Bile salt hydrolase gene mutant BSHg1294N81G and application thereof

Abstract

The invention relates to the technical field of enzyme gene engineering, in particular to a bile salt hydrolase gene mutant BSHg1294N81G and application of the bile salt hydrolase gene mutant BSHg1294N81G. The invention provides a bile salt hydrolase g1294 mutant BSHg1294N81G, compared with a wild type bile salt hydrolase g1294, the mutant comprises N81G mutation, and the amino acid sequence of the wild type bile salt hydrolase g1294 is as shown in SEQ ID NO. 1. The amino acid sequence of the bile salt hydrolase g1294 mutant BSHg1294N81G is as shown in SEQ ID NO. 2. Compared with wild type bile salt hydrolase g1294, the bile salt hydrolase g1294 mutant BSHg1294N81G provided by the invention has higher bile salt hydrolysis activity and wider specificity, not only can be used for hydrolyzing taurine bile salt, but also can be used for hydrolyzing glycine bile salt, and has a wider market prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme gene engineering, and particularly relates to a bile salt hydrolase gene mutant BSHg1294N81G and an application thereof. Background Art

[0002] The human intestine is a very large and complex microbial community. The intestinal microbiota is closely related to the host and is essential for many physiological processes, including the deconjugation of primary bile acids (Bas) by bile salt hydrolase (BSH). Host-derived bile acids are called "primary bile acids" and are bound to glycine or taurine through amide bonds. The bound bile acids are excreted into the proximal small intestine, where they are recognized and cleaved by bacterial bile salt hydrolases, releasing the bound amino acids and producing deconjugated bile acids. This step is key to downstream bile acid metabolism because deconjugated bile acids are precursors for intestinal microbial metabolic conversions to produce "secondary bile acids", thereby greatly expanding the chemical diversity of the bile acid pool. Therefore, the presence of active BSH has long been considered a selection criterion for defining potential probiotics.

[0003] Currently known bile salt hydrolases exist in multiple species, including Lactobacillus, Bifidobacterium, Enterococcus, etc. The enzymatic activity and substrate selectivity of bile salt hydrolases from different sources are significantly different. For example, BSH derived from Lactiplantibacillus plantarum WCFS1 and ST-Ⅲ have a preference for glycine bile acid (GCA), and BSH derived from Bifidobacterium animalis subsp. lactis Bi30 also has a preference for GCA, but BSH from Ligilactobacillus salivarius JCM1046 has a preference for taurine bile acid (TCA). Sometimes different bile salt hydrolases from the same source have different substrate specificities. For example, the two BSHs from Lactobacillus acidophilus NCFM have different substrate specificities. BSH A prefers GCA, while BSH B can hydrolyze both GCA and TCA. Matthew H. Foley reported that the substrate specificity of lactobacillus bile salt hydrolases determines bacterial adaptability and host colonization. Regarding the reasons for preference, AKBatta reported that bile salt hydrolases recognize the bile acid part, and SM Huijghebaert reported that they recognize the amino acid part. The catalytic mechanism of BSH includes the initial nucleophilic attack of Cys2 on the amide bond, followed by the formation of a tetrahedral intermediate, which is stabilized by the oxyanion hole formed by the peptide NH of Asn82 and the Nδ2 of Asn173; in this process, Arg18 stabilizes the negatively charged sulfhydryl group of Cys2, and the N-terminal Cys2 forms a catalytic Ntn-dimer with Asp21; Asn173 participates in substrate recognition through hydrogen bonds, while Arg226 contributes to the stabilization of the transition state. These sites are relatively conserved and are crucial for the catalytic activity of BSH.

[0004] The present invention finds that the laboratory-preserved Ligilactobacillus cholophilus BD7642 has strong bile salt hydrolase activity. Previous studies have found that the bile salt hydrolase g1294 derived from the strain contributes most to its bile salt hydrolase activity. It has a preference for taurine bile salts, but has low hydrolysis activity for glycine bile salts, a narrow specificity range, and limited applications. Summary of the invention

[0005] The purpose of the present invention is to provide a bile salt hydrolase gene mutant BSHg1294 N81GThe bile salt hydrolase g1294 from Ligilactobacillus cholophilus BD7642 is modified by random mutation and saturation mutation to obtain a bile salt hydrolase g1294 mutant, thereby solving the problems of low activity, low expression level, poor solubility, and limited application of the bile salt hydrolase g1294 in the prior art.

[0006] The first aspect of the present application provides a bile salt hydrolase g1294 mutant BSHg1294 N81G , the mutant comprises an N81G mutation compared to the wild-type bile salt hydrolase g1294, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The second aspect of the present application provides an isolated polynucleotide encoding the bile salt hydrolase g1294 mutant BSHg1294 N81G .

[0008] The third aspect of the present application provides a construct, which contains the isolated polynucleotide mentioned above.

[0009] The fourth aspect of the present application provides a host cell, which comprises the above-mentioned construct, or the above-mentioned polynucleotide is integrated into the genome of the host cell.

[0010] The fifth aspect of the present application provides a method for producing a bile salt hydrolase g1294 mutant BSHg1294N81G, the method comprising: N81G Under the expression conditions, the host cell is cultured to obtain the bile salt hydrolase g1294.

[0011] In a sixth aspect, the present application provides a bile salt hydrolase g1294 mutant BSHg1294 N81G , isolated polynucleotides, constructs or host cells in the preparation of medicines, health products or cosmetics.

[0012] The bile salt hydrolase g1294 mutant BSHg1294 provided in this specification N81G The possible beneficial effects include but are not limited to:

[0013] 1. The present invention obtains a bile salt hydrolase mutant BSHg1294 with high bile salt hydrolase activity by random mutation and saturation mutation of bile salt hydrolase g1294 N81GThe enzyme activity specificity of the mutant has changed, and it has stronger bile salt hydrolase activity and broader specificity. It can hydrolyze both taurine bile salts and glycine bile salts, which provides a reference for further understanding of bile salt hydrolase and also provides new material for the future transformation of probiotics.

[0014] 2. Bile salt hydrolase mutant BSHg1294 of the present invention N81G In the future, it can be applied to the fields of medicine, therapeutic food, animal husbandry, etc., such as application in pharmaceutical preparations, food additives, feed fermentation additives, etc. The development of bile salt hydrolase can provide targeted development of drugs for the in-depth understanding and treatment of diseases such as hypercholesterolemia and obesity in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figures are the results of enzyme activity analysis, A is the scanning spot plate result of bile salt hydrolase g1294 glycine mutants D21, N81, N174, R227, N267, E268, Q269, Q271 and E273; B is the PCR amplification electrophoresis of the gene encoding the bile salt hydrolase g1294 glycine mutant; C is a schematic diagram of the scanning HPLC results of the bile salt hydrolase g1294 glycine mutant. DETAILED DESCRIPTION

[0016] In order to make the invention purpose, technical scheme and beneficial effect of the present invention clearer, the present invention is further described below in conjunction with examples. It should be understood that the examples are only used to explain the present invention and are not used to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified, and those familiar with the technology can easily understand other advantages and effects of the present invention from the contents disclosed in this description.

[0017] The inventors of the present application obtained a bile salt hydrolase mutant BSHg1294 with high bile salt hydrolase activity by using random mutation and saturation mutation methods on bile salt hydrolase g1294. N81G , the bile salt hydrolase mutant BSHg1294 N81G Compared with the wild-type bile salt hydrolase g1294, it has stronger bile salt hydrolase activity and wider specificity, so it can be applied to the fields of medicine, physical therapy products and animal husbandry. On this basis, the present invention is completed.

[0018] The present application provides a bile salt hydrolase mutant BSHg1294 N81G , the mutant comprises an N81G mutation compared to the wild-type bile salt hydrolase g1294, the amino acid sequence of which is shown in SEQ ID NO.1.

[0019] In one embodiment, the bile salt hydrolase g1294 mutant BSHg1294 N81G The amino acid sequence is shown in SEQ ID NO.2.

[0020] SEQ ID NO.1:

[0021] MCTGLRFNDAEGNLYFGRNLDVESSYGEKVLVTPRNYELPYRFLENGKTTKAIIGMGIMAGDYPMYFDACNENGLCIAGLNFPRYAYFTEGPVDGKTNMAPYEFMVWLMEEFDTVAEAKKGLEELNLVDAPFSPQMPVAPLHWIISDKDASIVVEQTK ENGLKVYDNYVGVLTNNPDYAWHMTNLNNYAGLTPHDAKAQEWNKQEVRPLGVGTGSLGLPGDSIPASRFVKVAYLNANYPTTVEGEEANVAKFFNILKSVAMIDGSVINEQGQDEFTVYTACYSSNTKTYYYNRYDDFTMKSVQLTDENMNAKEVTLY*

[0022] SEQ ID NO.2:

[0023] MCTGLRFNDAEGNLYFGRNLDVESSYGEKVLVTPRNYELPYRFLENGKTTKAIIGMGIMAGDYPMYFDACNENGLCIAGLGFPRYAYFTEGPVDGKTNMAPYEFMVWLMEEFDTVAEAKKGLEELNLVDAPFSPQMPVAPLHWIISDKDASIVVEQTK ENGLKVYDNYVGVLTNNPDYAWHMTNLNNYAGLTPHDAKAQEWNKQEVRPLGVGTGSLGLPGDSIPASRFVKVAYLNANYPTTVEGEEANVAKFFNILKSVAMIDGSVINEQGQDEFTVYTACYSSNTKTYYYNRYDDFTMKSVQLTDENMNAKEVTLY*

[0024] The present invention obtained 9 bile salt hydrolase mutants through random mutagenesis and site-directed saturation mutagenesis screening, among which the mutant BSHg1294 N81G The degradation ability of glycine bile salt is significantly improved, and the specificity is more extensive. For mutations at other sites, the desired effect of enhancing the degradation ability cannot be obtained.

[0025] Bile salt hydrolase g1294 mutant BSHg1294 of the present application N81G The bile salt hydrolase g1294 mutant BSHg1294 N81G Compared with wild-type bile salt hydrolase g1294, it has higher activity.

[0026] The present application also provides an isolated polynucleotide encoding the bile salt hydrolase g1294 mutant BSHg1294. N81G .

[0027] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0028] In one embodiment, the isolated polynucleotide has a nucleotide sequence as shown in SEQ ID No.3.

[0029] SEQ ID No.3:

[0030] ATGTGTACAGGTTTACGTTTTAATGACGCAGAAGGTAACTTATATTTTGGTCGTAACCTTGATGTAGAATCATCATACGGTGAAAAAGTTCTTGTAACACCAAGAAATTACGAATTACCATACCGCTTTTTAGAAAATGGTAAAACAACAAAAGCAATTATCGGAATGGGTATCATGGCTGGTGACTATCCAATGTATTTTGATGCTTGCAACGAAAATGGCCTTTGCATTGCAGGATTAGGCTTCCCACGTTATGCATACTTTACAGAAGGAGCAAGTTGATGGCAAAACTAACATGGCACCATATGAATTTATGGTTTGGTTAATGGAAGAATTTGATACAGTTGCTGAAGCTAAAAAAGGTTTAGAAGAATTAAATCTTGTTGATGCACCATTCTCACCACAAATGCCAGTTGCTCCATTACACTGGATCATCAGTGATAAAGATGCTTCAATCGTTGTAGAACAAACAAAAG AAAACGGCTTAAAAGTTTATGATAACTATGTTGGCGTATTAACAAACAACCCAGATTATGCTTGGCATATGACAAACTTAAACAACTATGCTGGTTTAACACCACATGATGCAAAAGCTCAAGAATGGAATAAGCAAGAAGTTAGACCATTAGGTGTAGGTACTGGTAGTTTAGGTTTACCTGGTGACAGTATTCCTGCTTCACGTTTTGTTAAGGTTGCATACTTAAATGCTAACTATCCAACAGTAGAAGGCGAAGAAGCAAATGTTGCTAAATTCTTTAACATTCTTAAATCAGTTGCAATGATTGATGGTAGTGTAATTAACGAACAAGGTCAAGATGAATTTACAGTTTATACTGCTTGCTACTCATCAAATACTAAGACATACTACTACAACCGTTTACGATGACTTTACAATGAAGAGTGTTCAATTAACAGACGAAAACATGAACGCTAAAGAAGTAACTTTATACTAG

[0031] The present application also provides a construct, wherein the construct contains the isolated polynucleotide described above.

[0032] In certain embodiments of the present invention, the construct is constructed by inserting the isolated polynucleotide into the multiple cloning site of an expression vector. The expression vector in the present invention generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors. Preferably, it is pET302.

[0033] The present application also provides a host cell, wherein the host cell comprises the above construct, or the above polynucleotide is integrated into the host cell genome.

[0034] In some specific embodiments, the host cell includes a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be selected from Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Bacteroides ovale, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgaris, Bacteroides monomorpha, Lactobacillus casei, Bacteroides fragilis, Acinetobacter lwoffii, Fusobacterium nucleatum, Bacteroides johnsii, Bacteroides arabidopsis, Lactobacillus rhamnosus, Bacteroides massiliense, Bacteroides feces, Fusobacterium death, Bifidobacterium breve or Listeria; or, the eukaryotic cell can be selected from yeast cells, Streptomyces, Drosophila S2 or Sf9 insect cells, CHO, COS.293 cells, or Bowes melanoma cells. Preferably, the host cell is Escherichia coli BL21 (DE3). The above-mentioned Escherichia coli needs to be able to express the above-mentioned bile salt hydrolase g1294 mutant BSHg1294 N81G , thereby providing the bile salt hydrolase g1294 mutant BSHg1294 N81G The conditions for the presence of the above-mentioned E. coli should be known to those skilled in the art.

[0035] The present application also provides a bile salt hydrolase g1294 mutant BSHg1294 N81G The method comprises: introducing the aforementioned polynucleotide and the aforementioned construct into engineering cells, inducing the expression of the foreign gene, separating and purifying to obtain the bile salt hydrolase g1294 mutant BSHg1294 N81G .

[0036] In some specific embodiments, the engineered cell is selected from a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be selected from Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Bacteroides ovale, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgaris, Bacteroides monomorpha, Lactobacillus casei, Bacteroides fragilis, Acinetobacter lwoffii, Fusobacterium nucleatum, Bacteroides johnsii, Bacteroides thaliana, Lactobacillus rhamnosus, Bacteroides massiliense, Parabacteroides feces, Fusobacterium death, Bifidobacterium breve or Listeria; or, the eukaryotic cell can be selected from yeast cells, Streptomyces, Drosophila S2 or Sf9 insect cells, CHO, COS.293 cells or Bowes melanoma cells.

[0037] In some specific embodiments, the introduction method can be selected from one or more of DEAE-dextran-mediated transfection, liposome-mediated transfection, virus or phage infection, lipofection, transfection, conjugation, protoplast fusion, polyethyleneimine-mediated transfection, electroporation, calcium phosphate precipitation, gene gun, calcium phosphate precipitation, microinjection, and nanoparticle-mediated nucleic acid delivery.

[0038] In one embodiment, the inducing agent used to induce expression is selected from IPTG. IPTG induction is a conventional induction method for engineering bacteria to express target proteins in the prior art, and the technology is mature, reliable, and easy to operate.

[0039] The present application also provides a bile salt hydrolase g1294 mutant BSHg1294 N81G , isolated polynucleotides, constructs or host cells in medicines, health products or cosmetics.

[0040] The bile salt hydrolase g1294 mutant BSHg1294N81G of the present application can effectively improve the bile salt hydrolysis activity. Experimental data show that the mutation of aspartic acid at position 84 of bile salt hydrolase g1294 to glycine has stronger bile salt hydrolase activity and wider specificity compared with the wild-type bile salt hydrolase g1294, which helps to solve the specificity problem of bile salt hydrolase g1294 and has good industrialization prospects.

[0041] The invention of the present application is further described below by way of examples, but the scope of the present application is not limited thereby. Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0042] Example 1 - Construction of BSHg1294 mutant expression vector

[0043] Plasmid pET302 was selected as the expression vector for mutants. The expression plasmid pET302-g1294 of the wild enzyme has been successfully constructed and expressed. Based on this plasmid, the g1294 bile salt hydrolase mutant was constructed using the large fragment primer point mutation method according to the In-Fusion Snap Assembly Master Mix kit instructions, pET302-g1294 (D21G), pET302-g1294 (N81G), pET302-g1294 (N174G), pET302-g1294 (R227G), pET302-g1294 (E273G), pET302-g1294 (N267G), pET302-g1294 (E268G), pET302-g1294 (Q269G) and pET302-g1294 (Q271G) were constructed, and the primers are shown in Table 2. The saturation mutation of the N81 site was constructed using the random base primer method, and the designed primers are shown in Table 1. The above-constructed plasmids were sequenced and verified to be correct.

[0044] Table 1. Primer sequences

[0045]

[0046]

[0047] Note: F: upstream primer; R: downstream primer; the underlined part is the mutation site.

[0048] Example 2—Expression of BSHg1294 mutants

[0049] The mutant expression vector obtained above was transformed into Escherichia coli BL21 (DE3), and a single colony was picked and inoculated into 10 mL of fermentation medium at 37°C and 200 rpm for 24 h for activation; 2% of the inoculum was transferred to 10 mL of fermentation medium and cultured for 24 h as seed liquid; the obtained seed liquid was transferred to a 250 mL conical flask containing 50 mL of fermentation medium, and the starting biomass OD was controlled. 600 When the strain grows to OD600 of 0.8-1.0, add IPTG with a final concentration of 500 μM to induce at 20°C, and collect the bacteria after 12-16 hours.

[0050] Example 3 - Analysis of Enzyme Activity of BSHg1294 Mutants

[0051] The collected bacteria were resuspended in PBS buffer, 2 μL was taken for spot plate experiment, and the rest of the bacteria were placed on ice water and treated with ultrasonic disruptor (power 200 W, working 3S, interval 2S, total ultrasonic 15min), centrifuged at 12000rpm for 10min, and the supernatant was collected as the bacterial content. TM , Bis-Tris, 8%, 12wells protein precast gel instructions for use. The bacterial contents were subjected to SDS-PAGE gel electrophoresis. The activity of bile salt hydrolase was determined by high performance liquid chromatography: 6 bile salt standards were mixed with 100mM mixed sample, and the standard curve was drawn by liquid chromatography. The mixed bile salt standard was mixed with the bacterial contents in a ratio of 1:1, and the reaction was carried out at a constant temperature of 37℃ for 3h, and the final residual bile salt was quantified; the liquid chromatography procedure was as follows: the mobile phase was A: acetonitrile + 0.09% trifluoroacetic acid and B: water + 0.1% trifluoroacetic acid, gradient elution, 0-2min: A: B = 25:75, 2-10min: A: B = 55:45, 10-15min: A: B = 25:75. The flow rate of fluidity was 0.6mL / min, the detection temperature was 40℃, and the detection wavelength was 205nm.

[0052] According to the above method, the degradation rate of the unmutated bile salt hydrolase g1294 for degrading 6 bile salts was determined to be 99.31% for taurocholate, 88.41% for taurochenodeoxycholate, 90.06% for taurodeoxycholate, 13.86% for glycocholate, 0.12% for glycinechenodeoxycholate, and 0.82% for glycinedeoxycholate. The analysis results of the mutants are shown in Figure 1 , Figure 1 A It can be seen that the change of the amino acid at the mutation site significantly changed the bile salt hydrolase activity of g1294. The hydrolysis circles of D21G and R227G mutants on the plate disappeared; the hydrolysis circles of N174G, N267G and E273G were reduced and located below the bacteria; the hydrolysis circles of E268G, Q269 and Q271G were similar to those of the unmutated g1294; interestingly, the bile salt hydrolysis circle of the N81G mutant was significantly enlarged. HPLC analysis of the hydrolysis of six bile salts showed the following results. Figure 1C, consistent with the results of the spot plate, D21G and R227G almost lost the ability to degrade six bile salts, N267G and E273G had decreased taurine bile salt degradation ability, Q269 and Q271G had the same bile salt degradation ability as the unmutated g1294, and the E268G mutant had a slightly decreased ability to degrade taurodeoxycholic acid. However, it is interesting that the N81G mutant and the N174G mutant developed the ability to degrade glycine bile salts, especially the N81G mutant, which completely degraded three taurine bile salts and had a significantly improved ability to degrade glycine bile salts.

[0053] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0054] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0055] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0056] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A bile salt hydrolase g1294 mutant BSHg1294 N81G , characterized in that, Compared with the wild-type bile salt hydrolase g1294, the mutant includes an N81G mutation, and the amino acid sequence of the wild-type bile salt hydrolase g1294 is shown in SEQ ID NO.

1.

2. The bile salt hydrolase g1294 mutant BSHg1294 according to claim 1 N81G , characterized in that, The bile salt hydrolase g1294 mutant BSHg1294 N81G The amino acid sequence of the bile salt hydrolase g1294 mutant BSHg1294 is shown in SEQ ID NO.2; and / or N81G The activity is higher than that of wild-type bile salt hydrolase g1294.

3. An isolated polynucleotide, characterized in that The isolated polynucleotide encodes the bile salt hydrolase g1294 mutant BSHg1294 according to claim 1 or 2 N81G .

4. The polynucleotide according to claim 3, characterized in that The isolated polynucleotide has a nucleotide sequence as shown in SEQ ID No.

3.

5. A construct, characterized in that The construct contains the isolated polynucleotide of claim 3 or 4.

6. The construct according to claim 5, characterized in that The construct is constructed by inserting the isolated polynucleotide into the multiple cloning site of an expression vector. Preferably, the expression vector is pET302.

7. A host cell, characterized in that The host cell comprises the construct according to claim 4, or the host cell genome is integrated with the polynucleotide according to claim 3 or 4.

8. The host cell according to claim 7, characterized in that The host cell includes a prokaryotic cell or a eukaryotic cell. Preferably, the host cell is Escherichia coli BL21 (DE3).

9. A bile salt hydrolase g1294 mutant BSHg1294 N81G A production method comprising: After the aforementioned polynucleotide and the aforementioned construct are introduced into the engineering cells, the expression of the foreign gene is induced, and the bile salt hydrolase g1294 mutant BSHg1294 is obtained by separation and purification. N81G .

10. The bile salt hydrolase g1294 mutant BSHg1294 according to any one of claims 1 to 2 N81G , the isolated polynucleotide according to claim 3 or 4, the construct according to any one of claims 5 to 6, or the host cell according to any one of claims 7 to 8 in the preparation of medicines, health products or cosmetics.

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