7alpha-hydroxysteroid dehydrogenase from animal fecal metagenome and preparation method and application thereof

Abstract

This invention discloses a 7α-hydroxysteroid dehydrogenase derived from animal fecal metagenomics, its preparation method, and its applications, belonging to the field of genetic engineering technology. The amino acid sequence of the enzyme is shown in SEQ ID NO.2, and the nucleotide sequence encoding the enzyme is shown in SEQ ID NO.1. The enzyme has a specific activity of 444.15 ± 41.38 U / mg, an optimal temperature of 50℃, an optimal pH of 10.0, and retains over 90% of its activity after 12 hours of incubation at pH 10.0. It exhibits good alkali tolerance. EDTA enhances enzyme activity, while Ag+ and SDS completely inactivate the enzyme. This enzyme has broad substrate specificity, catalyzing the conversion of chenodeoxycholic acid to 7-keto-lithocholic acid, providing a highly efficient candidate enzyme for the biosynthesis of ursodeoxycholic acid, and has significant application value in the medical field.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a 7α-hydroxysteroid dehydrogenase derived from animal fecal metagenomics, its preparation method, and its application. Background Technology

[0002] 7α-hydroxysteroid dehydrogenase (7α-HSDH, EC: 1.1.1.159) is an enzyme that catalyzes the conversion of chenodeoxycholic acid (CDCA) to 7-keto-lithocholic acid (7K-LCA) with NAD or NADP as cofactors. This enzyme is widely present in the gut microbiota and plays an important role in host health and disease progression. Studies have shown that 7-keto-lithocholic acid is an intermediate in the formation of ursodeoxycholic acid, an important compound that has shown excellent therapeutic effects in treating gallstones, cholestasis, bile reflux gastritis, and primary biliary cirrhosis.

[0003] In recent years, reported 7α-hydroxysteroid dehydrogenases have mainly come from... Shewanella morhuae (Xue JT, You ZN, Yang BY, et al. Efficient synthesis of 7-oxo-lithocholic acid using a newly identified 7α-hydroxysteroid dehydrogenase [J]. Molecular Catalysis, 2024, 553: 113739.), Stenotrophomonas maltophilia (Tonin F et al., 2019): ToninF, Otten LG, Arends I. NAD(+) -Dependent Enzymatic Route for theEpimerization of Hydroxysteroids [J]. ChemSusChem, 2019, 12(13): 3192-203.), Brevundimonas sp.(Chen X, Cui Y, Feng J, et al. Flavin Oxidoreductase-Mediated Regeneration of Nicotinamide Adenine Dinucleotide with Dioxygen andCatalytic Amount of Flavin Mononucleotide for One-Pot Multi-EnzymaticPreparation of Ursodeoxycholic Acid [J]. Advanced Synthesis&Catalysis, 2019,361(11): 2497-504.), Clostridium absonum (Ferrandi E E, Bertolesi G M,Polentini F, et al. In search of sustainable chemical processes: cloning,recombinant expression, and functional characterization of the 7α- and 7β-hydroxysteroid dehydrogenases from Clostridium absonum [J]. AppliedMicrobiology and Biotechnology, 2012, 95(5): 1221-1233.), Bacteroides fragilis(Bennett MJ, McKnight SL, Coleman J P. Cloning and characterization of theNAD-dependent 7alpha-Hydroxysteroid dehydrogenase from Bacteroides fragilis[J]. Current Microbiology, 2003, 47(6): 475-484.), Clostridium sordellii(Coleman JP, Hudson LL, Adams M J. Characterization and regulation of theNADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridiumsordellii [J]. Journal Of Bacteriology, 1994, 176(16): 4865-4874.), Eubacterium sp. strain (Baron SF, Franklund CV, Hylemon P B. Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708 [J]. JBacteriol, 1991, 173(15): 4558-69.) These studies generally suffer from low heterologous expression efficiency and insufficient basic stability. The catalytic properties of 7α-HSDH directly determine the efficiency of the UDCA biosynthetic pathway; therefore, developing enzymes with superior performance is a current research focus. While existing molecular modification methods can optimize wild-type enzymes, their effectiveness is largely limited by the inherent properties of the enzyme molecules themselves. Therefore, exploring and discovering new microbial sources of 7α-HSDH to find enzymes with superior natural activity and stability remains a fundamental research direction that urgently needs to be promoted.

[0004] The animal gastrointestinal tract is a rich repository of microbial enzymes, but its microbial communities are complex, and the vast majority are difficult to culture using traditional methods. The emergence of metagenomics has overcome this limitation, enabling researchers to obtain genetic resources directly from the environment without relying on culture methods. At present, metagenomic technology has been used from black bears (Song C, Wang B, Tan J, et al. Discovery of tauroursodeoxycholic acid biotransformation enzymes from the gut microbiome of black bears using metagenomics [J]. Scientific reports, 2017, 7: 45495.; Tang S, Pan Y, Lou D, et al. Structural and functional characterization of a novel acidophilic 7α-hydroxysteroid dehydrogenase [J]. Protein Science, 2019, 28(5): 910-919.; Ji S et al. A novel 7α-hydroxysteroiddehydrogenase: Magnesium ion significantly enhances its activity and thermomostability [J]. International journal of biological molecules, 2021, 177: 111-8.; Lou D, Cao Y, Duan H, et al. Characterization of a macro NovelThermostable 7α-hydroxysteroid Dehydrogenase [J]. Some thermostable, acidophilic 7α-hydroxysteroid dehydrogenases were obtained from the metagenomics of Protein and peptideletters (2024). However, all the gut-derived 7α-HSDHs discovered so far have come from the single species of black bear, and similar resources in other animals still need to be explored.

[0005] Investigating the differences in enzyme activity and stability of 7α-HSDH from different animal sources and identifying enzymes with superior performance will help broaden the natural enzyme resource library of 7α-HSDH and provide new candidate enzymes for the biosynthesis of UDCA.

[0006] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a 7α-hydroxysteroid dehydrogenase derived from animal fecal metagenomics, which has more beneficial properties in terms of enzyme activity and stability.

[0008] To achieve the above objectives, the present invention provides a 7α-hydroxysteroid dehydrogenase derived from animal fecal metagenomics, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The present invention also provides a gene encoding the above-mentioned 7α-hydroxysteroid dehydrogenase, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] The present invention also provides a recombinant expression vector containing the above-mentioned coding gene.

[0011] Preferably, the recombinant expression vector is selected from pEASY-E2.

[0012] The present invention also provides a recombinant strain containing the above-mentioned recombinant expression vector.

[0013] Preferably, the recombinant strain is selected from Escherichia coli, more preferably from BL21(DE3).

[0014] The present invention also provides a set of primers for amplifying the above-mentioned coding gene, the nucleotide sequences of which are shown in SEQ ID NO. 3 and 4. The present invention also provides a method for preparing the above-mentioned 7α-hydroxysteroid dehydrogenase, comprising the following steps: 1) Using metagenomic DNA from fecal microorganisms of the western black-crested gibbon as a template, the target gene was amplified by PCR using primers with nucleotide sequences as shown in SEQ ID NO. 3 and 4; 2) The obtained target gene is ligated with the enzyme-digested vector to construct a recombinant expression vector; 3) The obtained recombinant expression vector was transformed into competent E. coli cells, and positive recombinant strains were screened. 4) Cultivate positive recombinant strains, induce expression with IPTG, and purify with Nickel-NTA Agarose to obtain 7α-hydroxysteroid dehydrogenase.

[0015] Preferably, in the above preparation method, the final concentration of IPTG in step 4) is 0.2 mmol / L, and the induction conditions are 16℃ and 110 rpm for 12 h.

[0016] The 7α-hydroxysteroid dehydrogenase provided by this invention can be used to catalyze the conversion of chenodeoxycholic acid to 7-keto-lithocholic acid.

[0017] Preferably, the optimal temperature for the catalytic reaction in the above-mentioned catalytic application is 50°C, and the optimal pH is 10.0.

[0018] The 7α-hydroxysteroid dehydrogenase provided by this invention can be used in the biosynthesis of ursodeoxycholic acid.

[0019] The present invention has the following advantages: This invention discloses a novel 7α-hydroxysteroid dehydrogenase derived from animal fecal metagenomics, the amino acid sequence of which is shown in SEQ ID NO.2, consisting of 258 amino acids, with a theoretical molecular weight of 27.03 kDa, and the nucleotide sequence encoding the enzyme is shown in SEQ ID NO.1.

[0020] This invention provides a heterologously expressed 7α-hydroxysteroid dehydrogenase with a specific activity of 444.15 ± 41.38 U / mg, significantly higher than other reported microbial-derived 7α-HSDH. The optimal temperature for this 7α-hydroxysteroid dehydrogenase was 50℃, and the optimal pH was 10.0. After incubation at pH 10.0 for 12 hours, it retained over 90% of its residual enzyme activity, demonstrating good alkali tolerance. EDTA significantly enhanced the dehydrogenase's activity, while SDS completely inactivated it. Substrate specificity analysis showed that the dehydrogenase provided by this invention also exhibited broad substrate specificity, capable of hydrolyzing not only CDCA but also bile acids and their derivatives. The unique properties of this enzyme, especially its high catalytic efficiency, are of great significance for promoting the application of 7α-hydroxysteroid dehydrogenase in medical and other fields. Attached Figure Description

[0021] Figure 1 This is the SDS-PAGE analysis result of the recombinant 7α-hydroxysteroid dehydrogenase expressed in Escherichia coli provided by the present invention.

[0022] Figure 2 This is the optimal pH determination result of the recombinant 7α-hydroxysteroid dehydrogenase provided by the present invention.

[0023] Figure 3 The results are the pH stability determination results of the recombinant 7α-hydroxysteroid dehydrogenase provided by this invention.

[0024] Figure 4 The results show the optimal temperature determination of the recombinant 7α-hydroxysteroid dehydrogenase provided by this invention.

[0025] Figure 5 The results are the temperature stability determination results of the recombinant 7α-hydroxysteroid dehydrogenase provided by this invention.

[0026] Figure 6 The results are the substrate-specific assay results of the recombinant 7α-hydroxysteroid dehydrogenase provided by this invention.

[0027] Figure 7 The results show the production yield of 7K-LCA from the recombinant 7α-hydroxysteroid dehydrogenase provided by this invention. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] Some of the experimental materials and reagents: 1. Strains and vectors: Western black-crested gibbon fecal microbial metagenomic DNA and expression vector pEASY-E2 were purchased from Beijing TransGen Biotech Co., Ltd. E. coli BL21(DE3) was purchased from Qingke Biotechnology (Beijing).

[0031] 2. Enzymes, kits and other biochemical reagents used in genetic engineering: Plasmid extraction kit and gel extraction and purification kit were purchased from Omega Laboratories, USA; all other reagents were of analytical grade.

[0032] 3. LB medium: Peptone 10 g, Yeast extract 5 g, NaCl 10 g, add distilled water to 1000 mL, pH natural (approximately 7). 7% (w / v) NaCl LB medium: Peptone 10 g, Yeast extract 5 g, NaCl 70 g, add distilled water to 1000 mL, pH natural (approximately 7). Solid medium is prepared by adding 2.0% (w / v) agar to the above.

[0033] Note: Molecular biology experimental methods not specifically described in the following examples were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0034] Example 1: Obtaining a novel 7α-hydroxysteroid dehydrogenase 1) Screening for 7α-hydroxysteroid dehydrogenase Based on gene prediction, functional annotation, and secretory protein prediction analysis, a 7α-hydroxysteroid dehydrogenase gene, denoted as NC3-2, was screened and annotated from the West Black Crested Gibbon fecal microbial metagenomic library. Its gene sequence is shown in SEQ ID NO.1, and the encoded enzyme sequence is shown in SEQ ID NO.2.

[0035] 7α-hydroxysteroid dehydrogenase encoding gene (SEQ ID NO.1): ATGTCGATAGAATCGCTTTTCGACCTTACCGGAAAGGTCGCTATTGTTACCGGCGGAGGAAACGGGATCGGTCGCCGGTGTTGCGAAACGCTCGCAGCGGCAGGGGCCGACATTGTGATCGGAGACCTGAAAGCCGAAGATGCCGAAACGGTGGCCCGCGGAATAAGGGAGAGCGGAGGGCGGGCGTTGAGCGTTCCGTGCAACGTGCTTGAAGATCATGACCTTGTCCGCCTGGTGGATAAGGCGGTCGAAACATTCGGAACCGTCAATATATTGGTGAACAATGCCGGCGGGGGCGGTGGCGGAAAAGAAAATCCGTTCGAAATAGATGTGGAGTATTTTGAAAAAATATTCAAACTCAATGTATTCAGTGTCTGGCGTCTCTGTCAGCTCGTCGTGCCTCACATGGCTAAGTCGGGATATGGCAGTATCGTCAATATTACGTCTATGGGCAGCATAAACAAAAGTCCCGGTATGAGCGCCTATGCCTCGTCGAAAGCCGCATTGAATCATATGGCGGCTAACCTGGCATACGATTTCGGCCCGATGAACGTAAGAATCAATAACGTAGGGCCTGGCGCGACCCGGACTCATGCGTTGTCCACGGTATTGACGCCCGACATCGAGGCCCGTATGCTGGCTCATACTCCTCTGAAGCGGCTCGGGGAAACGGCCGATATCGCAGGGGCCGTGTTATATTTCGCGGCTCCGGTTTCGCAATGGGTCAGCGGTCAGGTGCTGTTCGTAAACGGCGGCGGAGTGCAGACACTCGACTGA。

[0036] 7α-Hydroxysteroid dehydrogenase (SEQ ID NO.2): MSIESLFDLTGKVAIVTGGGNGIGRRCCETLAAAGADIVIGDLKAEDAETVARGIRESGGRALSVPCNVLEDHDLVRLVDKAVETFGTVNILVNNAGGGGGGKENPFEIDVEYFEKIFKLNVFSVWRLC QLVVPHMAKSGYGSIVNITSMGSINKSPGMSAYASSKAALNHMAANLAYDFGPMNVRINNVGPGATRTHALSTVLTPDIEARMLAHTPLKRLGETADIAGAVLYFAAPVSQWVSGQVLFVNGGGVQTLD.

[0037] 2) Cloning of the 7α-hydroxysteroid dehydrogenase gene NC3-2 Primer pairs NC3-2-F and NC3-2-R were designed for PCR amplification using fecal microbial metagenomic DNA of the western black-crested gibbon as a template. The PCR reaction mixture (10 μL) consisted of: 5 μL of 2×HieffCanace® PCRMasterMix high-fidelity enzyme premix, 0.4 μL each of the two 10 μm primers, 1 μL of fecal microbial metagenomic DNA, and ddH2O to a final volume of 10 μL.

[0038] The primer pairs have the following sequences, 5'-3' respectively: NC3-2-F (SEQ ID NO.3): TAAGAAGGAGATATACATATGGAATTGATGTCGATAGAATCGCTT; NC3-2-R (SEQ ID NO.4): GTGGTGGTGGTGGTGCTCGAGGTCGAGTGTCTGCACTC.

[0039] The PCR reaction parameters were as follows: 98℃ for 3 min; 98℃ for 10 s, 70-55℃ for 20 s, 72℃ for 30 s, 15 cycles, decreasing by 1℃ in each cycle; 98℃ for 10 s, 55℃ for 20 s, 72℃ for 30 s, 20 cycles; 72℃ for 5 min; 4℃ for 10 min, to amplify the target gene fragment.

[0040] 3) Construction of recombinant vectors Following the instructions for use of the Clon Express II One Step Cloning Kit and Exnase II, 2 μL of enzyme-digested pEASY-E2, 1 μL of the target gene NC3-2, 2 μL of CE buffer, 1 μL of Exnase II, and 4 μL of sterile water were mixed and reacted in a PCR instrument at 37℃ for 30 min to complete the ligation reaction. A recombinant expression vector pEASY-E2 / NC3-2 was obtained after verification.

[0041] The ligated recombinant expression vector was transformed into competent BL21(DE3) cells and incubated upside down at 37 ℃ for 12–16 h. Colony PCR was used to identify the recombinant plasmid, verifying the authenticity of the positive clone and ruling out false positives, resulting in a positive recombinant E. coli strain, E. coli-pEASY-E2 / NC3-2.

[0042] Example 2: Preparation of recombinant 7α-hydroxysteroid dehydrogenase NC3-2 The recombinant *E. coli* strain E. coli-pEASY-E2 / NC3-2 prepared in Example 1 was inoculated at 0.1% in LB broth (containing 100 μg / mL Amp) and cultured overnight at 37°C and 180 rpm. Then, this activated bacterial solution was inoculated at 1% in fresh LB broth (containing 100 μg / mL Amp) and cultured at 37°C and 180 rpm for approximately 3–4 h (until OD600 reached 0.6–0.8). IPTG was then added to a final concentration of 0.2 mmol / L for induction, and the culture was carried out at 16°C and 110 rpm for approximately 12 h. After induction, the bacterial solution was aliquoted and centrifuged at 6000 rpm for 10 min in a refrigerated centrifuge to collect the bacterial cells. The cells were resuspended in an appropriate amount of pH=8 buffer and then sonicated under ice bath conditions. The intracellular concentrated initial enzyme solution was centrifuged at 12,000 rpm and 4°C for 10 min. The supernatant was collected and the target protein was purified with Nickel-NTA Agarose to obtain recombinant 7α-hydroxysteroid dehydrogenase, which consists of 258 amino acids and has a theoretical molecular weight of 27.03 kDa.

[0043] The recombinant 7α-hydroxysteroid dehydrogenase NC3-2 expressed in *Escherichia coli* was purified and analyzed by SDS-PAGE. The results are shown in the figure below. Figure 1 As shown, where, Figure 1 Lane M in the diagram represents a low molecular weight protein marker; lane 1 represents the initial enzyme induced by *E. coli* containing only the pEasy-E2 vector; lane 2 represents the unpurified crude 7α-hydroxysteroid dehydrogenase; and lane 3 represents the purified recombinant 7α-hydroxysteroid dehydrogenase. Figure 1It can be seen that NC3-2 was expressed in Escherichia coli and was a single band after purification with Nickel-NTAAgarose.

[0044] Example 37: Determination of the properties of α-hydroxysteroid dehydrogenase NC3-2 In this embodiment, the enzyme activity assay was performed according to Tang SJ's method, as follows: Add 310 μL Tris-HCl buffer (pH=10.0), 30 μL CDCA, and 30 μL NAD+ to an EP tube. Preheat at 37℃ for 15 minutes, then add 30 μL of enzyme diluted appropriately. React for 2 minutes, then boil in water for 5 minutes to terminate the reaction. After centrifugation, transfer 200 μL of the supernatant to an ELISA plate and read the OD value at 340 nm using an ELISA reader. Replace the 30 μL enzyme solution with 30 μL buffer as a blank control. Include one blank control group in each reaction system and three parallel experimental groups, ensuring identical reaction conditions for each group. Calculate the 7α-hydroxysteroid dehydrogenase activity and specific enzyme activity based on the NADH standard curve. Using the enzyme activity measured under optimal reaction conditions as 100%, calculate the relative enzyme activity, and repeat the experiment three times.

[0045] Enzyme activity unit is defined as: under the optimal reaction conditions of an enzyme, the amount of enzyme required to reduce one enzyme activity unit (U) to NADH per mol of NAD+.

[0046] 1) Determination of the optimal pH and pH stability of 7α-hydroxysteroid dehydrogenase NC3-2 Determination of the optimal pH of the enzyme: The 7α-hydroxysteroid dehydrogenase purified in Example 2 was subjected to an enzymatic reaction in a buffer solution at 37°C and pH 5.0-12.0.

[0047] Determination of enzyme pH stability: The purified enzyme solution was placed in a buffer solution with pH=9.0-11.0 and incubated at 37℃ for 12h. Enzyme activity was measured every 2h, with untreated enzyme solution as a control.

[0048] The buffer solutions are: citrate-Na2HPO4 buffer solution with pH 5-6; Tris-HCl buffer solution with pH 7-8; and glycine-NaOH buffer solution with pH 9-12.

[0049] See Figure 2 and Figure 3 , Figure 2 The optimal pH determination results for 7α-hydroxysteroid dehydrogenase NC3-2 provided by this invention are as follows: Figure 3 The pH stability results of the 7α-hydroxysteroid dehydrogenase NC3-2 provided by this invention are presented. Figure 2 and Figure 3It is known that the optimal pH for 7α-hydroxysteroid dehydrogenase NC3-2 is 10.0, and the enzyme activity can still be maintained at more than 60% after treatment in the pH range of 9.0-11.0 for 12 hours.

[0050] 2) Determination of the optimal temperature and temperature stability of 7α-hydroxysteroid dehydrogenase NC3-2 Determination of the optimal temperature for enzymes: Enzymatic reactions were carried out at pH=10.0 and 0-70℃.

[0051] Enzyme temperature stability assay: The same amount of enzyme solution was placed in the set temperature (25, 37, 50, 60℃) for 72h. Enzyme activity was measured every 2h to 12h. Untreated enzyme solution was used as control.

[0052] See Figure 4 , Figure 5 , Figure 4 This is the result of the optimal temperature determination of 7α-hydroxysteroid dehydrogenase NC3-2 provided by the present invention. Figure 5 This invention presents the results of a temperature stability assay for the 7α-hydroxysteroid dehydrogenase NC3-2. The results show that after incubation at 25°C for 72 hours, NC3-2 retained approximately 90% of its initial enzyme activity, indicating good stability at room temperature. Incubation at 37°C for the same time reduced the remaining enzyme activity to 68%, indicating that the enzyme's structural stability gradually decreases with increasing temperature. When the temperature was raised to its optimal reaction temperature of 50°C and incubated for 72 hours, the enzyme activity further decreased to 40%, reflecting that prolonged treatment at this temperature leads to significant denaturation or degradation of the enzyme protein. More significantly, after incubation at 60°C for 48 hours, the activity of NC3-2 was completely lost.

[0053] 3) Determination of kinetic parameters of 7α-hydroxysteroid dehydrogenase NC3-2 Enzyme activity was determined using different concentrations of CDCA (2-26 mM) as substrates under optimal conditions of pH=10.0, 50℃, and the same reaction time. Kinetic parameters were measured, and Km and Vmax values ​​were calculated using the Lineweaver-Burk method.

[0054] The substrate specificity of recombinant 7α-hydroxysteroid dehydrogenase was determined as follows: Figure 6As shown, the enzyme contains: 1: chenodeoxycholic acid (CDCA); 2: taurine chenodeoxycholic acid (TCDCA); 3: glycochenodeoxycholic acid (GCDCA); 4: cholic acid (CA); 5: deoxycholic acid (DCA). The results indicate that the enzyme is active against chenodeoxycholic acid (CDCA), taurine chenodeoxycholic acid (TCDCA), glycochenodeoxycholic acid (GCDCA), and cholic acid (CA), but inactive against deoxycholic acid (DCA). The production yield of 7K-LCA by 7α-hydroxysteroid dehydrogenase is shown in [the table below]. Figure 7 As shown in the figure. The results indicate that product accumulation shows a trend of rapid accumulation followed by slower accumulation. After 2 hours of reaction, the concentration of 7K-LCA reached 0.43 mg / mL; with the extension of reaction time, the product formation rate gradually slowed down, reaching a concentration of 0.53 mg / mL at 6 hours. Further extension of the reaction time did not significantly increase the concentration of 7K-LCA, indicating that the reaction had reached equilibrium and the substrate conversion was essentially saturated. Considering both reaction efficiency and production cycle, in practical applications, controlling the reaction time to around 6 hours can achieve a better balance between product yield and time cost. Meanwhile, under optimal conditions, the Km value and Vmax value of this enzyme were (32.42±5.35) mM and (1021±107.87) U / mg, respectively.

[0055] 4) Determination of the effects of different metal ions and chemical reagents on the activity of 7α-hydroxysteroid dehydrogenase NC3-2 The effects of adding metal ions (final concentration 1.25 mM) and chemical reagents (final concentration 1%) to the enzymatic reaction system on enzyme activity were investigated. Enzyme activity was measured under optimal conditions of pH=10 and 50℃ (the enzymatic reaction without metal ions and chemical reagents was used as a control under the same conditions), and the results are shown in Table 1.

[0056] Table 1. Effects of metal ions and chemical reagents on recombinant 7α-hydroxysteroid dehydrogenase NC3-2 ; Note: During the search, it was found that the 7α-HSDH provided in this invention differs from the publicly annotated glucose-1-dehydrogenase (WP_256620805.1) only in the three amino acids at its N-terminus. However, no reports were found regarding the nucleic acid sequence and enzymatic properties of this glucose-1-dehydrogenase. According to other literature, glucose-1-dehydrogenase mainly participates in the pentose phosphate pathway, catalyzing the oxidation of aldoses at the C1 position. It has a broad substrate spectrum for various monosaccharides, is mostly derived from intracellular or fermentation environments of microorganisms, and has a pH range closer to neutral. Industrial applications focus on biosensing, coenzyme regeneration, and glycosyl chemical synthesis, pursuing high catalytic efficiency and operational stability. In contrast, 7α-HSDH mainly participates in the bile acid metabolism pathway, specifically acting on the hydroxyl / keto group at the C7 position of the steroid backbone. Due to the bile acid metabolism environment (the upper intestinal pH is alkaline), it has evolved an optimal alkaline pH. Its active site often contains alkaline-tolerant residues. Industrially, it is mainly aimed at the green biomanufacturing of steroid drugs, emphasizing the enzyme's alkaline tolerance, thermal stability, and stereoselective regulation. The two have distinct properties and different industrial applications.

[0057] In summary, this invention provides a novel 7α-hydroxysteroid dehydrogenase derived from animal fecal metagenomics. This enzyme exhibits high specific activity and good alkali tolerance, as well as broad substrate specificity. It can catalyze the conversion of chenodeoxycholic acid to 7-keto-lithocholic acid, providing a highly efficient candidate enzyme for the biosynthesis of ursodeoxycholic acid and possessing significant application value in the medical field.

[0058] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A 7α-hydroxysteroid dehydrogenase derived from animal fecal metagenomics, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding 7α-hydroxysteroid dehydrogenase as described in claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

3. A recombinant expression vector containing the encoding gene of claim 2.

4. A recombinant strain containing the recombinant expression vector of claim 3.

5. A set of primers for amplifying the gene encoding as described in claim 2, characterized in that, The nucleotide sequences of the primers are shown in SEQ ID NO.3 and 4.

6. The method for preparing 7α-hydroxysteroid dehydrogenase as described in claim 1, characterized in that, Includes the following steps: 1) Using metagenomic DNA from fecal microorganisms of the western black-crested gibbon as a template, the target gene was obtained by PCR amplification using primers with nucleotide sequences as shown in SEQ ID NO.3 and 4; 2) The target gene is ligated with the enzyme-digested vector to construct a recombinant expression vector; 3) The obtained recombinant expression vector was transformed into competent E. coli cells, and positive recombinant strains were screened. 4) The positive recombinant strain was cultured, and the 7α-hydroxysteroid dehydrogenase was obtained by IPTG induction expression and purification with Nickel-NTA Agarose.

7. The preparation method according to claim 6, characterized in that, In step 4), the final concentration of IPTG is 0.2 mmol / L, and the induction conditions are 16℃ and 110 rpm for 12 h.

8. The application of the 7α-hydroxysteroid dehydrogenase as described in claim 1 in catalyzing the conversion of chenodeoxycholic acid to 7-keto-lithocholic acid.

9. The application according to claim 8, characterized in that, The optimal temperature for the catalytic reaction is 50℃, and the optimal pH is 10.

0.

10. The application of the 7α-hydroxysteroid dehydrogenase as described in claim 1 in the biosynthesis of ursodeoxycholic acid.

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