7alpha-hydroxysteroid dehydrogenase mutant and application thereof in preparation of ursodesoxycholic acid

Abstract

The invention discloses a 7alpha-hydroxysteroid dehydrogenase mutant and application thereof in preparation of ursodesoxycholic acid, the enzyme mutant is obtained by mutating a wild type amino acid sequence as shown in SEQ ID NO: 1 through Q31A, H72Q, W111R or A178R, the mutated enzyme activity is high, 7-ketolithocholic acid can be prepared based on catalysis of a high-concentration substrate, and the 7alpha-hydroxysteroid dehydrogenase mutant can be applied to preparation of ursodesoxycholic acid. And the method has a significant meaning for industrial preparation of ursodesoxycholic acid under catalysis of the propulsor.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme mutation, in particular to 7α-hydroxysteroid dehydrogenase mutant and application thereof in preparation of ursodeoxycholic acid. BACKGROUND

[0002] Ursodeoxycholic acid, English name is Ursodeoxycholic Acid, chemical name is 3α, 7β-dihydroxy-5β-cholane-24-acid, molecular formula is C 24 H 40 O4, CAS number is 128-13-2, is used for treating various hepatobiliary diseases. Ursodeoxycholic acid is the only drug approved by the US Food and Drug Administration for the treatment of primary biliary cirrhosis disease, and has a broad market prospect.

[0003] There are three main methods for producing ursodeoxycholic acid at present: live bear bile, chemical synthesis method and biological transformation method. Live bear bile will affect animal welfare, cause physiological pain and psychological trauma to animals, and is not recommended. The chemical synthesis method has been industrialized, accounting for about 1 / 3 of the market share, but the route of chemical synthesis of ursodeoxycholic acid is mostly tedious, pollutes the environment, and involves high-temperature steps. The biological transformation method mainly prepares ursodeoxycholic acid through enzyme catalytic reaction. Compared with other production methods, enzyme catalytic reaction has the advantages of high specificity, fast reaction speed, and mild reaction conditions.

[0004] However, the traditional two-step enzyme method is based on chenodeoxycholic acid first catalyzed by 7α-hydroxysteroid dehydrogenase (7α-HSDH) to obtain 7-ketolithocholic acid (7-KLCA), and then catalyzed by 7β-hydroxysteroid dehydrogenase (7β-HSDH) to obtain 7-KLCA. Due to the low activity of 7α-HSDH, the low substrate concentration leads to high coenzyme dosage, long reaction time and complex post-treatment, which has been difficult to industrialize.

[0005] Therefore, it is of great significance to provide a high-activity 7α-hydroxysteroid dehydrogenase suitable for high-concentration substrate dosage for enzyme-catalyzed preparation of 7-ketolithocholic acid and further preparation of ursodeoxycholic acid. SUMMARY

[0006] The present application provides a 7α-hydroxysteroid dehydrogenase mutant with high activity, which is used to overcome the defects of low catalytic activity of 7α-hydroxysteroid dehydrogenase in the prior art, low substrate concentration, and difficulty in industrial production.

[0007] Therefore, the scheme of the present application is as follows: The first aspect of the present application is to provide a 7a-hydroxysteroid dehydrogenase mutant, which is obtained by mutating a wild-type 7a-hydroxysteroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO: 1, and the mutation is Q31A, H72Q, W111R or A178R.

[0008] The second aspect of the present application is to provide a gene encoding the 7a-hydroxysteroid dehydrogenase mutant of the first aspect.

[0009] The third aspect of the present application is to provide an expression vector containing the gene of the second aspect.

[0010] The fourth aspect of the present application is to provide a strain containing the expression vector of the third aspect.

[0011] The fifth aspect of the present application is to provide the use of the 7a-hydroxysteroid dehydrogenase mutant of the first aspect in catalyzing the 7a-hydroxy dehydrogenation reaction of chenodeoxycholic acid.

[0012] The sixth aspect of the present application is to provide a preparation method of 7-ketolithocholic acid, which is obtained by catalyzing the dehydrogenation reaction of chenodeoxycholic acid by the 7a-hydroxysteroid dehydrogenase mutant of the first aspect.

[0013] Preferably, the 7a-hydroxysteroid dehydrogenase mutant is A178R mutant.

[0014] Further, the catalytic oxidation process is carried out under the synergistic action of NAD(P) / NAD(P)H coenzyme cycle system.

[0015] Preferably, in the catalytic reaction process, the substrate chenodeoxycholic acid directly reacts with NAD + to produce 7-ketolithocholic acid and NADH, and under the action of glutamate dehydrogenase, NAD + is produced by adding alpha-ketoglutaric acid and NADH, and glutamic acid is formed by adding ammonium sulfate to provide ammonium ion, and the addition amount of alpha-ketoglutaric acid is 25-45%, and the addition amount of ammonium sulfate is 20-35%.

[0016] Further, the substrate concentration is 1-200 g / L; the catalytic process temperature is 20-30 ℃; and the catalytic oxidation process pH is 7.8-8.5. Under the above conditions, a reaction conversion rate of 7-ketolithocholic acid of more than 90% can be achieved. Preferably, when the substrate concentration is 50-150 g / L, the reaction temperature is 30 ℃, and the pH is controlled at 8.00 ± 0.05, the reaction conversion rate is more than 99%.

[0017] The seventh aspect of the present application provides a preparation method of ursodeoxycholic acid, which comprises adding 7beta-hydroxysteroid dehydrogenase to 7-ketolithocholic acid to reduce 7-keto group, wherein the 7-ketolithocholic acid is prepared by the preparation method of the sixth aspect.

[0018] Further, the 7beta-hydroxysteroid dehydrogenase can be selected from commercially available or self-developed enzymes, and preferably the 7beta-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO: 3.

[0019] Compared with the prior art, the present application has the following beneficial effects: The 7alpha-hydroxysteroid dehydrogenase mutant provided by the present application is obtained by gene engineering technology and computer-aided design to modify the active pocket of the enzyme, and the enzyme mutant has high activity in catalyzing the substrate chenodeoxycholic acid and can be used to catalytically prepare 7-KLCA based on high-concentration substrates, which has significant significance for promoting the industrialized preparation of ursodeoxycholic acid by enzyme catalysis. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The present application provides a reaction route diagram for preparing ursodeoxycholic acid from chenodeoxycholic acid by enzyme method. DETAILED DESCRIPTION

[0021] The technical solutions of the present application will be described in detail below in combination with preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0022] In the present application, the term "An" means that the nth amino acid A is changed to various amino acids, i.e. single-point saturation mutation; "AnB" means that the nth amino acid A is changed to amino acid B, such as Q108V, which means that the 108th amino acid Q is mutated to amino acid V, and so on.

[0023] In the following examples, the materials, reagents, etc. can be obtained from commercial channels unless otherwise specified.

[0024] Example 1

[0025] Synthesis of 7alpha-HSDH_Pb gene: The 7alpha-HSDH_Pb gene (derived from Pseudomonadota_bacterium ) is synthesized by gene synthesis (synthesized by Wuhan Qikexin Biotechnology Co., Ltd.), and the related amino acid sequence and nucleotide sequence are as follows: MDSVFRLDGEVALVTGAAAGIGRAIAETFAQAGAAVVVTDLKLDQAEEAASVIHQAGGRAIGLECNVTDEAHRAAAIEAAVRTFGKLSILVNNAGGGGPKPFDMPMSDFEWAYQLNVFAAFRLMQLAAPHMQQAGGGAILNISSMAGENKNTRMAAYGSSKAAVNHLTRNVAFDLGPAGIRVNAIAPGAIKTGALAKVLTPEIEAAMLKHTSLGRLGAPQDIAHAALFLCSPAAAWISGQVLTVSGGGVQELD (SEQ ID NO: 1) ATGGATAGCGTGTTTCGCCTGGATGGCGAAGTGGCGCTGGTGACCGGCGCGGCGGCGGGCATTGGCCGCGCGATTGCGGAAACCTTTGCGCAGGCGGGCGCGGCGGTGGTGGTGACCGATCTGAAACTGGATCAGGCGGAAGAAGCGGCGAGCGTGATTCATCAGGCGGGCGGCCGCGCGATTGGCCTGGAATGCAACGTGACCGATGAAGCGCATCGCGCGGCGGCGATTGAAGCGGCGGTGCGCACCTTTGGCAAACTGAGCATTCTGGTGAACAACGCGGGCGGCGGCGGCCCGAAACCGTTTGATATGCCGATGAGCGATTTTGAATGGGCGTATCAGCTGAACGTGTTTGCGGCGTTTCGCCTGATGCAGCTGGCGGCGCCGCATATGCAGCAGGCGGGCGGCGGCGCGATTCTGAACATTAGCAGCATGGCGGGCGAAAACAAAAACACCCGCATGGCGGCGTATGGCAGCAGCAAAGCGGCGGTGAACCATCTGACCCGCAACGTGGCGTTTGATCTGGGCCCGGCGGGCATTCGCGTGAACGCGATTGCGCCGGGCGCGATTAAAACCGGCGCGCTGGCGAAAGTGCTGACCCCGGAAATTGAAGCGGCGATGCTGAAACATACCAGCCTGGGCCGCCTGGGCGCGCCGCAGGATATTGCGCATGCGGCGCTGTTTCTGTGCAGCCCGGCGGCGGCGTGGATTAGCGGCCAGGTGCTGACCGTGAGCGGCGGCGGCGTGCAGGAACTGGATTAA (SEQ ID NO: 2) Example 2 Construction of 7a-HSDH_Pb mutants Based on 7a-HSDH_Pb, the binding free energy of virtual saturation mutation of residues on the substrate binding pocket site was calculated by stability prediction software, and the key sites that may affect the structure and function of 7a-HSDH_Pb were found, and 7a-HSDH_Pb_1~7a-HSDH_Pb_7 were modified as shown in Table 1.

[0026] Table 1: 7a-hydroxysteroid dehydrogenase mutant sequence

[0027] PCR reaction was carried out with 7α-HSDH_Pb as a template and the upstream and downstream primers in the table, and the reaction procedure was as follows: (1) Pre-denaturation: 98 ℃, 2:00; (2) Denaturation: 95 ℃, 0:20; (3) Annealing: 55 ℃, 0:20; (4) Extension: 72 ℃, 0:10; (5) Cycle: GOTO step 2, 25x; (6) Complete extension: 72 ℃, 5:00; (7) Preservation: 4 ℃, ∞.

[0028] After the PCR reaction, a small amount of the PCR product was detected by 1% agarose gel electrophoresis. The detection result showed that the PCR amplification product was a single band and the bp number was correct. The remaining PCR product was recovered and purified and used Dpn Ⅰ treatment to remove the template DNA.

[0029] The constructed 7α-HSDH_Pb mutant was transformed into the competent cells of Escherichia coli BL21 (DE3), and the transformants were plated on LB solid selective medium (formula: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar) containing 50 μg / mL concentration of kanamycin resistance. The transformants were picked on the LB solid selective medium plate and cultured in LB liquid medium (formula: 1% tryptone, 0.5% yeast extract, 1% sodium chloride) at 32 ℃ for 16 h. The bacterial liquid was subjected to PCR identification and sequencing verification, and the bacterial liquid with correct sequencing results was preserved in a glycerol freezing tube for subsequent heterologous expression.

[0030] Example 3 Heterologous expression of 7α-hydroxysteroid dehydrogenase gene 7α-HSDH_Pb and its mutant in Escherichia coli BL21 (DE3)

[0031] The synthesized expression plasmid pET-26b-7α-HSDH_Pb containing 7α-hydroxysteroid dehydrogenase gene 7α-HSDH_Pb was transformed into the competent cells of Escherichia coli BL21 (DE3), and plated on LB solid selective medium plate containing 50 μg / mL concentration of kanamycin resistance.

[0032] The transformant BL21(DE3)-pET-26b-7α-HSDH_Pb was picked on LB solid selective medium plate, and seed liquid was prepared in LB liquid medium (37 °C, 220 rpm, 16 h). The bacterial liquid was transferred to a 250 mL conical flask containing 50 mL of LB liquid medium at a seeding amount of 0.5 mL, and was cultured at 37 °C and 220 rpm until the OD 600 = 0.6-0.8, 0.5 mM IPTG was added, and induction was carried out at 18 °C for 20 h. The induced bacterial liquid was centrifuged at 8000 g and 4 °C for 15 min, the supernatant was discarded, and 0.1 M PBS (pH 8.0) buffer was added for washing and resuspension. Then, ultrasonic crushing was carried out to obtain a clear whole-cell lysate. The supernatant obtained by centrifugation at 12000 rpm and 4 °C was the crude enzyme liquid of 7α-hydroxysteroid dehydrogenase. The crude enzyme liquid of the 7α-hydroxysteroid dehydrogenase mutant was obtained by the same method.

[0033] Example 4: Establishment of enzyme activity method and standard curve

[0034] The 200 µL reaction system was composed of the following components: 170 µL PBS (100 mM, pH = 8.0), 10 µL chenodeoxycholic acid solution (10 mg / mL, dissolved in anhydrous ethanol), 10 µL NAD + (10 mM), and 10 µL diluted 7α-hydroxysteroid dehydrogenase enzyme liquid.

[0035] The change in absorbance at 340 nm within 4 min was measured at 30 °C, and the standard curve was calculated using Excel to calculate y = Kx + b, y being the absorbance A340, x being the time (min), and the slope K of the change in absorbance within 4 min, i.e., the 7α-hydroxysteroid dehydrogenase enzyme activity, was calculated. The calculation formula of the 7α-hydroxysteroid dehydrogenase enzyme activity is as follows: Enzyme activity (U / mg) = wherein K is the slope of the change in absorbance, A is the slope of the standard curve, N is the dilution multiple of the enzyme liquid, V0 is the reaction volume (200 µL in this method), V1 is the enzyme liquid volume (10 µL in this method), and C is the enzyme liquid configuration concentration (protein concentration). The protein concentration was determined by the Coomassie brilliant blue method.

[0036] The standard curve was prepared as follows: 2 mM NADH stock solution was diluted with 100 mM pH 8.0 PBS to a final concentration of 0.1 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, and the absorbance at 340 nm was measured (zero point marked with deionized water), and the standard curve y = Ax + b was made using Excel, y is the absorbance A340, x is the NADH concentration (mM), and A is the slope of the standard curve.

[0037] Example 5 Enzyme activity determination of 7a-hydroxysteroid dehydrogenase 7a-HSDH_Pb and its mutants

[0038] Based on the methods of Examples 1-3, 7a-HSDH_Pb and its mutants were synthesized, constructed and expressed, and the crude enzyme solutions of 7a-HSDH_Pb, 7a-HSDH_Pb_1, 7a-HSDH_Pb_2, 7a-HSDH_Pb_3, 7a-HSDH_Pb_4, 7a-HSDH_Pb_5, 7a-HSDH_Pb_6, 7a-HSDH_Pb_7 were obtained.

[0039] Based on the enzyme activity detection method of 7a-hydroxysteroid dehydrogenase in Example 4, the enzyme activity was detected, and the results are shown in Table 2. As can be seen from Table 2, there are four mutants with higher enzyme activity than the wild type, including 7a-HSDH_Pb_2, 7a-HSDH_Pb_3, 7a-HSDH_Pb_4, 7a-HSDH_Pb_5, wherein the relative enzyme activity of mutant 7a-HSDH_Pb_5 is the highest, which is 5.49 times that of wild type 7a-HSDH_Pb.

[0040] Table 2: Enzyme activity results of 7a-HSDH_Pb and its mutants

[0041] Example 6

[0042] 1) Enzymatic preparation of 7-ketolithocholic acid

[0043] In 100 mL reaction system, 5 g, 10 g, 15 g, 20 g chenodeoxycholic acid substrate was added respectively in a flask containing 50 mL pH 8.0 phosphate buffer, placed in a 30 ℃ water bath, then 25% substrate ratio ketoglutaric acid, 20% substrate ratio ammonium sulfate, 6 M sodium hydroxide solution was added, and the pH value was controlled at 8.00 ± 0.05, the reaction system was supplemented to 100 mL, and the chenodeoxycholic acid concentration was 50 g / L, 100 g / L, 150 g / L, 200 g / L respectively, stirred for 30 min, then 5% substrate ratio 7α-hydroxysteroid dehydrogenase (obtained by expressing 7α-HSDH_Pb_5 in Escherichia coli BL21 (DE3)), 1% substrate ratio glutamate dehydrogenase (4000 U / mg) and 0.1% substrate ratio NAD + After 1 h of reaction, sample was taken for HPLC detection to analyze the content of chenodeoxycholic acid and 7-ketolithocholic acid in the reaction solution, so as to determine the conversion rate.

[0044] Table 3 Effect of substrate concentration on conversion rate

[0045] 2) 7-ketolithocholic acid post-treatment

[0046] In the reaction solution with a substrate concentration of 150 g / L and a conversion rate of 99.44%, 3 M hydrochloric acid was added to control the pH value of the reaction solution at 5.0-5.5, then the temperature was raised to 95-100 ℃, and stirred for 1 h. After stirring, the filtered product was 7-ketolithocholic acid.

[0047] 3) Enzymatic preparation of ursodeoxycholic acid

[0048] In a flask containing 50 mL pH 7.0 phosphate buffer, 7-ketolithocholic acid prepared in the previous step was added, placed in a 30 ℃ water bath, then 10% of the system (10 mL) of ethanol, 10% substrate ratio glucose was added, stirred for 30 min, then 1% substrate ratio 7β-hydroxysteroid dehydrogenase (self-made, amino acid sequence is SEQ ID NO: 3, nucleotide sequence is SEQ ID NO: 4, enzyme activity is about 30 U / mg), 5% substrate ratio glucose dehydrogenase (20 U / mg) and 0.1% substrate ratio NADP + After the reaction system was supplemented to 100 mL, 6 M NaOH was added to control the pH value at 6.8-7.0 by a pH self-control system, and sample was taken for HPLC detection after 2 h of reaction. The conversion rate was 98.97%, and the substrate residue was 0.18%.

[0049] 4) Post-treatment of ursodeoxycholic acid

[0050] The reaction liquid was rotary evaporated at 60°C, and the ethanol in the reaction liquid was rotary evaporated. Then 10% of the substrate was added to the diatomite, and stirred at 60°C for 1 h. After stirring, the filter cake was filtered into a flask, 150 mL of methanol was added, and rotary evaporation was performed at 60°C. When there was about 30 mL of methanol left in the flask, 300 mL of water was added and stirred to precipitate. After water precipitation, rotary evaporation was continued at 60°C. The methanol in the reaction liquid was rotary evaporated, and after rotary evaporation, the filter cake was collected and dried in an oven at 60-70°C. The product, ursodeoxycholic acid, was obtained by drying to constant weight, with a purity of 99.01% and a yield of 96.25% based on chenodeoxycholic acid.

[0051] The process of obtaining 7-ketolithocholic acid from chenodeoxycholic acid by catalytic reaction, and then obtaining ursodeoxycholic acid by further enzymatic catalytic reaction is shown in the following scheme: Figure 1

[0052] Example 7: Scale-up reaction

[0053] 1) Enzymatic preparation of 7-ketolithocholic acid

[0054] In a 1000 mL reaction system, 150 g of chenodeoxycholic acid substrate was added to a flask containing 500 mL of pH 8.0 phosphate buffer, and placed in a 30°C water bath. Then 25% of the substrate was added as ketoglutaric acid, 20% of the substrate was added as ammonium sulfate, 6 M sodium hydroxide solution was added to control the pH at 8.00 ± 0.05, the reaction system was supplemented to 1000 mL, stirred for 30 min, 5% of the substrate was added as 7α-hydroxysteroid dehydrogenase (obtained by expression of 7α-HSDH_Pb_5 in E. coli BL21 (DE3)), 1% of the substrate was added as glutamate dehydrogenase (4000 U / mg), and 0.1% of the substrate was added as NAD+. After 1 h of reaction, the sample was detected by HPLC, the conversion rate was 99.36%, and the substrate residue was 0.13%.

[0055] 2) Post-treatment of 7-ketolithocholic acid

[0056] 3 M hydrochloric acid was added to the reaction liquid to control the pH of the reaction liquid at 5.0-5.5, then heated to 95-100°C and stirred for 1 h. After stirring, the product was filtered as 7-ketolithocholic acid.

[0057] 3) Enzymatic preparation of ursodeoxycholic acid

[0058] ​In a flask containing 500 mL pH 7.0 phosphate buffer, 7-ketolithocholic acid prepared in the previous step was added, placed in a 30°C water bath, then 10% of the system (100 mL) of ethanol, 10% of the substrate proportion of glucose, stirred for 30 min, 1% of the substrate proportion of 7β-hydroxysteroid dehydrogenase (30 U / mg), 5% of the substrate proportion of glucose dehydrogenase (20 U / mg), and 0.1% of the substrate proportion of NADP + After the reaction system was supplemented to 1000 mL, 6 M NaOH was added to control the pH to 6.8-7.0 by the pH self-control system, and after 2 h of reaction, sampling was performed for HPLC detection, the conversion rate was 98.87%, and the substrate residue was 0.45%.

[0059] 4) Post-treatment of ursodeoxycholic acid

[0060] The reaction liquid was rotary evaporated at 60°C, and the ethanol in the rotary evaporated reaction liquid was then removed, then 10% of the substrate proportion of diatomite was added, and stirred at 60°C for 1 h, after stirring, filtration was performed, the filter cake was put into a flask, 1500 mL of methanol was added, rotary evaporation was performed at 60°C, when about 300 mL of methanol remained in the flask, 3000 mL of water was added and stirred for water extraction, after water extraction, rotary evaporation was continued at 60°C, the methanol in the rotary evaporated reaction liquid was removed, after rotary evaporation, filtration was performed, the filter cake was collected and dried in an oven at 60-70°C, dried to constant weight, and the UDCA product was collected, the purity was 98.94%, and the yield was 93.68% based on chenodeoxycholic acid.

[0061] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, the scope of the present application being defined by the appended claims and their equivalents.

Claims

1. 7α-hydroxysteroid dehydrogenase mutant, characterized in that, It is obtained by mutation of wild-type 7α-hydroxysteroid dehydrogenase with an amino acid sequence as shown in SEQ ID NO: 1, wherein the mutation is Q31A, H72Q, W111R or A178R.

2. The gene encoding the 7α-hydroxysteroid dehydrogenase mutant of claim 1.

3. An expression carrier, characterized in that, It contains the gene described in claim 2.

4. A bacterial strain, characterized in that, It contains the expression vector as described in claim 3.

5. The application of the 7α-hydroxysteroid dehydrogenase mutant according to claim 1 in the catalytic 7α-hydroxy dehydrogenation reaction of chenodeoxycholic acid.

6. A method for preparing 7-ketolithocholic acid, characterized in that, It is obtained by dehydrogenation catalyzed by the 7α-hydroxysteroid dehydrogenase mutant as described in claim 1, using chenodeoxycholic acid as a substrate.

7. The preparation method according to claim 6, characterized in that, The 7α-hydroxysteroid dehydrogenase mutant is the A178R mutant.

8. The preparation method according to claim 6, characterized in that, The catalytic oxidation process is carried out under the synergistic effect of the NAD(P) / NAD(P)H coenzyme cycle system.

9. The preparation method according to claim 6, characterized in that, The substrate concentration is 1-200 g / L; And / or, the temperature of the catalytic oxidation process is 20-30 °C; And / or, the pH of the catalytic oxidation process is 7.8-8.

5.

10. A method for preparing ursodeoxycholic acid, comprising reducing the 7-keto group of 7-ketolithocholic acid as a substrate by adding 7β-hydroxysteroid dehydrogenase, wherein the 7-ketolithocholic acid is prepared by the method described in any one of claims 6-9.

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