Rational modification of 7β-hydroxysteroid dehydrogenase coenzyme-dependent efficient synthesis of ursodeoxycholic acid

Abstract

The present invention discloses a rational modification of 7β-hydroxysteroid dehydrogenase coenzyme-dependent efficient synthesis of ursodeoxycholic acid, belonging to the field of biocatalytic engineering. The present invention obtains Hs7β-HSDH mutant A22T / Q23E / L45A / N46E with improved enzyme activity and improved affinity for cofactor NADH, the enzyme activity is increased by 65.2 times, the catalytic efficiency is increased by 44.9 times, and the yield of UDCA catalyzed by the mutant can reach 77.6% with 7K-LCA as substrate and NADH as coenzyme, which is 13.9 times higher than that of the wild-type enzyme. After coupling with Bm7α-HSDH, only 0.5mM NADH needs to be added to catalyze the one-pot synthesis of UDCA by substrate CDCA, and the yield of the mutant reaches 90.1%, which is 11.5 times higher than that of the wild-type enzyme.

Description

Technical Field

[0001] The invention relates to rationally transforming 7β-hydroxysteroid dehydrogenase coenzyme-dependent efficient synthesis of ursodeoxycholic acid, and belongs to the field of biocatalytic engineering. Background Art

[0002] Ursodeoxycholic acid (UDCA), also known as 3α,7β-dihydroxy-5β-cholestane-24-acid, is a hydrophilic bile acid with multiple biological activities. It can promote the excretion of endogenous bile acids, increase the proportion of hydrophilic bile acids, protect hepatocytes and bile duct cells from the toxicity of hydrophilic bile acids, prevent hydrophobic and hydrophilic bile acids from damaging mitochondrial membranes and their cytotoxicity, inhibit hepatocyte apoptosis, significantly enhance liver function, and exhibit immunomodulatory and cytoprotective effects. It is one of the most commonly prescribed drugs in the clinical treatment of cholestatic diseases.

[0003] There are three main synthetic pathways for UDCA: the first pathway involves using primary hydrophilic bile acid as a substrate, and then undergoing a hydroxyl dehydrogenation reaction through chemical oxidation to produce the first intermediate 3α, 7β, 12-oxolithocholic acid, and then under the action of 3α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase (7β-HSDH), the second intermediate 12-ketolithocholic acid is produced, and finally converted into UDCA through the Wolff-Kishner reduction reaction; the second pathway is through 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 12α-hydroxy Sterol dehydrogenase catalyzes the production of 7,12-oxolithocholic acid, and then 7β-HSDH catalyzes 7,12-oxolithocholic acid to produce the second intermediate 12-ketolithocholic acid, and finally converts 12-ketolithocholic acid into UDCA through Wolff-Kishner reduction reaction; the third pathway 7α-HSDH catalyzes chenodeoxycholic acid (CDCA) to produce the intermediate 7-keto-lithocholic acid (7K-LCA), and then 7β-HSDH catalyzes 7K-LCA to produce UDCA (reaction structure shown in the figure), which has become the preferred scheme for UDCA biosynthesis. Regardless of the chosen pathway, 7β-HSDH is the key enzyme in UDCA synthesis.

[0004] Hydroxysteroid dehydrogenases are a class of oxidoreductases that use NAD(H) or NADP(H) as cofactors. They play a key role in bile acid substance conversion and cholesterol metabolism, and show significant potential in industrial biocatalysis. In the reaction pathway of synthesizing UDCA from CDCA, a large number of studies have shown that 7α-HSDH exhibits high catalytic activity and stability, using NADH as a cofactor. However, 7β-HSDH often has lower enzyme activity, poor substrate tolerance and stability, and mainly uses NADPH as a cofactor. Compared with NADPH, NADH exhibits higher economic efficiency and relative stability. Therefore, it is of great significance to obtain highly active NADH-dependent 7β-HSDH through targeted modification, and to achieve efficient and low-cost one-pot synthesis from CDCA to UDCA through dual enzyme coupling of 7α-HSDH and 7β-HSDH, and based on the coenzyme self-cycle of these two enzymes. Summary of the invention

[0005] The present invention uses 7β-hydroxysteroid dehydrogenase (Hs7β-HSDH) from Hyphomicrobium sp. as a template enzyme, and obtains a recombinant Hs7β-HSDH mutant strain with significantly improved NADH affinity and enzyme activity through structural analysis and virtual mutation screening. The cofactor dependence of Hs7β-HSDH is transformed from NADPH to NADH, thereby reducing the cost of the coenzyme. The enzymatic properties of Hs7β-HSDH and its mutants are characterized in detail. At the same time, Bm7α-HSDH is coupled, and only a small amount of NADH is added to start the coenzyme cycle regeneration, thereby realizing the one-pot synthesis of UDCA from CDCA. The present invention lays a solid foundation for the industrialized and efficient preparation of ursodeoxycholic acid.

[0006] The present invention provides an Hs7β-HSDH mutant, wherein the mutant is obtained by mutating at least one of positions 22, 23, 45, and 46 of Hs7β-HSDH; the Hs7β-HSDH is an Hs7β-HSDH having an amino acid sequence as shown in SEQ ID NO.1, or an Hs7β-HSDH having a sequence identity of at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, with the sequence as shown in SEQ ID NO.1.

[0007] In one embodiment of the present invention, the Hs7β-HSDH mutant comprises one or more amino acid substitutions at positions 22 / 23 / 45 / 46, wherein the amino acid at position 22 is substituted with T or D, the amino acid at position 23 is substituted with E or A, the amino acid at position 45 is substituted with A, and the amino acid at position 46 is substituted with E.

[0008] In one embodiment of the present invention, the catalytic efficiency of the Hs7β-HSDH mutant is measured by using 7K-LCA as a substrate and NADH as a cofactor to react, and detecting the change in the absorbance of NADH at 340 nm using an enzyme reader. One unit of enzyme activity (U) is defined as the amount of 1 μmol of NADH oxidized to NAD per minute under the measurement conditions. + The amount of enzyme.

[0009] The present invention provides a Hs7β-HSDH mutant, wherein the Hs7β-HSDH mutant is obtained by mutating the 22nd and / or 23rd and / or 45th and / or 46th position of the Hs7β-HSDH with an amino acid sequence as shown in SEQ ID NO.1.

[0010] The parent enzyme sequence of the Hs7β-HSDH mutant (SEQ ID NO.1) is:

[0011] MSYDAFADFFRMIGHNVIITGGAQNIGAGIARTLSGAGAKVMIADLNGDMAKETAAGIQKDTGNDCRGIKCDVTSLDDINAVVAETVKAFGGISTLVNNVGWGGRHDDPAAVTEEDFIASYKLNAISAYRMSM ACLPHLLKTKNATITNSGSFSASAPAYDILAYGTAKAALNQMMVSLAHMLAKKVRVNSVLIGTVMTAGYADAGIDPEMQERLSHPDNLTGRSGKPEDIANAMLWLCSPASGWVSGQIINVHGGGDVVRLFGE

[0012] In one embodiment of the present invention, the nucleotide sequence encoding the Hs7β-HSDH wild-type enzyme is shown as SEQ ID NO.2.

[0013] SEQ ID NO.2:

[0014] ATGAGCTACGATGCCTTTGCAGATTTCCGTATGATTGGCCACAACGTTATCATCACTGGCGGTGCTCAGAACATTGGTGCGGGTATCGCGCGTACCCTGTCTGGTGCTGGCGCGAAAGTAATGATCGCCGACCTGAACGGCGATATGGCGAAAGAAACCGCGGCGGGCATTCAGA AAGACACGGGTAACGACTGCCGTGGCATCAAATGTGACGTGACGTCCCTGGATGACATCAACGCTGTTGTGGCGGAGACCGTTAAGGCATTCGGTGGCATTTCTACCCTGGTTAACAATGTTGGTTGGGGCGGCCGTCATGATGATCCGGCTGCTGTCACTGAGGAAGACTTCATCGCGTCTTACAAACTGAACGCAATCTCCGCGTACCGCATGAGCATGGCCTGCCTGCCGCATCTGCTGAAAACCAAAAACGCCACCATCACCAACTCCGGTTCCTTTAGCGCTTCCGCACCTGCGTACGACATTCTGGCTTATGGCACCGCCAAAGCTGCTCTGAACCAAATGATGGTCTCTCTGGCTCATATGCTGGCGAAAAAAGTTCGCGTGAACTCCGTGCTGATCGGTACTGTAATGACGGCCGGCTATGCTGATGCAGGCATCGACCCGGAAATGCAAGAACGTCTGAGCCATCCGGACAATCTGACCGGCCGTTCTGGTAAGCCAGAGGATATCGCGAACGCAATGCTGTGGCTGTGTTCCCCGGCGTCTGGCTGGGTTTCCGGCCAGATTATCAACGTTCATGGTGGTGGTGACGTAGTTCGTCTGTTCGGTGAA

[0015] The present invention provides an Hs7β-HSDH mutant, which is a mutant obtained by mutating Hs7β-HSDH with the amino acid sequence shown in SEQ ID NO.1: A22T, A22D, Q23E, Q23A, L45A, N46E, A22D / Q23A / L45A / N46E, or A22T / Q23E / L45A / N46E.

[0016] In one embodiment of the present invention, the mutant is obtained by mutating the alanine at position 22 of 7Hs7β-HSDH as shown in SEQ ID NO.1 to threonine, and is named A22T;

[0017] Or the mutant is obtained by mutating the alanine at position 22 of Hs7β-HSDH shown in the amino acid sequence of SEQ ID NO.1 to aspartic acid, and is named A22D;

[0018] Or the mutant is obtained by mutating the glutamine at position 23 of Hs7β-HSDH as shown in SEQ ID NO.1 to glutamic acid, and is named Q23E;

[0019] Or the mutant is obtained by mutating the glutamine at position 23 of Hs7β-HSDH as shown in SEQ ID NO.1 to alanine, and is named Q23A;

[0020] Or the mutant is obtained by mutating the leucine at position 45 of Hs7β-HSDH shown in the amino acid sequence of SEQ ID NO.1 to alanine, and is named L45A;

[0021] Or the mutant is obtained by mutating the asparagine at position 46 of the Hs7β-HSDH shown in the amino acid sequence of SEQ ID NO.1 to glutamic acid, and is named N46E;

[0022] Or the mutant is obtained by mutating the alanine at position 22 of Hs7β-HSDH as shown in SEQ ID NO.1 to glutamic acid, mutating the glutamine at position 23 to alanine, mutating the leucine at position 45 to alanine, and mutating the asparagine at position 46 to glutamic acid, and is named A22D / Q23A / L45A / N46E;

[0023] Or the mutant is obtained by mutating the alanine at position 22 of Hs7β-HSDH as shown in SEQ ID NO.1 to threonine, glutamine at position 23 to glutamic acid, leucine at position 45 to alanine, and asparagine at position 46 to glutamic acid, and is named A22T / Q23E / L45A / N46E;

[0024] The present invention also provides a gene encoding the mutant.

[0025] The present invention also provides a recombinant vector carrying the mutant or the gene.

[0026] In one embodiment of the present invention, the recombinant vector is an expression vector using pET series vectors such as pET-28a plasmid, pET-21a plasmid, pRSF series vectors such as pRSF-Duet1 plasmid, and pGEX series vectors such as pGEX-6p-1 plasmid.

[0027] In one embodiment of the present invention, the recombinant vector uses pET28a plasmid, pRSF-Duet1 plasmid, pET21a plasmid or pGEX-6P-1 plasmid as an expression vector.

[0028] The present invention also provides a recombinant cell expressing the mutant, carrying the gene, or carrying the recombinant vector.

[0029] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0030] In one embodiment of the present invention, the recombinant cell uses Escherichia coli as an expression host.

[0031] The invention provides a recombinant vector carrying Hs7β-HSDH.

[0032] In one embodiment of the present invention, the recombinant vector uses pET28a, pRSF-Duet1, pET-Duet1, pACY-Duet1, pET21a or pGEX-6P-1 as an expression vector.

[0033] The present invention also provides the enzymatic properties of the engineered Hs7β-HSDH, including optimum temperature, pH, temperature stability, pH stability and kinetic parameters.

[0034] The present invention also provides a recombinase catalyst containing the above-mentioned Hs7β-HSDH mutant sequence, which is any one of the following forms:

[0035] (1) culturing the recombinant expression transformant and isolating transformant cells containing the recombinase;

[0036] (2) culturing the recombinant expression transformant, isolating transformant cells containing the recombinase, and disrupting the transformant cells containing the recombinase to obtain a cell disrupted liquid;

[0037] (3) culturing the recombinant expression transformant, isolating transformant cells containing the recombinant enzyme, disrupting the transformant cells containing the recombinant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant enzyme to obtain a lyophilized enzyme powder.

[0038] The present invention also provides a method for preparing UDCA, wherein CDCA or 7K-LCA is used as a substrate, NAD + Or NADH as a cofactor, the above recombinant cells or the above recombinant enzyme preparations and recombinant cells containing 7α-HSDH are used to jointly catalyze the conversion to prepare UDCA.

[0039] In one embodiment of the present invention, the nucleotide sequence encoding the Bm7α-HSDH is shown as SEQ ID NO.3.

[0040] SEQ ID NO.3:

[0041] ATGAGCTACGAAAGCCCGTTCCATCTGAACGACGCAGTGGCCATTGTTACCGGCGCAGCCGCTGGTATTGGTCGTGCAATTGCCGGTACCTTTGCCAAAGCCGGTGCAAGCGTGGTGGTGACCGATCTGAAAAGCGAAGGCGCAGAAGCAGTGGCAGCCGCAATTCGCCAAGC TGGTGGTAAAGCCATCGGTCTGGAGTGTAACGTGACCGATGAACAGCATCGCGAGGCCGTGATTAAAGCAGCTTTAGATCAGTTCGGCAAGATTACCGTGCTGGTGAACAATGCTGGTGGTGGCGGCCCGAAACCGTTTGACATGCCG ATGAGCGACTTCGAATGGGCCTTTAAGCTGAATTTATTCTCTTTATTTCGTTTAAGCCAGCTGGCAGCCCCGCACATGCAGAAAGCCGGTGGCGGCGCAATTCTGAACATCAGCAGCATGGCTGGTGAGAATACCAACGTGCGCATGG CCAGCTATGGTAGCAGCAAAGCCGCCGTGAACCATTTAACACGTAATATCGCCTTCGATGTGGGTCCGATGGGCATCCGCGTGAATGCAATCGCCCCGGGTGCCATTAAAACCGATGCACTGGCCACCGTGCTGACCCCGGAAATTGA ACGCGCAATGTTAAAACACACACCGCTGGGTCGTCTGGGTGAAGCCCAAGATATTGCAAACGCCGCACTGTTTTTATGTAGCCCCGCTGCAGCATGGATCAGTGGTCAAGTTCTGACCGTGAGCGGTGGTGGTGTTCAAGAACTGGAT

[0042] In one embodiment of the present invention, the reaction system contains 7K-LCA and NADH.

[0043] In one embodiment of the present invention, the concentration of 7K-LCA is at least 5 g / L, preferably, the concentration of 7K-LCA is 5 g / L.

[0044] In one embodiment of the present invention, the concentration of NADH is 12 mM.

[0045] In one embodiment of the present invention, the reaction system contains CDCA, NAD + .

[0046] In one embodiment of the present invention, the concentration of CDCA is at least 5 g / L, preferably, the concentration of CDCA is 5 g / L.

[0047] In one embodiment of the present invention, the NAD + The concentration is 0-1mM, not 0.

[0048] In one embodiment of the present invention, the reaction conditions are pH 7-8 and temperature 30°C.

[0049] The present invention also provides the use of the above Hs7β-HSDH mutant or the above gene or recombinant vector, or the above recombinant cell, or the above recombinant enzyme catalyst, or the above recombinant Escherichia coli, or the above method in preparing UDCA or a product containing UDCA.

[0050] In one embodiment of the present invention, the product is a chemical.

[0051] Beneficial Effects

[0052] (1) The present invention explored Hs7β-HSDH from Hyphomicrobium sp. as a template enzyme, performed structural analysis and virtual mutation screening on it, and obtained Hs7β-HSDH mutants A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E with significantly improved affinity for NADH.

[0053] (2) The present invention studied the enzymatic properties of Hs7β-HSDH and its mutants A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E, determined the optimal temperature, pH and kinetic parameters of the enzyme, and verified that the enzyme activity and catalytic efficiency of the mutants were significantly improved. The enzyme activity of the mutants was increased by 65.2 and 48.3 times, respectively, and the catalytic efficiency (k cat / K m ) were increased by 44.9 and 29.3 times respectively.

[0054] (3) Based on homology modeling, molecular docking analysis, affinity determination of ligand-enzyme complexes and dynamic simulation, the present invention analyzed the molecular mechanism of improved catalytic performance of Hs7β-HSDH mutants A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E, and provided a general technology for improving the cofactor affinity of hydroxysteroid dehydrogenase.

[0055] (4) The present invention transforms the dependence of Hs7β-HSDH on NADPH into dependence on NADH. When reacted with 5 g / L 7K-LCA and 12 mM NADH, the yields of mutants A22D / Q23A / L45A / N46E and A22T / Q23E / L45A / N46E reached 77.6% and 65.3%, respectively, which were 13.9 times and 11.5 times higher than those of the wild-type enzyme, respectively.

[0056] (5) The method of the present invention was used to construct a "one-pot" reaction system, which was coupled with Bm7α-HSDH and only 0.5 mM NADH was added to achieve one-pot synthesis of UDCA from CDCA. The yields of the mutants reached 90.1% and 79.6%, respectively. Compared with the wild-type enzyme, the yields of the mutants A22D / Q23A / L45A / N46E and A22T / Q23E / L45A / N46E increased by 11.5 times and 10.2 times, respectively.

[0057] (6) The "one-pot" reaction system constructed by the present invention uses a whole-cell biocatalytic reaction to complete the conversion of chenodeoxycholic acid to ursodeoxycholic acid in one step, which significantly reduces the amount of cofactors added, does not require the addition of other co-substrates, does not require cumbersome steps, does not generate any unnecessary by-products, has mild reaction conditions, is environmentally friendly, and is a green and efficient method for the biosynthesis of ursodeoxycholic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 :(a) Sequence conservation analysis of Hs7β-HSDH; (b) Multiple sequence alignment and secondary structure analysis of Hs7β-HSDH.

[0059] Figure 2 :(a) Key amino acid residues of Hs7β-HSDH in the NADPH binding pocket; (b) Key amino acid residues of Hs7β-HSDH in the NADH binding pocket; (c) Superposition of key residues of Hs7β-HSDH and two dominant mutants; (d) Substrate and cofactor channels of Hs7β-HSDH.

[0060] Figure 3 :(a) Energy change when the amino acid near NADH in the binding pocket mutates to alanine;(b) Energy change analysis of virtual saturation mutations at potential key amino acid residue sites;(ce) Heat map analysis of virtual combination mutations at potential key amino acid residue sites;(f) Energy change analysis of combined virtual saturation mutations at potential key amino acid residue sites.

[0061] Figure 4: SDS-PAGE analysis of wild-type and mutant enzyme expression. (a) SDS-PAGE analysis of wild-type and mutant supernatants; (b) SDS-PAGE analysis of wild-type and mutant pure enzymes.

[0062] M, color pre-stained protein maker; 1, E. coli / pGEX-6p-Hs7β-hsdh; 2, E. coli / pGEX-6p-TEAE; E. coli / pGEX-6p-DAAE.

[0063] Figure 5 :Comparative enzyme activity determination of wild-type and mutant Hs7β-HSDH. (a) Optimum pH value; (b) Optimum temperature; (c) Thermal stability; (d) Changes in binding energy and binding entropy of Hs7β-HSDH and mutant with NADH.

[0064] Figure 6 : Dynamic simulation of Hs7β-HSDH and mutants. (a) Changes in root mean square deviation (RMSD); (b) Changes in root mean square fluctuation (RMSF); (c) Types of forces acting on amino acid residues in the binding pocket.

[0065] Figure 7 : Time curve of UDCA synthesis catalyzed by Hs7β-HSDH and its mutants.

[0066] Figure 8 :Hs7β-HSDH and its mutants coupled with Bm7α-HSDH using CDCA as substrate and 0.5 mM NAD + As a cofactor, UDCA is synthesized by one-pot catalysis. DETAILED DESCRIPTION

[0067] Technical terms:

[0068] Ursodeoxycholic acid: 3α,7β-dihydroxy-5β-cholestane-24-acid, is a hydrophilic bile acid with multiple biological activities. It can promote the excretion of endogenous bile acids, increase the proportion of hydrophilic bile acids, protect hepatocytes and bile duct cells from the toxicity of hydrophilic bile acids, prevent hydrophobic and hydrophilic bile acids from damaging mitochondrial membranes and their cytotoxicity, inhibit hepatocyte apoptosis, significantly enhance liver function, and exhibit immunomodulatory and cytoprotective effects. It is one of the most commonly used prescription drugs in the clinical treatment of cholestatic diseases.

[0069] Expression: The term "expression" includes any step involved in the production of the 7β-hydroxysteroid dehydrogenase mutant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0070] Expression vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a 7β-hydroxysteroid dehydrogenase mutant of the present invention and operably linked to control sequences that provide for its expression.

[0071] Fragment: The term "fragment" means a polypeptide that lacks one or more (e.g., several) amino acids at the amino and / or carboxyl terminus of the polypeptide; wherein the fragment has 7β-hydroxysteroid dehydrogenase activity. In one aspect, the fragment contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% of the number of amino acids 1 to 440 (i.e., not including the length of the zymogen region sequence) shown in SEQ ID NO.1.

[0072] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0073] The host cell can be any cell useful in the recombinant production of a 7β-hydroxysteroid dehydrogenase mutant, such as a prokaryotic cell or a eukaryotic cell.

[0074] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanic Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Mycobacterium, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0075] The host cell may also be a eukaryotic organism, such as a mammalian, insect, plant, or fungal cell.

[0076] CDCA (chenodeoxycholic acid) and 7K-LCA (7-ketolithocholic acid) involved in the following examples were purchased from: Sinopharm Chemical Reagent Co., Ltd., CAS No.: 474-25-9, 4651-67-6, respectively, and the structural formula is as follows:

[0077]

[0078] Reaction formula of the present invention is as follows:

[0079]

[0080] The culture medium involved in the following examples is as follows:

[0081] LB liquid medium (g / L): peptone 10, yeast powder 5, sodium chloride 10, natural pH.

[0082] The primer sequences involved in the following examples are shown in Table 1:

[0083] Table 1: Primer design for Hs7β-HSDH mutant enzyme gene

[0084]

[0085]

[0086] The detection methods involved in the following embodiments are:

[0087] The detection method of 7β-hydroxysteroid dehydrogenase activity is:

[0088] The enzyme activity of 7β-HSDH reducing 7K-LCA was measured at 30°C by measuring the absorbance change rate of NADH or NADPH at 340nm. Enzyme activity assay system: 250μL reaction volume, 2mM NADH and 10mM 7K-LCA were added to 0.1M PBS buffer (pH7.5), incubated in a 30°C metal bath for 3min, 50μL of pure enzyme at an appropriate concentration was added, and the absorbance change at 340nm was scanned using the Bio-Tek Cytation 5 Cell Imaging Multifunctional Detection System.

[0089] Enzyme activity is defined as: under the above conditions, catalyzing the oxidation of 1 μmol NADH or NADPH to NAD per minute. + The amount of enzyme is defined as one unit U.

[0090] The calculation formula of enzyme activity is: enzyme activity (U) = EW × V × 10 3 / (6220×0.625)

[0091] The calculation formula of specific activity is: specific activity (U·mg -1 ) = enzyme activity (U) / protein content (mg)

[0092] Where, EW: change in absorbance at 340 nm within 1 min; V: volume of reaction solution (mL); 6220: molar extinction coefficient L·mol -1 cm -1 );0.5:optical path distance(cm).

[0093] Determination of kinetic parameters:

[0094] According to the Michaelis equation v = V max ×[S] / K m +[S], calculate the K of the enzyme for different substrates m value.

[0095] Where, v: reaction rate (U·mg -1 );V max :Maximum reaction rate (U·mg -1 ); [S]: substrate concentration (mM); K m :The reaction speed v reaches half 1 / 2V max The substrate concentration at .

[0096] When the substrate concentration is saturated, k cat =V max / Et, calculate k cat value.

[0097] Among them, V max : maximum reaction rate; Et: concentration of enzyme sites.

[0098] Determination of kinetic parameters:

[0099] The total reaction volume was 250 μL. 0.1 M PBS buffer (pH 7.5) and 10 mM 7K-LCA were added. The temperature was kept at 30 °C for 2 min. 50 μL of pure enzyme at an appropriate concentration was added. The specific enzyme activity under different concentrations (0.1-2 mM) of cofactor NADH or NADPH was measured. The enzyme kinetic curve was fitted by GraphPad software. The substrate concentration and specific enzyme activity were used as the horizontal and vertical coordinates, respectively. The Michaelis-Menten equation model was selected for nonlinear fitting to obtain V max Value and K m Value, obtained by data conversion cat Value and k cat / K m Values ​​(Table 2).

[0100] The high performance liquid chromatography (HPLC) detection method of ursodeoxycholic acid content is:

[0101] Agilent 1260 liquid chromatograph and Accucore-C18 LC column were used for detection, the mobile phase was 60% acetonitrile solution with a flow rate of 1 mL / min, the detection temperature was 30°C, and the detection was performed at UV 195 nm. The substrate and product standards were dissolved in methanol or acetonitrile solution for detection, and the standard curves of UDCA, CDCA and 7K-LCA were obtained.

[0102] Example 1: Structural analysis of 7β-hydroxysteroid dehydrogenase and virtual screening of key residues for cofactor-dependent mutations The specific steps are as follows:

[0103] (1) Discovery of Hs7β-HSDH

[0104] The results of amino acid sequence comparison and conservation analysis are as follows: Figure 1 As shown, 7β-hydroxysteroid dehydrogenase (Hs7β-HSDH) from Hyphomicrobium sp. exhibits a highly conserved catalytic domain (Ser-(Xaa)13-Tyr-(Xaa)4-Lys) and cofactor binding region (Thr-Gly-(Xaa)3-Gly-(Xaa)1-Gly), and this enzyme was selected as the research object.

[0105] (2) Structural analysis of Hs7β-HSDH

[0106] By docking Hs7β-HSDH with NADPH and NADH, the results are as follows Figure 2 As shown, several semi-conserved sites existing in the cofactor binding region, such as A22, Q23, N24, G26, D44, L45, and N46, were selected as potential key amino acid residues affecting cofactor binding, and these sites were selected for virtual mutation.

[0107] (3) Virtual screening of key residues of Hs7β-HSDH mutation

[0108] By virtually mutating the above amino acid residues to alanine and performing virtual saturation mutation screening, the results are as follows Figure 3 As shown, the mutants A22T / Q23E, A22D / Q23A, L45A / N46E, A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E showed a significant decrease in binding energy to protein molecules, proving that these mutants have increased affinity for NADH.

[0109] Example 2: Construction and protein expression of 7β-hydroxysteroid dehydrogenase and its mutants

[0110] The specific steps are as follows:

[0111] (1) Construction of pGEX-6p-Hs7β-hsdh

[0112] The 7β-hydroxysteroid dehydrogenase Hs7β-HSDH from Hyphomicrobium sp. (amino acid sequence as shown in SEQ ID NO.1) was codon-optimized (nucleotide sequence as shown in SEQ ID NO.2) and connected to the plasmid pGEX-6p-1 (which can be sent to the company for synthesis), and named pGEX-6p-Hs7β-hsdh.

[0113] (2) Construction of recombinant vector containing mutants

[0114] First, pGEX-6p-Hs7β-hsdh was used as a template and primers such as L45A / N46E-F, L45A / N46E-R, A22T / Q23E-F, A22T / Q23E-R, A22D / Q23A-F, and A22D / Q23A-R (Table 1) were used for amplification to obtain a plasmid containing multiple mutants A22T / Q23E, A22D / Q23A, L45A / N46E, L45A / N46E / A22T / Q23E and L45 Recombinant vectors of A / N46E / A22D / Q23A: pGEX-6p-A22T / Q23E, pGEX-6p-A22D / Q23A, pGEX-6p-L45A / N46E, pGEX-6p-A22T / Q23E / L45A / N46E / (named: pGEX-6p-TEAE) and pGEX-6p-A22D / Q23A / L45A / N46E / (named: pGEX-6p-DAAE).

[0115] (3) Construction of recombinant strains

[0116] The recombinant vectors containing mutants obtained in step (2) (pGEX-6p-A22T / Q23E, pGEX-6p-A22D / Q23A, pGEX-6p-L45A / N46E, pGEX-6p-TEAE and pGEX-6p-DAAE) and the recombinant vector pGEX-6p-Hs7β-hsdh containing wild-type β-hydroxysteroid dehydrogenase obtained in step (1) were transformed into E. coli BL21 (DE3) competent cells after sequencing verification.

[0117] The recombinant strains were prepared respectively:

[0118] E.coli BL21 / pGEX-6p-Hs7β-hsdh, E.coli BL21 / pGEX-6p-A22T / Q23E, E.coliBL21 / A22D / Q23A, E.coli BL21 / L45A / N46E, E.coli BL21 / pGEX-6p-TEAE and E.coliBL21 / pGEX-6p-DAAE.

[0119] (4) Expression and purification of Hs7β-HSDH and its mutants

[0120] The recombinant bacteria obtained in step (3) were inoculated into LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C, 200 rpm, with shaking for 8 to 10 h to obtain seed solution. The obtained seed solution was inoculated into LB medium containing 100 μg / mL ampicillin at an inoculum volume of 1% (v / v), and cultured at 37°C, 200 rpm, with shaking until OD 600 After reaching 0.6-0.8 (about 2 hours), add 0.1mM IPTG and continue to culture at 20℃ for 16 hours. Collect the bacteria by centrifugation at 10,000×g and 4℃ for 10 minutes. Resuspend the bacteria with PBS buffer, disrupt the cells with an ultrasonic disruptor in an ice bath, centrifuge the disrupted liquid at 10,000×g and 4℃ for 30 minutes, take the supernatant for SDS-PAGE analysis, and purify the supernatant with GSTrap HP affinity column. After purification, add PreScission protease and digest at 4℃ for 10 hours to cut and remove the GST tag, and perform SDS-PAGE analysis on the purified protein. The results are as follows Figure 4 As shown, the pure enzyme was obtained for the next step of analysis.

[0121] (4) Detection of enzyme activity

[0122] The enzyme activity of the pure enzyme solution obtained in step (3) was tested respectively, and the results were as follows:

[0123] When NADH is used as a cofactor, the enzyme activity data of the wild type and mutant are as follows:

[0124] The enzyme activity of the wild-type enzyme is 0.02U / mg, the enzyme activity of mutant A22D / Q23 is 0.82U / mg, the enzyme activity of mutant A22T / Q23E is 1.02U / mg, the enzyme activity of mutant L45A / N46E is 1.23U / mg, the enzyme activity of mutant A22D / Q23A / L45A / N46E is 3.25U / mg, and the enzyme activity of mutant A22T / Q23E / L45A / N46E is 4.18U / mg, which are 41.0 times, 51.0 times, 61.5 times, 162.5 times, and 181.7 times higher than that of the wild type, respectively.

[0125] When NADPH is used as a cofactor, the enzyme activity data of the wild type and mutant are as follows:

[0126] The enzyme activity of the wild-type enzyme was 5.22U / mg, the enzyme activity of mutant A22D / Q23A was 4.82U / mg, the enzyme activity of mutant A22T / Q23E was 4.31U / mg, the enzyme activity of mutant L45A / N46E was 2.23U / mg, the enzyme activity of mutant A22D / Q23A / L45A / N46E was 0.25U / mg, and the enzyme activity of mutant A22T / Q23E / L45A / N46E was 0.18U / mg, which were reduced to 92.3%, 82.6%, 42.7%, 4.8% and 3.4% of the wild-type, respectively.

[0127] Example 3: Enzymatic properties of 7β-hydroxysteroid dehydrogenase and its mutants

[0128] The enzymatic properties of the mutant and wild-type enzymes obtained in Example 2 were tested respectively, and the specific steps were as follows:

[0129] (1) Optimum pH

[0130] The optimal pH of Hs7β-HSDH and its mutants was determined by measuring the specific enzyme activity (with NADH as cofactor) at pH 6.0-9.0 (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0) and 25°C.

[0131] The results are as follows Figure 5 As shown in a, Hs7β-HSDH and its mutants were most active at pH 7.5. The maximum enzyme activities of mutants A22D / Q23A, A22T / Q23E / , L45A / N46E, A22D / Q23A / L45A / N46E and A22T / Q23E / L45A / N46E with NADH as cofactor were 1.43U / mg, 1.94U / mg, 2.16U / mg, 5.39U / mg and 8.45U / mg, respectively, which were 19.1 times, 25.86 times, 28.8 times, 71.8 times and 112.6 times higher than those of the wild type.

[0132] When the pH was higher than 7.5, the enzyme activity decreased significantly.

[0133] (2) Optimum temperature

[0134] The optimum temperature of Hs7β-HSDH and its mutants was determined by measuring the specific enzyme activity (with NADH as cofactor) at pH 7.5 in the temperature range of 20-45°C (20, 25, 30, 35, 40, 45).

[0135] The results are as follows Figure 5 As shown in b, Hs7β-HSDH and its mutants were most active at 30℃. Compared with the wild type, the enzyme activities of the mutants were significantly improved. The maximum enzyme activities of mutants A22D / Q23A, A22T / Q23E / , L45A / N46E, A22D / Q23A / L45A / N46E and A22T / Q23E / L45A / N46E with NADH as cofactor were 1.69U / mg, 2.35U / mg, 2.63U / mg, 6.39U / mg and 8.38U / mg, respectively, which were 13.0 times, 18.0 times, 20.2 times, 48.3 times and 65.2 times higher than those of the wild type.

[0136] (3) Stability

[0137] The enzyme stability was evaluated at pH 7.5 and 30°C (with NADH as cofactor).

[0138] The results are as follows Figure 5 As shown in c, compared with the wild type, the enzyme activity of the mutants was improved, but the half-life showed similar properties, with the enzyme activity decreasing by about 50% in about 3 hours and the enzyme being completely inactivated after 8 hours.

[0139] (4) Kinetic parameters

[0140] Kinetic data were obtained using a BioTek-Cytation-5 cell imaging multimode reader at 30°C and pH 7.5. The assay was performed in 10 mM PBS (pH 7.5) containing 10.0 mM 7K-LCA and using 50 μM enzyme and varying concentrations of cofactors NADH or NADPH (0.1-10.0 mM). All values ​​represent the average of three replicates.

[0141] The results are shown in Table 2.

[0142] Table 2: Kinetic parameters of 7β-hydroxysteroid dehydrogenase and its mutants

[0143]

[0144]

[0145] Compared with the wild type, the enzyme activity of the mutant A22T / Q23E / L45A / N46E increased by 65.2 times, and the catalytic efficiency (k cat / K m ) increased by 44.9 times; the enzyme activity of mutant A22D / Q23A / L45A / N46E increased by 48.3 times, and the catalytic efficiency of NADH (k cat / K m) increased by 29.3 times.

[0146] Example 4: Binding energy determination of 7β-hydroxysteroid dehydrogenase and its mutants

[0147] The binding energy and binding entropy of the wild type and mutant with the cofactor NADH were measured using Discovery Studio. Figure 5 As shown in d.

[0148] The results show:

[0149] Compared with the wild type, the binding free energy (ΔG) of the mutants A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E was significantly reduced to -71.4 kcal / mol and -85.7 kcal / mol, respectively, while that of the wild type was -52.0 kcal / mol, indicating that the affinity of the mutants to NADH was significantly improved.

[0150] In contrast, the changes in the complex entropy of the two mutants were very small, and the binding entropy of the wild type and the two best mutants A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E were all about -21.5 kcal / mol, indicating that the mutants have similar thermal stability to NADH.

[0151] Example 5: Molecular dynamics simulation of Hs7β-HSDH and its mutants

[0152] The specific steps are as follows:

[0153] (1) Root-mean-square deviation analysis

[0154] The results are as follows Figure 6 As shown in a, the root mean square deviation (RMSD) values ​​of the A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E mutant enzymes and the wild-type enzyme reached equilibrium at approximately 20 ns.

[0155] In contrast, the RMSD fluctuation of the wild-type Hs7β-HSDH complex was larger.

[0156] It is noteworthy that the RMSD values ​​of these two mutants are always lower than that of the wild-type complex, indicating that the mutants have better stability relative to the wild-type enzyme.

[0157] (2)Root Mean Square Fluctuation analysis

[0158] The results are as follows Figure 6 As shown in b, the RMSF values ​​of the A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E mutant enzymes deviate slightly from the RMSF values ​​of the wild-type enzyme, but the rigidity within the loop region is slightly enhanced, which leads to a slight improvement in the stability of the enzyme.

[0159] (3) Analysis of the interaction between residues and ligands

[0160] The results are as follows Figure 6 As shown in (c), compared with the wild type, the hydrophobic interaction of the A22T / Q23E / L45A / N46E mutant at the A22 / Q23 site was significantly enhanced, and the hydrogen bond interaction at the Q23 site was also increased to about 0.4.

[0161] The hydrophobic interaction at the D22 site of the A22D / Q23A / L45A / N46E mutant increased to approximately 2.0.

[0162] In addition, mutations at the L45 / N46 sites in both mutants resulted in enhanced hydrophobic interactions at the D44 site, increasing to approximately 2.2 and 0.5, respectively.

[0163] This indicates that the mutant has a stronger affinity with NADH.

[0164] Example 6: Synthesis of ursodeoxycholic acid using NADH as a cofactor

[0165] The specific steps are as follows:

[0166] (1) Preparation of substrate solution and culture of wet cells:

[0167] 5 g / L 7K-LCA solution: Dissolve 7K-LCA in 200 mM Tris-NaCl buffer and adjust the pH to 7.5;

[0168] Wet cell preparation:

[0169] The recombinant bacteria obtained in step (3) of Example 2 were inoculated into LB liquid culture medium containing 100 μg / mL ampicillin, and cultured at 37°C, 200 rpm, with shaking for 10 h to obtain a seed solution. The obtained seed solution was inoculated into LB culture medium containing 100 μg / mL ampicillin at an inoculum volume of 1% (v / v), and cultured at 37°C, 200 rpm, with shaking until the OD 600 After reaching 0.6-0.8 (about 2 hours), add 0.1 mM IPTG and continue to culture at 20°C for 16 hours to induce the expression of the target protein to obtain a fermentation broth;

[0170] The obtained fermentation broth was centrifuged at 12000×g and 4°C and then filtered to obtain wet cells;

[0171] (2) Preparation of ursodeoxycholic acid:

[0172] The reaction mixture (10 mL) included: 200 mM Tris-NaCl buffer (pH 7.5), 12 mM NADH, 5 g / L 7K-LCA solution and 20 g / L wet cells obtained in step (1).

[0173] The reaction was carried out at 30°C and 200 rpm for 2 h, then heated in boiling water for 10 min to terminate the reaction, and saturated NaOH solution was added to the reaction mixture to adjust the pH to 11 to ensure that the substrate and product were completely dissolved in the solution. The mixture was centrifuged at 12,000 × g for 10 min to remove denatured cells, and the supernatant was used to detect the product by HPLC.

[0174] The results are as follows Figure 7 As shown, after 2 hours of reaction using 5 g / L 7K-LCA and 12.5 mM NADH as cofactors, the yields of mutants A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E reached 77.6% and 65.3%, respectively, which were 13.9 times and 11.5 times higher than those of the wild-type enzyme (5.6%).

[0175] Example 7: Construction of a “one-pot” efficient synthesis system for ursodeoxycholic acid

[0176] The Hs7β-HSDH mutant was coupled with the Bm7α-HSDH to achieve one-pot synthesis of UDCA from CDCA while also being able to regenerate the coenzyme NAD in situ + and NADH to reduce the amount of coenzymes used.

[0177] The specific steps are as follows:

[0178] (1) Preparation of reaction substrate:

[0179] 1) 5 g / L CDCA: Dissolve CDCA in 200 mM Tris-NaCl buffer and adjust the pH to 7.5;

[0180] 2) Preparation of wet cells:

[0181] The recombinant bacteria obtained in step (3) of Example 2 were inoculated into LB liquid culture medium containing 100 μg / mL ampicillin, and cultured at 37°C, 200 rpm, with shaking for 10 h to obtain a seed solution. The obtained seed solution was inoculated into LB culture medium containing 100 μg / mL ampicillin at an inoculum volume of 1% (v / v), and cultured at 37°C, 200 rpm, with shaking until the OD 600After reaching 0.6-0.8 (about 2 hours), add 0.1 mM IPTG and continue to culture at 20°C for 16 hours to obtain fermentation broth;

[0182] The obtained fermentation broth was centrifuged at 12000×g and 4°C and then filtered to obtain wet cells expressing the Hs7β-HSDH mutant or wild-type enzyme;

[0183] 3) Preparation of recombinant cells expressing Bm7α-HSDH:

[0184] 7α-HSDH (SEQ ID NO. 3) from Brucella was constructed into pET28a vector to construct recombinant vector pET28a-Bm7α-HSDH. The recombinant vector was transformed into E. coli BL21 (DE3) competent cells to obtain recombinant cells E. coli / pET28a-Bm7α-HSDH.

[0185] The recombinant bacteria after sequencing verification were inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 200 rpm, with shaking for 10 h to obtain seed solution. The obtained seed solution was inoculated into LB medium containing 50 μg / mL kanamycin at an inoculum volume of 1% (v / v), and cultured at 37°C, 200 rpm, with shaking until OD 600 After reaching 0.6-0.8 (about 2 hours), add 0.1 mM IPTG and continue to culture at 20°C for 16 hours to obtain fermentation broth;

[0186] The obtained fermentation broth was centrifuged at 12000×g and 4°C and then filtered to obtain wet cells expressing Bm7α-HSDH;

[0187] (2) Reaction system:

[0188] The reaction mixture (10 mL) included 200 mM Tris-NaCl buffer (pH 7.5), 0.5 mM NADH, 5 g / L LCDCA, 10 g / L wet cells obtained in step (1) and 10 g / L cultured wet cells expressing Bm7α-HSDH.

[0189] The reaction was carried out at 30 °C with shaking at 200 rpm for 2 h, then heated in boiling water for 10 min to terminate the reaction, and saturated NaOH solution was added to the reaction mixture to adjust the pH to 11 to ensure that the substrate and product were completely dissolved in the solution. The mixture was centrifuged at 12,000 × g for 10 min to remove denatured cells, and the supernatant was used for HPLC to detect the product content.

[0190] The results are as follows Figure 8As shown, the yields of mutants A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E reached 90.1% and 79.6%, respectively, which were increased by 11.5 times and 10.2 times compared with the wild-type enzyme (7.8%).

[0191] The same reaction system as Example 6: After 2 hours of reaction using 5 g / L 7K-LCA and 12.5 mM NADH as cofactors, the yields of mutants A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E reached 77.6% and 65.3%, respectively. In comparison, when 5 g / L CDCA and Bm7α-HSDH were used for the reaction, the yields of mutants A22T / Q23E / L45A / N46E and A22D / Q23A / L45A / N46E were increased by 12.5% ​​and 14.3%, respectively.

[0192] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A 7β-hydroxysteroid dehydrogenase mutant, characterized in that: The mutant is obtained by mutating the alanine at position 22 of the 7β-hydroxysteroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 to threonine, the glutamine at position 23 to glutamic acid, the leucine at position 45 to alanine, and the asparagine at position 46 to glutamic acid; Or the mutant is obtained by mutating the alanine at position 22 of the 7β-hydroxysteroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 to aspartic acid, the glutamine at position 23 to alanine, the leucine at position 45 to alanine, and the asparagine at position 46 to glutamic acid.

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

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

4. 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.

5. The recombinant cell according to claim 4, characterized in that The recombinant cell uses bacteria or fungi as expression hosts.

6. A recombinant enzyme catalyst comprising the 7β-hydroxysteroid dehydrogenase mutant sequence of claim 1, characterized in that: The recombinase catalyst is any one of the following forms: (1) culturing a recombinant expression transformant containing the 7β-hydroxysteroid dehydrogenase mutant, and isolating transformant cells containing the recombinant 7β-hydroxysteroid dehydrogenase mutant enzyme; (2) culturing an expression transformant containing the recombinant 7β-hydroxysteroid dehydrogenase mutant, isolating transformant cells containing the recombinant 7β-hydroxysteroid dehydrogenase mutant enzyme, and disrupting the transformant cells containing the recombinant 7β-hydroxysteroid dehydrogenase mutant enzyme to obtain a cell disrupted liquid; (3) culturing a recombinant expression transformant containing the 7β-hydroxysteroid dehydrogenase mutant, isolating transformant cells containing the recombinant 7β-hydroxysteroid dehydrogenase mutant enzyme, disrupting the transformant cells containing the recombinant 7β-hydroxysteroid dehydrogenase mutant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant 7β-hydroxysteroid dehydrogenase mutant enzyme to obtain a freeze-dried enzyme powder.

7. A method for increasing the affinity of 7β-hydroxysteroid dehydrogenase for coenzyme NADH, characterized in that: The method is: The amino acid sequence of 7β-hydroxysteroid dehydrogenase as shown in SEQ ID NO.1 is mutated from alanine at position 22 to threonine, glutamine at position 23 to glutamic acid, leucine at position 45 to alanine, and asparagine at position 46 to glutamic acid; Or the alanine at position 22 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 is mutated to aspartic acid, the glutamine at position 23 is mutated to alanine, the leucine at position 45 is mutated to alanine, and the asparagine at position 46 is mutated to glutamic acid.

8. A method for preparing ursodeoxycholic acid, characterized in that: The method comprises the following steps: using chenodeoxycholic acid as a substrate and NAD + As a cofactor, the mutant according to claim 1 or the recombinant cell according to claim 4 or 5 or the recombinant enzyme catalyst according to claim 6 and a recombinant cell containing 7α-hydroxysteroid dehydrogenase are used to jointly catalyze the substrate to convert and prepare ursodeoxycholic acid; Alternatively, the method is to use 7-keto-lithocholic acid as a substrate, NADH as a cofactor, and the mutant described in claim 1 or the recombinant cell described in claim 4 or 5 or the recombinant enzyme catalyst described in claim 6, and a recombinant cell containing 7α-hydroxysteroid dehydrogenase to jointly catalyze the substrate and convert it to prepare ursodeoxycholic acid.

9. The method according to claim 8, characterized in that The reaction system contains at least 5g / L 7-ketolithocholic acid and 10-15mM NADH; or at least 5g / L chenodeoxycholic acid and 0-1mM NAD + 、NAD + The concentration is not 0; the reaction conditions are pH 6-8; and the temperature is 25-35°C.

10. Use of the 7β-hydroxysteroid dehydrogenase mutant according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, the recombinant cell according to claim 4 or 5, the recombinant enzyme catalyst according to claim 6, or the method according to any one of claims 7 to 9 in the preparation of ursodeoxycholic acid or a product containing the above ursodeoxycholic acid.

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