Bile salt hydrolase as well as preparation method and application thereof

Abstract

The invention discloses a bile salt hydrolase as well as a preparation method and application thereof. The amino acid sequence of the bile salt hydrolase is shown as SEQ ID NO.2, and the nucleotide sequence of a gene for coding the bile salt hydrolase is shown as SEQ ID NO.1. The bile salt hydrolase has good acid resistance, the optimum pH value is 5.0, and after the bile salt hydrolase is treated for 1 h within the pH value range of 3.0-9.0, the residual enzyme activity is 80% or above; the optimum temperature is 40 DEG C, the strain can tolerate for 1 h under the conditions of 30 DEG C and 40 DEG C, and the residual enzyme activity is 60% or above. The recombinant strain E.coli / NCbsh3 is subjected to induced expression in a cholesterol-containing culture medium for 16 hours, and the cholesterol degradation rate reaches 56%.

Description

Technical Field

[0001] The invention relates to a hydrolase, in particular to a bile salt hydrolase and a preparation method and application thereof. Background Art

[0002] The information provided in this section is for the purpose of generally presenting the background of the present disclosure. To the extent described in this section, the work of the presently named inventors and aspects of the description that may not constitute prior art at the time of filing are neither explicitly nor implicitly admitted to be prior art to the present disclosure.

[0003] Bile salt hydrolase (EC 3.5.1.24, BSH), belongs to the N-terminal nucleophilic hydrolase family, and is an enzyme that catalyzes the hydrolysis of glycine / taurine-bound bile acids into unbound bile acids and amino acid residues. Current studies have shown that BSH is mainly distributed in the intestinal flora, and the intestines and feces of humans and mammals are the main sources of BSH-producing strains. Bile salt hydrolases have been detected or purified in many microorganisms, such as Clostridium, Bacteroides, Lactobacillus, Listeria, Enterococcus, Bifidobacterium, etc. The enzymatic activity and substrate selectivity of bile salt hydrolases from different sources are significantly different, and different strains also have different types and quantities of bile salt hydrolases.

[0004] Bile salts, as a defense mechanism of the gastrointestinal tract, are toxic to intestinal microorganisms. The free bile salts released by bile salt hydrolases are less toxic, thereby reducing the inhibitory ability of bile on bile salt hydrolase-producing probiotics and enhancing the ability of strains to colonize in the intestine. In addition, bile salt hydrolases can increase the tension of bacterial cell membranes and change the fluidity and permeability of membranes, thereby providing potential protection for cell membranes. BSH produces free bile acids by hydrolyzing bound bile acids. Free bile acids can precipitate together with cholesterol, dynamically regulate bile acid metabolism, and reduce cholesterol levels in the body, which is of great significance for maintaining host health.

[0005] At present, the research on bile salt hydrolase mainly involves directly screening bile salt hydrolase-producing strains from environmental samples, extracting single strain genomic DNA, and designing primers to clone bile salt hydrolase genes. However, most microorganisms in the environment are uncultivable, so the screening of new BSHs by traditional isolation and culture methods greatly limits the extensiveness, effectiveness, and safety of the screening. Metagenomics avoids the problem of microbial isolation and culture, and directly sequences all microorganisms in environmental samples, greatly expanding the utilization space of microbial resources. There are tens of thousands of microorganisms in the gastrointestinal tract of animals, and many of them are closely related to diseases. Therefore, the animal gastrointestinal metagenome provides a new research strategy for finding and discovering new functional genes and new targets for cholesterol-lowering drugs—bile salt hydrolase. Summary of the invention

[0006] The purpose of the present invention is to provide a bile salt hydrolase and a preparation method and application thereof. The bile salt hydrolase of the present invention has good acid resistance, and the recombinant bile salt hydrolase strain has an in vitro cholesterol-lowering function.

[0007] In order to achieve the above object, the present invention provides a bile salt hydrolase, the amino acid sequence of the bile salt hydrolase is shown in SEQ ID NO.2.

[0008] The second object of the present invention is to provide a gene encoding the bile salt hydrolase, the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] The third object of the present invention is to provide a recombinant vector containing the gene.

[0010] Preferably, the expression vector used is selected from pEASY-E2.

[0011] The fourth object of the present invention is to provide a recombinant bacterium containing the gene.

[0012] Preferably, the host cell used is selected from Escherichia coli BL21 (DE3).

[0013] The fifth object of the present invention is to provide the use of the recombinant bacteria in lowering cholesterol. Specifically, the recombinant bacteria can lower cholesterol in vitro.

[0014] The sixth object of the present invention is to provide a method for preparing the bile salt hydrolase, the method comprising:

[0015] 1) transforming a recombinant vector containing the gene into a host cell to obtain a recombinant strain, culturing the recombinant strain, and inducing expression of the recombinant bile salt hydrolase;

[0016] 2) Recovering and purifying the expressed bile salt hydrolase.

[0017] The seventh object of the present invention is to provide the use of the bile salt hydrolase in hydrolyzing glycine-bound bile salts.

[0018] Preferably, the bile salt hydrolase performs a hydrolysis reaction under the conditions of pH 4-7 and 30-40° C.; or / and, during the hydrolysis, DTT is added to promote the hydrolysis reaction; or / and, during the hydrolysis, at least one of metal ions, SDS, Tween-80, EDTA and anhydrous ethanol is added to inhibit the hydrolysis reaction.

[0019] More preferably, the metal ion is selected from Na + , Ca 2+ , Cu 2+ , Mn 2+ , Fe 2+ 、Ni 2+ 、Al 3+ , Li + 、Co 2+ 、Zn 2+ At least one of them.

[0020] More preferably, the final concentration of the metal ion is 5 mM; or / and, the final concentration of DTT is 1%; or / and, the final concentration of SDS, Tween-80, EDTA and anhydrous ethanol is 1%.

[0021] The bile salt hydrolase of the present invention and its preparation method and application have the following advantages:

[0022] The present invention uses metagenomics technology to directly screen BSH genes from the fecal microbial metagenomics of the western black-crested gibbon, designs primers to amplify BSH gene fragments, and heterologously expresses them in Escherichia coli to explore their enzymatic properties. The optimal pH of the bile salt hydrolase NCbsh3 of the present invention is 5.0; after being treated with a buffer solution of pH 3.0 to 9.0 for 1 hour, the remaining enzyme activity is more than 80%; the optimal temperature is 40°C, and the remaining enzyme activity is more than 60% at 30 or 40°C for 1 hour; at 40°C and pH 5.0, K m and V max 243.3±35.38mM, 29.19±2.264μmol / L / min; Na + , Ca 2+ , Cu 2+ , Mn 2+ , Fe 2+ 、Ni 2+ 、Al 3+ , Li + 、Co 2+ 、Zn 2+ SDS, Tween 80, EDTA, and anhydrous ethanol will inhibit the activity of the enzyme, among which Na + 、Al3+ , Cu 2+ 、Zn 2+ DTT almost completely inhibits enzyme activity, and has an activating effect on the recombinant enzyme, increasing its activity by nearly two times.

[0023] The above properties show that the bile salt hydrolase prepared by the present invention has a wide pH tolerance and outstanding acid resistance. The recombinant strain E.coli / NCbsh3 was induced to express in a cholesterol-containing medium for 16 hours, and the cholesterol degradation rate reached 56%, which has potential application value in the development of cholesterol-lowering drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the SDS-PAGE analysis of the bile salt hydrolase NCbsh3 expressed in Escherichia coli in Experimental Example 2 of the present invention, wherein M: low molecular weight protein Marker; 3: purified recombinant bile salt hydrolase.

[0025] Figure 2 This is the optimum pH of the bile salt hydrolase of Experimental Example 3 of the present invention.

[0026] Figure 3 This is the pH stability of the bile salt hydrolase in Experimental Example 3 of the present invention.

[0027] Figure 4 It is the optimum temperature of bile salt hydrolase in Experimental Example 3 of the present invention.

[0028] Figure 5 This is the temperature stability of the bile salt hydrolase in Experimental Example 3 of the present invention.

[0029] Figure 6 This is the substrate specificity of the bile salt hydrolase in Experimental Example 3 of the present invention.

[0030] Figure 7 This is the in vitro cholesterol degradation rate of the recombinant strain E. coli / NCbsh3 in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Note: The molecular biology experimental methods not specifically described in the following experimental examples are all carried out in accordance with the specific methods listed in the book Molecular Cloning Experiment Guide (3rd Edition) by J. Sambrook, or in accordance with the kits and product instructions. The materials and reagents used in the following experimental examples, unless otherwise specified, can be obtained from commercial channels.

[0033] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as values, quantities, contents and concentrations, are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0034] The features mentioned in the present invention can be combined arbitrarily, as long as there is no contradiction in the combination of these features, all possible combinations should be considered as the scope of this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0035] The test materials and reagents used in the present invention are as follows:

[0036] 1) Samples, strains and vectors:

[0037] Western black-crested gibbon fecal microbial metagenomic DNA; expression vector pEASY-E2 was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; BL21 (DE3) was purchased from Beijing Qingke Xinye Biotechnology Co., Ltd.

[0038] 2) Genetic engineering enzymes, kits and other biochemical reagents:

[0039] Restriction endonucleases, DNA polymerases, and ligases were purchased from TaKaRa; plasmid extraction kits and gel recovery and purification kits were purchased from Omega Corporation of the United States; total cholesterol (T-CHO) test kits were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.; other reagents were of analytical grade.

[0040] 3) LB medium:

[0041] Peptone 10g, Yeast extract 5g and NaCl 10g, add distilled water to 1000mL, pH natural (about 7). Solid medium plus 2.0% (w / v) agar on the above basis.

[0042] Experimental Example 1 Obtaining the bile salt hydrolase gene NCbsh3

[0043] 1) Screening of bile salt hydrolase genes from the fecal microbial metagenomics of the western black-crested gibbon

[0044] Based on the results of gene prediction, functional annotation and secretory protein prediction analysis from the constructed western black crested gibbon fecal microbial library, genes annotated as bile salt hydrolase were screened to obtain the bile salt hydrolase gene NCbsh3, the gene sequence of which is shown in SEQ ID NO.1.

[0045] The bile salt hydrolase gene NCbsh3 sequence (SEQ ID NO.1) is as follows:

[0046] atgtgtactgcggcaacatacaaggcagacgatgcatactttggcagaacgcttgattacgaattttcatacggagacgagataacgattacgccgagaaattataaatttcatttcagacatacagaagatattaaaagtcattatgctataataggtatggcgcacatagcggaaaattatccgctttactatgatgcaatcaatgaaatgggtgtcggtatggcggggcttaactttgtcggaaacgcattctatcccgagccgagtgagaataaaatctctgttgctcagtttgagtttataccttgggttctctcactttgtaaaaatattgaggaagtaaaagaactgctgaaaaatgcagagatagtgtgcacgcctttcagcgaaaaactgccgggtgctcaacttcactggataattgcagataaaaacagttcgattgttgttgaatcaacagcagacggaatgcacatatatgacaaccctgtcggtgttctgactaacaatccgccgtttccgattcaaatgtttaaccttaacaactatatgaatctttcgccaaagcagcccgaaaacaagctttctcaaagcctcgaccttaacgcatacagcagaggaatgggagcaatgggactgcccggtgacttatcatcggcatcacgttttataagggttgcttttgtaaaactcaattcaaaatcaggcaaggacgaaaacgaagcagtaagccaattctttcacattctcggcagcgtagaccaacaaagaggctgttgcgaagtggctgacggcaagtatgaaataacaatttacacctcctgttgcaatctcgacaagggtatttactactacacaacctattcaaaccaccaaatcaccgcagttgatatgcacaacgaggacttagaaaaatcagagctgataagctatgagccgatagcaacgcaacaaataatgcgacaaaactaa。

[0047] 2) Cloning of bile salt hydrolase gene NCbsh3

[0048] PCR amplification was performed using NCbsh3-F1 and NCbsh3-R1 as primer pairs and metagenomic DNA of fecal microorganisms of the western black-crested gibbon as template.

[0049] The NCbsh3-F1 sequence (SEQ ID NO.3) is as follows:

[0050] 5'-taagaaggagatatacatatggaattgatgtgtacagcagcgacttatcaga-3';

[0051] The NCbsh3-R1 sequence (SEQ ID NO.4) is as follows:

[0052] 5'-gtggtggtggtggtgctcgagattcgcccagcgaatctgt-3'.

[0053] PCR reaction system (20.0 μL): 0.5 μL of metagenomic library DNA, 10 μL of PrimeSTARMax, 10.5 μL of NCbsh3-F, 10.5 μL of NCbsh3-R, and ddH2O to make up to 20.0 μL.

[0054] The PCR reaction parameters were: 98°C for 10 s; 55°C for 15 s; 72°C for 1 min and 30 s; 30 cycles; 72°C for 10 min; and 4°C for 10 min.

[0055] The PCR results showed that the target gene NCbsh3 was obtained.

[0056] Experimental Example 2 Preparation of bile salt hydrolase NCbsh3

[0057] The bile salt hydrolase gene NCbsh3 prepared in Example 1 was connected to the plasmid pEASY-E2 to obtain the recombinant expression vector pEASY-E2-NCbsh3, and then transformed into Escherichia coli BL21 (DE3) to obtain the recombinant Escherichia coli strain E. coli / NCbsh3.

[0058] Take the E. coli strain E. coli / NCbsh3 containing the recombinant expression vector pEasy-E2-NCbsh3, inoculate it into LB (containing 100μg / mLAmp) culture medium at a 0.1% inoculum, and culture it at 37°C, 180rpm overnight. Then inoculate the activated bacterial solution into fresh LB (containing 100μg / mLAmp) culture medium at a 1% inoculum, and culture it at 37°C, 180rpm for about 3-4h (OD 600After reaching 0.8-1.0), add IPTG with a final concentration of 1.0mmol / L for induction and culture at 16℃ and 180rpm for about 16h. Centrifuge at 5000rpm for 10min to collect the bacteria. Suspend the bacteria with an appropriate amount of pH 6.0 phosphate buffer and disrupt the bacteria with ultrasound in an ice bath. After the above intracellular concentrated primary enzyme solution is centrifuged at 12,000rpm and 4℃ for 10min, the supernatant is aspirated and the target protein is purified with Nickel-NTA Agarose to obtain bile salt hydrolase NCbsh3, a total of 324 amino acids, with a theoretical molecular weight of 35.64kDa.

[0059] The purified target protein was analyzed by SDS-PAGE. Figure 1 , Figure 1 This is the SDS-PAGE analysis of the recombinant bile salt hydrolase expressed in Escherichia coli provided in Experimental Example 2 of the present invention, wherein M: protein marker; 3: purified recombinant bile salt hydrolase. Figure 1 It can be seen that the recombinant bile salt hydrolase was expressed in Escherichia coli and was a single band after Nickel-NTAAgarose purification.

[0060] The sequence of the obtained bile salt hydrolase NCbsh3 (SEQ ID NO.2) is as follows:

[0061] MCTAATYKADDAYFGRTLDYEFSYGDEITITPRNYKFHFRHTEDIKSHYAIIGMAHIAENYPLYYDAINEMGVGMAGLNFVGNAFYPEPSENKISVAQFEFIPWVLSLCKNIEEVKELLKNAEIVCTPFSEKLPGAQLHWIIADKNSSIVVESTADGMHIYD NPVGVLTNNPPFPIQMFNLNNYMNLSPKQPENKLSQSLDLNAYSRGMGAMGLPGDLSSASRFIRVAFVKLNSKSGKDENEAVSQFFHILGSVDQQRGCCEVADGKYEITIYTSCCNLDKGIYYYTTYSNHQITAVDMHNEDLEKSELISYEPIATQQIMRQN.

[0062] Experimental Example 3 Determination of properties of bile salt hydrolase NCbsh3

[0063] The enzyme activity was determined by referring to the method of Zhao Ruifeng et al., with slight modifications as follows:

[0064] 10 μL enzyme solution and 10 μL 200 mmol / L sodium glycocholate were added to 180 μL 0.01 mol / L phosphate buffer (pH 6.0), mixed gently and thoroughly, and then placed in a 37°C constant temperature water bath for reaction for 30 min. After the reaction, 15% trichloroacetic acid was immediately added to terminate the reaction (trichloroacetic acid was added to the blank group before the reaction), centrifuged at 10000 r / min for 10 min, and the supernatant was taken to determine the amino acid content using the ninhydrin method.

[0065] Determination of amino acid content by ninhydrin method: Take 50 μL of supernatant and add 950 ninhydrin colorimetric solution, boil in water for 15 min, and measure absorbance at 570 nm. Actual absorbance of sample = absorbance - control absorbance. Calculate the amount of generated amino acids according to the glycine standard curve, and calculate the enzyme activity of BSH in the enzyme solution. Take the enzyme activity measured under the optimal reaction conditions as 100%, calculate the relative enzyme activity, and repeat the experiment three times.

[0066] The enzyme activity units are defined as follows:

[0067] One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of glycine within 1 min.

[0068] 1) Determination of the optimal pH and pH stability of bile salt hydrolase NCbsh3

[0069] Determination of the optimum pH of the enzyme: The pure enzyme NCbsh3 was subjected to enzymatic reaction in a buffer solution of pH 3-9 at 37°C for 30 min.

[0070] Determination of enzyme pH stability: Place the purified enzyme solution in a buffer solution with a pH of 3 to 9, incubate at 37°C for 60 min, take the enzyme solution every 30 min to determine the enzyme activity, and use the untreated enzyme solution as a control.

[0071] The buffer solution is: buffer solution (pH 3-9) 0.01 mol / L phosphate buffer solution.

[0072] Sodium glycocholate was used as substrate to measure the enzymatic properties of purified bile salt hydrolase.

[0073] Results Figure 2 and Figure 3 , Figure 2 The optimal pH of bile salt hydrolase provided in the experimental examples of the present invention, Figure 3 The pH stability of bile salt hydrolase provided in the experimental example of the present invention. Figure 2 and Figure 3 It can be seen that the optimum pH of the bile salt hydrolase is 5.0. After being treated in the pH range of 3.0 to 9.0 for 1 hour, the remaining enzyme activity is more than 80%.

[0074] 2) Determination of the optimal temperature and temperature stability of bile salt hydrolase NCbsh3

[0075] Determination of the optimum temperature of the enzyme: Carry out the enzymatic reaction at pH 5.0, at 20-80°C for 30 min.

[0076] Determination of enzyme temperature stability: The enzyme solution with the same amount of enzyme was placed at a set temperature (20-80°C) for 1 hour, and the enzyme activity was measured at pH 5.0 and 40°C every 30 minutes. The enzymatic reaction time was 30 minutes, and the untreated enzyme solution was used as a control.

[0077] Results Figure 4 , Figure 5 , Figure 4 is the optimum temperature of the bile salt hydrolase provided in the experimental example of the present invention, Figure 5 The temperature stability of bile salt hydrolase provided by the experimental example of the present invention is shown in the results: the optimum temperature is 40°C, and the enzyme can be tolerated at 30 or 40°C for 1 hour with a residual enzyme activity of more than 60%.

[0078] 3) Determination of kinetic parameters of bile salt hydrolase NCbsh3

[0079] The enzyme activity was determined under the optimal conditions of pH 5.0, 40°C and first-order reaction time using different concentrations of sodium glycocholate as substrate (0-300 mM). The kinetic parameters were determined and K was calculated according to the Lineweaver-Burk method. m and V max value.

[0080] It was determined that under the optimal conditions, the K m and V max They were 243.3±35.38mM and 29.19±2.264μmol / L / min respectively.

[0081] 4) Determination of the effects of different metal ions and chemical reagents on the activity of bile salt hydrolase NCbsh3

[0082] Metal ions (final concentration of 5 mM) and chemical reagents (final concentration of 1%) were added to the enzyme reaction system to study their effects on enzyme activity. Enzyme activity was measured under the optimum conditions of pH 5.0 and 40°C (the enzyme reaction without metal ions and chemical reagents was used as a control under the same conditions). The results are shown in Table 1.

[0083] Table 1 shows that Na + , Ca 2+ , Cu 2+ , Mn 2+ , Fe 2+ 、Ni 2+ 、Al 3+ , Li+ 、Co 2+ 、Zn 2+ , SDS (sodium dodecyl sulfate), Tween-80, EDTA (ethylenediaminetetraacetic acid), and anhydrous ethanol all inhibit the activity of bile salt hydrolase NCbsh3, among which Na + 、Al 3+ , Cu 2+ 、Zn 2+ It almost completely inhibits the activity of bile salt hydrolase NCbsh3. DTT (dithiothreitol) activates bile salt hydrolase NCbsh3 and increases its activity by nearly two times.

[0084] Table 1 Effects of metal ions and chemical reagents on the activity of bile salt hydrolase NCbsh3

[0085]

[0086]

[0087] 5) Determination of the degradation of different substrates by bile salt hydrolase NCbsh3

[0088] Under the optimal conditions of pH 5.0 and 40°C, different substrates including sodium glycocholate (GCA), sodium glycodeoxycholate (GDCA), sodium glycochenodeoxycholate (GCDCA), sodium taurocholate (TCA), sodium taurodeoxycholate (TDCA) and sodium taurochenodeoxycholate (TCDCA) were added to the above enzyme activity assay system at the same concentration to determine the enzyme activity (the enzyme activity with sodium glycocholate as substrate was used as a control, and the enzymatic reaction time was 30 min). The results showed that the recombinant bile salt hydrolase could only hydrolyze glycine-bound bile salts.

[0089] Results Figure 6 The recombinant bile salt hydrolase has strong substrate specificity and has almost no enzymatic activity against taurine-conjugated bile salts.

[0090] Experimental Example 4 Verification of the cholesterol-lowering function of the recombinant strain E. coli / NCbsh3 in vitro

[0091] LB liquid culture medium with different cholesterol contents was prepared as follows:

[0092] Weigh 0.1g cholesterol, 0.2g ox bile salt and 0.11g sucrose fatty acid ester, dissolve with 1mL Tween-80 and 5mL anhydrous ethanol, filter and sterilize to prepare cholesterol micelle solution. Add cholesterol micelle solution to LB medium according to the final cholesterol concentration of 0.15mg / mL (bsh3-1), 0.20mg / mL (bsh3-2), and 0.30mg / mL (bsh3-3), and vortex to mix to obtain LB medium with different cholesterol content.

[0093] The recombinant E. coli / NCbsh3 was inoculated into cholesterol-LB medium with different concentrations at an inoculum volume of 1% and cultured at 37°C and 180 rpm for about 3-4 h (OD 600 After reaching 0.8-1.0), add IPTG with a final concentration of 1.0 mmol / L for induction, and culture at 16°C and 180 rpm for about 20 h. Use a total cholesterol kit according to the detection method described in the instructions to measure the cholesterol content in the culture medium at 0 h, 12 h, 16 h, and 20 h, respectively. Take the cholesterol content in the culture medium at 0 h as the control, and calculate the degradation rate of cholesterol in the culture medium by the recombinant bacteria E. coli / NCbsh3 at different times.

[0094] Results Figure 7 The recombinant bacteria E.coli / NCbsh3 has the function of lowering cholesterol, and when the cholesterol content in the LB medium is 0.15 mg / mL, the cholesterol-lowering ability of the recombinant bacteria E.coli / NCbsh3 is relatively higher.

[0095] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A bile salt hydrolase, characterized in that The amino acid sequence of the bile salt hydrolase is shown in SEQ ID NO.

2.

2. The gene encoding the bile salt hydrolase according to claim 1, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. A recombinant vector containing the gene as claimed in claim 2.

4. A recombinant bacterium containing the gene as claimed in claim 2.

5. Use of the recombinant bacteria as claimed in claim 4 in lowering cholesterol.

6. The method for preparing the bile salt hydrolase according to claim 1, wherein The preparation method comprises: 1) transforming a recombinant vector containing the gene as claimed in claim 2 into a host cell to obtain a recombinant strain, culturing the recombinant strain, and inducing the expression of the recombinant bile salt hydrolase; 2) Recovering and purifying the expressed bile salt hydrolase.

7. Use of the bile salt hydrolase according to claim 1 in hydrolyzing glycine-bound bile salts.

8. The use according to claim 7, characterized in that: The bile salt hydrolase performs a hydrolysis reaction at pH 4-7 and 30-40°C; or / and, in the hydrolysis, adding DTT to promote the hydrolysis reaction; Or / and, during the hydrolysis, at least one of metal ions, SDS, Tween-80, EDTA and anhydrous ethanol is added to inhibit the hydrolysis reaction.

9. The use according to claim 8, characterized in that: The metal ion is selected from Na + , Ca 2+ , Cu 2+ , Mn 2+ , Fe 2 + 、Ni 2+ 、Al 3+ , Li + 、Co 2+ 、Zn 2+ At least one of them.

10. The use according to claim 8, characterized in that: The final concentration of the metal ion is 5 mM; Or / and, the final concentration of DTT is 1%; Or / and, the final concentration of the SDS, Tween-80, EDTA and anhydrous ethanol is 1%.

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