Variants of 7 beta-hydroxysteroid dehydrogenase and related application

Abstract

The invention belongs to the field of biology, discloses a variant of 7 beta-hydroxysteroid dehydrogenase and related application, and aims to improve the catalytic efficiency and stability of catalyzing 7-ketolithocholic acid to be converted into ursodeoxycholic acid, and particularly, the variant is obtained by performing single mutation or multipoint combined mutation on 18th, 40th, 93th, 94th, 95th, 145th, 150th, 153th, 193th, 196th, 197th and 199th sites of an amino acid sequence shown as SEQ ID NO.1.

Description

Technical Field

[0001] The present invention belongs to the biological field, and particularly relates to a variant of 7β-hydroxysteroid dehydrogenase and corresponding applications. Background Art

[0002] Ursodeoxycholic acid (UDCA) is an important bile acid with multiple pharmacological effects, including choleretic, hepatoprotective, and lipid-lowering properties, and is widely used to treat hepatobiliary diseases. Currently, UDCA production relies primarily on extraction from bear bile, but this method is plagued by limited resources, high costs, and animal welfare issues.

[0003] In recent years, enzymatic synthesis of UDCA has become a potential alternative method. 7β-Hydroxysteroid dehydrogenase (7β-HSDH) is a key enzyme that catalyzes the conversion of 7-ketolithocholic acid (7-KLCA) to UDCA (e.g. Figure 1 However, naturally occurring 7β-HSDH often suffers from low catalytic efficiency and poor stability, which limits its application in industrial production. Summary of the Invention

[0004] The present invention provides a variant of 7β-hydroxysteroid dehydrogenase and related applications, with the purpose of serving as a key enzyme for catalyzing the conversion of 7-ketolithocholic acid (7-KLCA) to ursodeoxycholic acid (UDCA), thereby improving the catalytic efficiency and stability of the catalytic reaction and enhancing the industrial production of ursodeoxycholic acid. To this end, the present invention provides the following technical solutions.

[0005] The present invention provides a variant of 7β-hydroxysteroid dehydrogenase, characterized in that: the variant is obtained by single mutation or multi-point combined mutation at positions 18, 40, 93, 94, 95, 145, 150, 153, 193, 196, 197, 198, and 199 of the amino acid sequence shown in SEQ ID NO.1.

[0006] The variant of 7β-hydroxysteroid dehydrogenase provided by the present invention also has the following characteristics:

[0007] The amino acid after the mutation at position 18 is one of the following: threonine (T), cysteine ​​(C), valine (V); the amino acid after the mutation at position 40 is one of the following: lysine (K), valine (V), tryptophan (W); the amino acid after the mutation at position 93 is one of the following: phenylalanine (F); the amino acid after the mutation at position 94 is one of the following: valine (V), isoleucine (I); the amino acid after the mutation at position 95 is one of the following: leucine (L), isoleucine (I), cysteine ​​(C); the amino acid after the mutation at position 145 is one of the following: cysteine ​​(C), alanine (A), isoleucine (I), valine (V); the amino acid after the mutation at position 150 is one of the following: The amino acid after mutation is one of the following: cysteine ​​(C), valine (V), leucine (L), isoleucine (I); the amino acid after mutation at position 153 is one of the following: leucine (L), methionine (M), cysteine ​​(C); the amino acid after mutation at position 193 is methionine (M); the amino acid after mutation at position 196 is one of the following: methionine (M), leucine (L); the amino acid after mutation at position 197 is isoleucine (I); the amino acid after mutation at position 198 is phenylalanine (F); the amino acid after mutation at position 199 is one of the following: leucine (L), methionine (M), cysteine ​​(C); or,

[0008] The present invention provides a variant of 7β-hydroxysteroid dehydrogenase, the amino acid sequence of which is shown in any one of SEQ ID NOs: 2-7, or the amino acid sequence of the variant has at least 85%, 90%, 95% or 99% identity with any one of SEQ ID NOs: 2-7.

[0009] The present invention also provides a nucleic acid characterized by encoding a variant of the aforementioned 7β-hydroxysteroid dehydrogenase.

[0010] The present invention also provides a vector, characterized in that it contains the aforementioned nucleic acid.

[0011] The present invention also provides a genetically engineered strain, characterized in that it contains the aforementioned vector or the aforementioned nucleic acid is integrated into its cell genome.

[0012] The present invention also provides the use of the aforementioned nucleic acid, the aforementioned vector or the aforementioned genetically engineered strain in preparing the aforementioned variant of 7β-hydroxysteroid dehydrogenase.

[0013] The present invention also provides the use of the aforementioned variant of 7β-hydroxysteroid dehydrogenase, the nucleic acid of claim 3, the vector of claim 4 or the genetically engineered strain of claim 5 in catalyzing the conversion of 7-ketolithocholic acid into ursodeoxycholic acid.

[0014] The present invention also provides the use of the aforementioned variant of 7β-hydroxysteroid dehydrogenase, the aforementioned nucleic acid, the aforementioned vector or the aforementioned genetically engineered strain in the biocatalytic preparation of ursodeoxycholic acid.

[0015] The present invention also provides a method for catalyzing the conversion of 7-ketolithocholic acid into ursodeoxycholic acid, characterized in that: a variant of the aforementioned 7β-hydroxysteroid dehydrogenase is used to catalyze the conversion of 7-ketolithocholic acid into ursodeoxycholic acid in a buffer solution with a pH of 7.0-8.5; preferably, the buffer solution is a phosphate solution.

[0016] The method for catalyzing the conversion of 7-ketolithocholic acid into ursodeoxycholic acid provided by the present invention also has the following characteristics: the reaction temperature range is 30-45° C.; and the reaction time range is 6-8 hours.

[0017] Beneficial effects:

[0018] The 7β-hydroxysteroid dehydrogenase variant provided by the present invention is obtained by single mutation or multi-point combined mutation at positions 18, 40, 93, 94, 95, 145, 150, 153, 193, 196, 197, 198, and 199 of the amino acid sequence shown in SEQ ID NO. 1. It has been verified that, compared with the wild type, in the process of catalyzing the conversion of 7-ketolithocholic acid to ursodeoxycholic acid, the yield can reach at least nearly 95%, and even as high as 99.6%, which is significantly improved compared to 55.8% of the wild type. This greatly reduces the production cost of catalyzing the conversion of 7-ketolithocholic acid to ursodeoxycholic acid, and has certain significance for the industrial production of ursodeoxycholic acid.

[0019] Furthermore, it has been verified that the variant provided by the present invention can adapt to a wider range of temperature and pH catalytic conditions compared to the wild type, and the reaction can maintain a certain stability in a wider range, which is further beneficial for application in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the reaction process from chenodeoxycholic acid (CDCA) via 7-ketolithocholic acid to UDCA;

[0021] Figure 2 The gel images of the wild-type 7β-hydroxysteroid dehydrogenase and several variants obtained in Example 1 are shown;

[0022] Figure 3 This is the peak diagram of the ursodeoxycholic acid standard in Example 2;

[0023] Figure 4 This is the HPLC standard curve diagram involved in Example 2. DETAILED DESCRIPTION

[0024] The following is a detailed description of the specific embodiments of the present invention. For the specific methods or materials used in the examples, those skilled in the art can make conventional replacements based on the technical ideas of the present invention and existing technologies, and are not limited to the specific description of the embodiments of the present invention.

[0025] Unless otherwise specified, the methods used in the examples are conventional methods; the materials and reagents used are all commercially available unless otherwise specified.

[0026] The variant herein refers to a variant that has different amino acids than the wild type but retains the basic properties of the wild type.

[0027] The wild-type 7β-hydroxysteroid dehydrogenase herein is derived from Collinsella and has a sequence of SEQ ID NO.1.

[0028] In this article, single mutation refers to the mutation of amino acids at only one position; multi-point combined mutation refers to the simultaneous mutation of amino acids at any several positions.

[0029] Example 1 Enzyme expression and purification

[0030] The genes encoding the amino acid sequences shown in SEQ ID NOs: 1-7 (the specific sequences are shown in Table 1) (the specific sequences are shown in SEQ ID NOs: 8-14) were cloned into the expression vector pET28a(+) (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.), transformed into Escherichia coli BL21 (DE3) (purchased from General Biotechnology (Anhui) Co., Ltd.) for expression, and purified by affinity chromatography and other methods to obtain 7β-hydroxysteroid dehydrogenase or its variants.

[0031] (1) Recombinant E. coli culture and crude enzyme solution preparation experiments

[0032] 1. Transform the plasmid into Escherichia coli BL21 (DE3): On a clean bench, take 2 μL of 50 mg / L plasmid and add it to 100 μL of BL21 (DE3) competent cell suspension. Flick it by hand or mix it with a gun, and place it on ice for 30 minutes; heat shock it in a 42°C water bath for 90 seconds, and quickly cool it on ice for 5 minutes after heat shock. Be careful not to shake it. On a clean bench, heat 0.9 mL of LB liquid culture medium to the cell suspension, mix it, and then shake it at 37°C for 45 minutes at a speed of 150-225 rpm to restore the cells to normal growth. Centrifuge it at 4000 rpm for 1 minute, aspirate the supernatant on the clean bench until 200 uL of bacterial liquid remains, and mix it by pipetting;

[0033] 2. Spreading: Apply 200 μL of the solution to a plate containing kanamycin-containing LB medium and spread evenly with a disposable spreading rod. Seal the plate with sealing film and place it upright until the bacterial solution is fully absorbed. Invert the plate and incubate at 37°C for 12-24 hours until transformants appear.

[0034] 3. Cultivate the primary seed solution: Use 10 mL of LB liquid medium with a final concentration of 50 μg / mL of kanamycin, pick a single clone, and culture the primary seed solution at 37°C, 200 rpm, for 12-18 hours.

[0035] 4. Send for gene sequencing: Take 2 mL of the cultured primary seed solution and send it for gene sequencing to verify whether the sequence of the target gene fragment on the plasmid is correct;

[0036] 5. Glycerol preservation: On a clean bench, open a sterile sterile tube and add 0.5 mL of first-grade seed solution using a pipette, followed by 0.5 mL of sterilized 50% glycerol. Mix thoroughly using a pipette and secure the lid. Store in a -80°C freezer.

[0037] 6. Prepare a primary seed culture using glycerol stock: After sequencing verification, culture the primary seed culture in 10 mL of LB liquid medium, with a final concentration of 50 μg / mL of kanamycin and 10 μL of 7βHSDH or a variant glycerol stock. Culture conditions: 37°C, 200 rpm, for 12-18 hours.

[0038] 7. Cultivate the secondary seed solution: Add 200 mL of LB medium, add 10 mL of the 7β-HSDH variant primary seed solution, and culture the secondary seed solution at 37°C and 180 rpm. During the culture, sample and measure the OD600nm until the OD reaches 0.6-0.8.

[0039] 8. Induction: After the secondary seed solution is cultured, add IPTG aqueous solution to the secondary seed solution on a clean bench to achieve a final IPTG concentration of 0.5 mM. Culture conditions: 25°C, 180 rpm, 16-24 hours;

[0040] 9. Collect bacteria by low-temperature centrifugation: After induction, place 40 mL of the culture solution in a 50 mL centrifuge tube and centrifuge at 8000 rpm for 5 minutes at 4°C. Remove the supernatant and retain the bacterial sludge.

[0041] 10. Washing: Add 5 mL of Tris-HCl buffer (50 mM, pH 6.8-7.2) to each centrifuge tube, mix thoroughly by pipetting, combine several tubes into one tube, vortex, and centrifuge at 8000 rpm for 5 min. Remove the supernatant and retain the bacterial sludge.

[0042] 11. Ultrasonic disruption: To the washed bacterial sludge, add 15 mL of Tris-HCl buffer (50 mM, pH 6.8-7.2) to a 50 mL centrifuge tube. Resuspend by pipetting, vortex to mix, and set the ultrasonic disruptor to 230 W. Ultrasonicate for 3 seconds, pause for 7 seconds, for a total of 40 minutes. Keep the centrifuge tube in an ice-water bath during the ultrasonic disruption phase to begin disrupting the bacterial cells.

[0043] 12. Centrifugation to remove precipitate: After ultrasonic disruption, centrifuge the bacterial suspension at 4°C, 10,000 rpm for 40 minutes. Remove the precipitate and retain the supernatant to obtain the crude enzyme solutions of 7β-hydroxysteroid dehydrogenase and variant enzyme, respectively.

[0044] (2) Affinity chromatography purification of enzyme products

[0045] Technical solution:

[0046] 1. Affinity Chromatography Column Preparation

[0047] When using a Ni-NTA affinity chromatography column (suitable for His-tagged fusion proteins), the buffer should contain 20 mM imidazole to reduce nonspecific adsorption.

[0048] 2. Sample Processing

[0049] The crude enzyme solution was centrifuged (12,000 × g, 15 min) and filtered through a 0.45 μm filter membrane before loading, with the flow rate controlled at 1 mL / min (column volume 5 mL).

[0050] 3. Washing and elution

[0051] Wash buffer: contains 50 mM phosphate (pH 7.4), 300 mM NaCl, and 40 mM imidazole to remove contaminating proteins.

[0052] Elution buffer: Increase the imidazole concentration to 250-500 mM and collect the target protein.

[0053] 4. Dialysis and Storage

[0054] The eluate was concentrated using a 10 kDa ultrafiltration tube and dialyzed into PBS (pH 7.2). The enzyme activity and purity were determined (verified by SDS-PAGE) to obtain the target enzyme products (numbered as: wild type, variant 1, variant 2, variant 3, variant 4, variant 5 and variant 6). The results are shown in Figure 2. Figure 2 As shown, Figure 2 In the figure, in addition to the wild type, from left to right, they are variant 1, variant 2, variant 3, variant 4, variant 5 and variant 6.

[0055]

[0056]

[0057]

[0058]

[0059] Example 2 Catalytic reaction

[0060] Catalytic reaction: Seven experiments were performed using the wild-type 7β-hydroxysteroid dehydrogenase and six groups of its variants obtained in Example 1. For each experiment, 8.0 g of 7-ketolithocholic acid was dissolved in 100 mL of pH 7.5 phosphate buffer solution, followed by the addition of 7.0 g of the wild-type 7β-hydroxysteroid dehydrogenase or its variant, 10.0 g of glucose, 6.0 g of GDH, and 0.15 g of NADP. The reaction was incubated at 37°C for 6-8 hours.

[0061] After the reaction is completed, the content of ursodeoxycholic acid in the reaction product is detected and analyzed by high performance liquid chromatography (HPLC). The HPLC detection method of ursodeoxycholic acid is as follows:

[0062] 1. Chromatographic conditions

[0063] Chromatographic column: C18 reverse phase column (4.6×250 mm, 5 μm).

[0064] Mobile phase:

[0065] Acetonitrile: phosphate buffer (adjusted to pH=3 with phosphoric acid): methanol = 3:3.7:4.

[0066] Flow rate: 0.8 mL / min; detector: differential detector; column temperature: 40°C ± 1°C; detection temperature: 35°C ± 1°C.

[0067] 2. Sample Processing

[0068] The sample to be tested (reaction product) was diluted with methanol (1:10) and filtered through a 0.22 μm filter membrane (injection volume 15 μL).

[0069] 3. Standard Curve and Quantification

[0070] Prepare a series of ursodeoxycholic acid standard concentrations (0.1-100 mg / mL) and analyze the peak area (e.g. Figure 4 Draw the standard curve (R 2 ≥0.99), quantified by external standard method, the standard curve equation is: y=15672x-26.012.

[0071] Based on the test results of each sample, the calculation results are shown in Table 2. The results show that the ursodeoxycholic acid produced by the mutant (yield at least close to 95%) is significantly higher than that produced by the wild type (yield 55.8%).

[0072] Yield in this article: The corresponding actual ursodeoxycholic acid concentration is obtained by substituting the HPLC peak area of ​​each sample into the standard curve equation, yield = actual ursodeoxycholic acid concentration / theoretical ursodeoxycholic acid concentration.

[0073] The theoretical ursodeoxycholic acid concentration is: (8000*392.58) / (390.56*100)=80.41 mg / mL.

[0074]

[0075]

[0076] Example 3 Comparison of different catalytic conditions

[0077] According to the catalytic reaction method of Example 2, other conditions remain unchanged, and the catalytic temperature and pH conditions are adjusted respectively. Several groups of comparisons are carried out, and the catalytic results are analyzed in the same manner as in Example 2. The results are shown in Table 3:

[0078]

[0079] As can be seen from Table 3, the variant obtained by the modification of the present invention has a wider range of reaction temperature and pH conditions than the wild type, is easier to maintain reaction stability, and is conducive to industrialization.

[0080] While the preferred embodiments of the present invention have been described in detail above, it should be understood that, after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention. Such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A variant of 7β-hydroxysteroid dehydrogenase, characterized in that: The variant is obtained by single mutation or multi-point combined mutation at positions 18, 40, 93, 94, 95, 145, 150, 153, 193, 196, 197, 198, and 199 of the amino acid sequence shown in SEQ ID NO.

1.

2. A variant according to claim 1, characterized in that: The amino acid after mutation at position 18 is one of the following: threonine (T), cysteine ​​(C), or valine (V); The amino acid after mutation at position 40 is one of the following: lysine (K), valine (V), or tryptophan (W); The amino acid after mutation at position 93 is: phenylalanine (F); The amino acid after mutation at position 94 is one of the following: valine (V), isoleucine (I); The amino acid after mutation at position 95 is one of the following: leucine (L), isoleucine (I), cysteine ​​(C); The amino acid after mutation at position 145 is one of the following: cysteine ​​(C), alanine (A), isoleucine (I), valine (V); The amino acid after mutation at position 150 is one of the following: cysteine ​​(C), valine (V), leucine (L), isoleucine (I); The amino acid after mutation at position 153 is one of the following: leucine (L), methionine (M), cysteine ​​(C); The amino acid after mutation at position 193 is: methionine (M); The amino acid after mutation at position 196 is one of the following: methionine (M), leucine (L); The amino acid after mutation at position 197 is: isoleucine (I); The amino acid after mutation at position 198 is: phenylalanine (F); The amino acid after mutation at position 199 is one of the following: leucine (L), methionine (M), cysteine ​​(C); or The amino acid sequence of the variant is shown in any one of SEQ ID NO: 2-7, or The amino acid sequence of the variant is at least 85%, 90%, 95% or more identical to any one of SEQ ID NOs: 2-7.

3. A nucleic acid, characterized in that: A variant encoding the 7β-hydroxysteroid dehydrogenase according to claim 1 or 2.

4. A carrier, characterized in that: Containing the nucleic acid according to claim 3.

5. A genetically engineered strain, characterized in that: A vector comprising claim 4 or a cell genome thereof in which the nucleic acid according to claim 3 is integrated.

6. Use of the nucleic acid of claim 3, the vector of claim 4 or the genetically engineered strain of claim 5 in preparing the variant of 7β-hydroxysteroid dehydrogenase according to claim 1 or 2.

7. Use of the variant of 7β-hydroxysteroid dehydrogenase according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4 or the genetically engineered strain according to claim 5 in catalyzing the conversion of 7-ketolithocholic acid into ursodeoxycholic acid.

8. Use of the variant of 7β-hydroxysteroid dehydrogenase according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4 or the genetically engineered strain according to claim 5 in the biocatalytic production of ursodeoxycholic acid.

9. A method for catalyzing the conversion of 7-ketolithocholic acid into ursodeoxycholic acid, characterized in that: Using the variant of 7β-hydroxysteroid dehydrogenase according to claim 1 or 2, catalyzing the conversion of 7-ketolithocholic acid into ursodeoxycholic acid in a buffer solution of pH 7.0-8.5; Preferably, the buffer solution is a phosphate solution.

10. The method according to claim 9, characterized in that: The reaction temperature range is 30-45°C; The reaction time range is 6-8 hours.

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