, the strain was inverted and cultured on a kanamycin-resistant LB solid plate for 12-16 h. Positive transformants were selected for DNA sequencing verification. The correct transformants were genetically engineered strains for 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase.

[0029] Example 2 Obtaining 7β-hydroxysteroid dehydrogenase or its mutant

[0030] 1. Co Construction of a 7β single-site saturation mutant library

[0031] Based on the molecular docking and rational analysis of wild-type 7β-hydroxysteroid dehydrogenase, a total of 4 sites were designed according to the phosphate coordination residues at the cofactor binding position under the NADH system. 放线杆菌纲 Four sites in the amino acid sequence of 7β-hydroxysteroid dehydrogenase (GenBank No.: WP_331880262.1) were modified, namely positions 17, 39, 40, and 41. A single-site saturation mutant library was constructed for each site, as shown in Table 1.

[0032] Table 1. 7β-Hydroxysteroid Dehydrogenase Co Single-site saturation mutant library constructed by 7β

[0033] Note: Table 1 contains primers for multiple single-stranded DNAs, including primers T17-F, G39-F, R40-F, and R41-F. The single-stranded DNAs were mixed in the order of NDT:VMA:ATG:TGG in the table at a molar ratio of 12:6:1:1. The degenerate bases N represent A, T, G, or C, D represent G, A, or T, V represent G, A, or C, and M represent A or C.

[0034] To obtain 7β-hydroxysteroid dehydrogenase Co 7β mutant, perform the following experiments: PCR amplification reaction system (50 μL): KOD Mix (2×) 25 μL, template Co7β 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL. The specific sequences of forward and reverse primers are shown in Table 1.

[0035] PCR amplification reaction conditions: 98°C: 2 min, (98°C: 10 s, 55°C: 15 s, 72°C: 4 min) 30 cycles, 72°C: 4 min.

[0036] The obtained PCR product was subjected to the following operation: 1 μL of Dpn I enzyme was used to digest the plasmid template and treated at 37 °C for 2 h. 5 μL of the digested PCR product was electroporated into 100 μL 大肠杆菌 In BL21 (DE3) competent cells, the electroporated 大肠杆菌 The BL21 (DE3) bacterial solution was evenly spread on a kanamycin-resistant LB plate (concentration of 50 μg / mL) and cultured at 37°C for 14 hours to produce a single colony, which was 7β-hydroxysteroid dehydrogenase. Co7β gene mutant engineered strain. The strain contains a recombinant plasmid with an undirected or directed mutation of the target nucleotide, which is the strain that expresses 7β-hydroxysteroid dehydrogenase. Co Expression vector for 7β gene mutant.

[0037] 2. Co Screening of 7β single-site saturation mutant library

[0038] After successful sequencing of the single-point saturation mutant library, 5 μL of the digested PCR product was electroporated to 大肠杆菌 BL21 (DE3) competent cells were plated on Kan resistant plates. Co Using the wild-type 7β as a control, sterilely selected positive and negative control monoclonal clones were transferred to a 96-well deep-well culture plate containing 400 µL LB medium containing 50 µg / mL kanamycin. After sealing, the plate was shaken at 800 rpm at 37°C for 12 h. 700 µL TB medium was added to the 96-well deep-well culture plate, along with IPTG and kanamycin to a final concentration of 0.5 mM and 50 µg / mL, respectively. The plate was shaken at 800 rpm at 30°C for 15 h for protein expression. The expressed cells were collected by centrifugation at 4000 rpm for 10 min at 4°C, and the mixed cells were resuspended in 500 µL of 50 mM potassium phosphate buffer, pH 7.0. 20 µL of the culture solution was then taken to measure the OD value. 600 The pellet was then centrifuged and the precipitate was collected. The pellet was mixed with 300 µL of 3 mg / mL lysozyme (dissolved in pH 7.0 PBS) by vortexing. After shaking at 37°C for 1.5 h, the cells were placed in a -80°C freezer and frozen and thawed twice every 30 min to disrupt the cells. The supernatant was then centrifuged and the enzyme activity was determined.

[0039] Enzyme activity assay: Prepare a 200 µL reaction system consisting of the following components: 170 µL PBS (100 mM, pH = 8.0), 10 µL 7K-LCA solution (10 mM, dissolved in MeOH), 10 µL NADH (8 mM) and Co 7β mutant crude enzyme solution (1 mg / mL). Under 30°C conditions, measure the change in absorbance at 340 nm within 3 minutes. Use Excel to create a standard curve and calculate y=kx+b, where y is the absorbance value A340 and x is the time (min). Calculate the slope k of the absorbance change within 3 minutes to measure Co 7β mutant enzyme activity. Single clones with enzyme activity higher than that of the wild type were selected and sequenced for identification. The enzyme activity was determined according to the enzyme activity calculation formula in Example 2. Co 7β mutant enzyme activity.

[0040] Obtained through preliminary screeningCo 7β-T17E, Co 7β-T17A, Co 7β-G39R, Co 7β-G39D, Co 7β-G39Q, Co 7β-R40H, Co 7β-R40T, Co 7β-R41N, a total of 8 mutants with relatively high activities, all of which have improved 7K-LCA substrate conversion rates compared with the wild type. The results are shown in Table 2.

[0041] Table 2: Ursodeoxycholic acid production ratio of recombinant engineering strains

[0042] As shown in Table 2, the above method is used to determine the activity of 7β-hydroxysteroid dehydrogenase. Co By mutating a specific site of 7β, the resulting mutant enzyme activity was significantly improved, and the yield of ursodeoxycholic acid catalyzed by it was significantly increased, which was 1.6-3.9 times that of the starting strain.

[0043] Example 3 7β-Hydroxysteroid Dehydrogenase Co Construction of 7β combination saturation mutants or acquisition of their mutants

[0044] 1. Co Construction of 7β combinatorial mutant library

[0045] Eight mutants, T17E, T17A, G39R, G39D, G39Q, R40H, R40T, and R41N, screened from four single-point saturation mutant libraries were subjected to combined mutagenesis. The primer designs are shown in Table 3 below.

[0046] Table 3, based on 7β-hydroxysteroid dehydrogenase Co Combinatorial saturation mutant library constructed by 7β

[0047] Note: In Table 3, there are multiple single-stranded DNA primers, the degenerate base M represents A or C, and R represents A or G.

[0048] To obtain 7β-hydroxysteroid dehydrogenase Co 7β mutant, perform the following experiments: PCR amplification reaction system (50 μL): KOD Mix (2×) 25 μL, template Co 7β 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL. The specific sequences of forward and reverse primers are shown in Table 1.

[0049] PCR amplification reaction conditions: 98°C: 2 min, (98°C: 10 s, 55°C: 15 s, 72°C: 4 min) 30 cycles, 72°C: 4 min.

[0050] Overlap extension PCR process: In the first round, R-1 and R-2 were mixed in a ratio of 1:2 to form downstream primers R, F, and R to amplify a fragment (about 114 bp). In the second round, the fragment was excised and recovered from the gel as a large primer to amplify the entire plasmid using the plasmid as a template.

[0051] The obtained PCR product was subjected to the following operation: 1 μL of Dpn I enzyme was used to digest the plasmid template and treated at 37 °C for 2 h. 5 μL of the digested PCR product was electroporated into 100 μL 大肠杆菌 In BL21 (DE3) competent cells, the electroporated 大肠杆菌 The BL21 (DE3) bacterial solution was evenly spread on a kanamycin-resistant LB plate (concentration of 50 μg / mL) and cultured at 37°C for 14 hours to produce a single colony, which was 7β-hydroxysteroid dehydrogenase. Co 7β gene mutant engineered strain. The strain contains a recombinant plasmid with an undirected or directed mutation of the target nucleotide, which is 7β-hydroxysteroid dehydrogenase. Co Expression vector for 7β gene mutant.

[0052] 2 Co Screening of 7β combinatorial saturation mutant library

[0053] After successful sequencing of the combined saturation mutant library, 5 μL of the digested PCR product was electroporated into 大肠杆菌 BL21 (DE3) competent cells were plated on Kan resistant plates. CoWild-type 7β served as a control. A single positive and negative control colony was aseptically picked and transferred to a 96-well deep-well plate containing 400 µL of LB medium containing 50 µg / mL kanamycin. After sealing, the plate was shaken at 800 rpm at 37°C for 12 hours. To the 96-well plate, 700 µL of TB medium was added, along with IPTG and kanamycin to final concentrations of 0.5 mM and 50 µg / mL, respectively. Protein expression was then performed by shaking at 800 rpm at 30°C for 15 hours. The expressed cells were harvested by centrifugation at 4000 rpm for 10 minutes at 4°C. The mixture was resuspended in 500 µL of 50 mM potassium phosphate buffer (pH 7.0). 20 µL of the culture suspension was then collected for OD600 measurement and the pellet was then centrifuged. The pellet was mixed thoroughly with 300 µL of 3 mg / mL lysozyme (dissolved in pH 7.0 PBS) and shaken at 37°C for 1.5 h. The cells were then frozen and thawed twice every 30 min at -80°C to disrupt the cells. The supernatant was then centrifuged and the enzyme activity was determined.

[0054] Enzyme activity assay: Prepare a 200 µL reaction system consisting of the following components: 170 µL PBS (100 mM, pH = 8.0), 10 µL 7K-LCA solution (10 mM, dissolved in MeOH), 10 µL NADH (8 mM) and Co 7β mutant crude enzyme solution (1 mg / mL). Under 30°C conditions, measure the change in absorbance at 340 nm within 3 minutes. Use Excel to create a standard curve and calculate y=kx+b, where y is the absorbance value A340 and x is the time (min). Calculate the slope k of the absorbance change within 3 minutes to measure Co 7β mutant enzyme activity. Single clones with enzyme activity higher than that of the wild type were selected and sequenced for identification. The enzyme activity was determined according to the enzyme activity calculation formula in Example 2. Co 7β mutant enzyme activity.

[0055] Obtained by filtering Co 7β-T17E, Co 7β-T17A, Co 7β-G39R, Co 7β-G39D, Co 7β-G39Q, Co 7β-R40H, Co 7β-R40T, Co 7β-R41N, a total of 7 mutants with relatively high activities, all of which have improved 7K-LCA substrate conversion rates compared with the wild type. The results are shown in Table 4.

[0056] Table 4: Ursodeoxycholic acid production ratio of recombinant engineering strains

[0057] In summary, based on the above Co 7β was subjected to combined saturation mutagenesis, and 7 multi-site combined mutants were obtained, whose enzyme activity was significantly improved and the production of ursodeoxycholic acid was significantly increased, which was 7.4-17.2 times that of the starting strain.

[0058] Co Amino acid sequence of 7β_7 (SEQ ID NO: 5) for: MNLREKYGEWGLILGAAEGVGKAFCEKIAAGGMNVVMVDRNEEKLNVLAGEIRETYGVETKVVRADFSQPGAAETVFAATEGLDMGFMSYVACLHSFGKIQDTPWEKHEAMINVNVVTFLKCFHHYMR IFATQDRGAVINVSSMTGISSSPWNGQYGAGKAFILKMTEAVACECEGTGVDVEVITLGTTLTPSLLSNLPGGPQGEAVMKIALTPEECVDEAFEKLGKELSVIAGQRNKDSVHDWKANHTEDEYIRYMGSFYRD

[0059] CoThe nucleotide sequence of 7β_7 (SEQ ID NO: 6) is: atgaacctgcgcgaaaaatatggcgaatggggcctgattctgggcgcggcggaaggcgtgggcaaagcgttttgcgaaaaaattgcggcgggcggcatgaacgtggtgatggtggatcgcaatgaagaaaaactgaacgtgctggcgggcgaaattcgcgaaacctatggcgtggaaaccaaagtggtgcgcgcggattttagccagccgggcgcggcggaaaccgtgtttgcggcgaccgaaggcctggatatgggctttatgagctatgtggcgtgcctgcatagctttggcaaaattcaggataccccgtgggaaaaacatgaagcgatgattaacgtgaacgtggtgacctttctgaaatgctttcatcattatatgcgcatttttgcgacccaggatcgcggcgcggtgattaacgtgagcagcatgaccggcattagcagcagcccgtggaacggccagtatggcgcgggcaaagcgtttattctgaaaatgaccgaagcggtggcgtgcgaatgcgaaggcaccggcgtggatgtggaagtgattaccctgggcaccaccctgaccccgagcctgctgagcaacctgccgggcggcccgcagggcgaagcggtgatgaaaattgcgctgaccccggaagaatgcgtggatgaagcgtttgaaaaactgggcaaagaactgagcgtgattgcgggccagcgcaacaaagatagcgtgcatgattggaaagcgaaccataccgaagatgaatatattcgctatatgggcagcttttatcgcgat

[0060] Example 4 7α-Hydroxysteroid Dehydrogenase Rb Determination of 7α Enzyme Activity

[0061] The recombinant strain constructed in Example 1 Rb7α was streaked onto LB plates (containing 50 µg / mL kanamycin), and a single colony was picked and transferred to 5 mL of LB liquid medium containing 50 µg / mL kanamycin. The culture was incubated at 37°C and 220 rpm for 12 h. The culture was then inoculated into TB liquid medium containing 50 µg / mL kanamycin at a 1% inoculum size and cultured at 37°C until the OD600 reached 0.6-0.8. IPTG was then added to a final concentration of 0.5 mmol / L and induced for 16 h at 20°C and 220 rpm. The culture was centrifuged at 8000 rpm at 4°C for 5 min, and the bacterial pellet (i.e., whole cells) was collected and resuspended in PBS buffer (100 mmol / L, pH 8.0). The bacterial cells were disrupted by high pressure (4°C) to obtain a whole-cell lysate (i.e., crude enzyme solution).

[0062] Enzyme activity assay: Prepare a 200 µL reaction system consisting of the following components: 170 µL PBS (100 mM, pH = 8.0), 10 µL CDCA solution (10 mM, dissolved in MeOH), 10 µL NADPH + (8 mM) and Rb 7α crude enzyme solution (1 mg / mL). Under 30°C conditions, measure the change in absorbance at 340 nm within 3 minutes. Use Excel to make a standard curve and calculate y=kx+b, where y is the absorbance value A340 and x is the time (min). Calculate the slope k of the absorbance change within 3 minutes to measure Rb 7α enzyme activity was determined according to the enzyme activity calculation formula in Example 2 Rb The 7α enzyme activity was 32.864 U / mg.

[0063] Example 5 Rb 7α、 Co One-step enzymatic synthesis of ursodeoxycholic acid by 7β mutant

[0064] like Figure 1 As shown, this embodiment provides a method for using chenodeoxycholic acid as a substrate. Rb 7α and Co The 7β mutant is enzymatically synthesized into ursodeoxycholic acid in one step. The specific steps are as follows: Add 6 g of substrate chenodeoxycholic acid and 1 g of sodium hydroxide to a 500 mL reaction bottle, dissolve it in 100 mM pH 8.0 PBS buffer to 150 mL, place it in a 30 °C water bath, stir until fully dissolved, and then add 82.6 U / mL of Rb 7α crude enzyme solution, 47.2 U / mL Co 7β_7 mutant crude enzyme solution, 10% isopropanol and 0.05 g / mL coenzyme NAD +The reaction was stirred for 16 hours using a magnetic stirrer. After the reaction, 500 μL of the reaction solution was extracted with 500 μL of ethyl acetate, vortexed, and centrifuged at 12,000 rpm / min for 1 minute. The upper organic layer was analyzed by TLC, and a sample was taken for HPLC analysis.

[0065] The TLC developing solvent ratio was dichloromethane:acetone:petroleum ether = 1:1:2, with the addition of 3 drops of glacial acetic acid. The liquid chromatography detection conditions were: LC column: ZORBAX Eclipse Plus C18 Analytical (4.6 mm × 250 mm, 5 µm), flow rate: 0.8 mL / min at 40°C. The injection volume was 50 µL, and the mobile phase was acetonitrile: 0.78 g / L sodium dihydrogen phosphate (pH = 3) aqueous solution: methanol = 3:3:4, with equal ratios for elution over 60 min. The liquid chromatography results showed that at a substrate concentration of 40 g / L and a conversion time of 16 hours, Rb 7α、 Co The conversion rate of 7β_7 in one-pot reaction was 99.1%.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A 7β-hydroxysteroid dehydrogenase mutant having an amino acid sequence as shown in SEQ ID NO:

5.

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

3. An expression vector, characterized in that Containing the gene according to claim 2.

4. A strain characterized in that Contains the expression vector according to claim 3.

5. Use of a 7β-hydroxysteroid dehydrogenase mutant in the reduction reaction of 7-carbonyl-lithocholic acid, characterized in that: The amino acid sequence of the 7β-hydroxysteroid dehydrogenase is shown in SEQ ID NO:

5.

6. A method for preparing ursodeoxycholic acid, characterized in that: The product is obtained by catalytic reduction of 7β-hydroxysteroid dehydrogenase mutant according to claim 1 with 7-ketolithocholic acid as substrate.

7. The preparation method according to claim 6, characterized in that The 7-ketolithocholic acid is obtained from chenodeoxycholic acid via 7α-hydroxysteroid dehydrogenase catalysis.

8. The preparation method according to claim 7, characterized in that The amino acid sequence of the 7α-hydroxysteroid dehydrogenase is shown in SEQ ID NO:

1.

9. The preparation method according to claim 7, characterized in that The catalytic reduction process is carried out under the synergistic action of the NAD(P) / NAD(P)H coenzyme circulation system.

10. The preparation method according to claim 7, characterized in that The concentration of chenodeoxycholic acid is 1-60 g / L.

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