CN116590268A

CN116590268A - Bile salt hydrolase with enhanced thermal stability, preparation method and application

Info

Publication number: CN116590268A
Application number: CN202310555862.XA
Authority: CN
Inventors: 吴齐龙; 唐宗兴; 何兆海; 胡德祥
Original assignee: Chongqing Jize Biotechnology Co ltd
Current assignee: Chongqing Jize Biotechnology Co ltd
Priority date: 2023-05-16
Filing date: 2023-05-16
Publication date: 2023-08-15

Abstract

The application relates to the technical field of enzyme engineering, in particular to a bile salt hydrolase with enhanced thermal stability, and a preparation method and application thereof. The amino acid sequence of the thermal stability enhanced bile salt hydrolase is shown as SEQ ID NO. 3, and the thermal stability enhanced bile salt hydrolase is formed by mutating glycine at position 80 of a wild-type enzyme into alanine. The scheme changes the hydrophobicity of the molecular surface by means of gene mutation, and solves the technical problem of low thermal stability of the existing bile salt hydrolase. The bile salt hydrolase is suitable for large-scale production, and the obtained bile salt hydrolase has the characteristic of high thermal stability, can increase the application scene of the enzyme, and widens the industrial application range of the bile salt hydrolase.

Description

Bile salt hydrolase with enhanced thermal stability, preparation method and application

Technical Field

The application relates to the technical field of enzyme engineering, in particular to a bile salt hydrolase with enhanced thermal stability, a preparation method and application thereof.

Background

The thermal stability of enzymes is one of the important properties of enzymes, and highly thermally stable enzymes have been the focus of research in the field of enzyme engineering. The obtained enzyme with high thermal stability has great significance for improving the efficiency of enzyme catalytic reaction, simplifying the catalytic process, reducing the production cost based on enzyme catalytic reaction, and the like. In the process of developing a novel enzyme preparation, the heat stability of the enzyme is an important index for measuring the quality and application range of the enzyme preparation.

Bile salt hydrolase (bile salt hydrolase BSH) is commonly found in the gastrointestinal flora of mammals and is capable of hydrolysing conjugated bile salts to amino acids and bile acids in the free form, thereby reducing the cholesterol content in the serum. In the biological circulation, bile acid is taken as a signal substance to participate in the biochemical metabolism and regulation of body lipid, cholesterol and sugar, and the change of the composition and flow of the bile acid can be taken as a new direction for treating metabolic syndrome. For bile salt hydrolase derived from bifidobacterium longum (Bifidobacterium longum), the research on amino acid residues or peptide fragments with heat stabilization effect of the enzyme molecule is insufficient at present, and information about the structure of the enzyme molecule, which can be used for increasing the heat stability of the enzyme by site-directed mutagenesis, cannot be obtained, so that mutants with obviously improved heat stability are difficult to obtain. There is a need to develop a new bile salt hydrolase with high thermal stability suitable for industrial mass production, thereby improving the quality of the enzyme preparation and widening the application range thereof.

Disclosure of Invention

The application aims to provide a bile salt hydrolase with enhanced thermal stability, so as to solve the technical problem that the thermal stability of the existing bile salt hydrolase does not meet the application requirement.

In order to achieve the above purpose, the application adopts the following technical scheme:

a heat stability enhanced bile salt hydrolase has an amino acid sequence shown in SEQ ID NO. 3.

The principle and the advantages of the scheme are as follows: the scheme specifically carries out mutation of single amino acid on glycine at position 80 of wild BSH enzyme (SEQ ID NO: 5) to obtain alanine. The heat stability of the enzyme obtained after the mutation of the amino acid is greatly improved. The reason why the thermal stability of the enzyme after mutation is improved is that alanine is a nonpolar amino acid relative to glycine, the hydrophobicity of the surface of the enzyme molecule is changed, the three-dimensional structure of the enzyme is finely adjusted, and the thermal stability of the bile salt hydrolase is improved. The bile salt hydrolase obtained by the scheme overcomes the defects that the wild bile salt hydrolase has low thermal stability and is unfavorable for mass production. In the bile salt hydrolase, the site affecting the enzymatic properties is amino acid 2 (cysteine) of wild-type BSH. In the scheme, glycine in a non-enzymatic center is changed into alanine, and generally, the influence of mutation of amino acid in the non-enzymatic center on the function of the enzyme is not too great, but the mutant form of the scheme greatly improves the thermal stability of the enzyme, improves the temperature by about sixty percent, and obtains unexpected technical effects.

Although there are operations of achieving a change in thermostability by gene mutation in the prior art, there has been NO report about the manipulation of gene mutation on the bile salt hydrolase (derived from bifidobacterium longum Bifidobacterium longum) having the amino acid sequence shown in SEQ ID NO. 5 of the present embodiment. Because there is no reference to the related art, great difficulty is presented to genetic engineering of enzymes and selection of key mutation sites. The inventors have conducted extensive studies and screening to obtain a very desirable effect of increasing the thermostability of the enzyme by mutating the bile salt hydrolase of the present embodiment to glycine at position 80 and mutating it to alanine. The mutation of other sites or the mutation of glycine at the 80 th site into other amino acids cannot achieve the ideal effect of enhancing the thermal stability.

Further, the nucleotide sequence of the bile salt hydrolase with enhanced thermostability is shown as SEQ ID NO. 4. The gene with the sequence shown as SEQ ID NO. 4 is suitable for replication and expression in an escherichia coli system through codon transformation.

Further, a method for preparing the bile salt hydrolase with enhanced thermal stability comprises the following steps in sequence:

s1: integrating a bile salt hydrolase gene with a nucleotide sequence shown as SEQ ID NO. 4 onto an empty plasmid to obtain an expression vector;

s2: transforming competent cells of the escherichia coli by using an expression vector to obtain engineering bacteria;

s3: inducing the engineering bacteria to express a protein with an amino acid sequence shown as SEQ ID NO. 3 to obtain an enzyme thallus;

s4: and crushing the enzyme thalli, taking supernatant generated after centrifugation, and purifying the supernatant to obtain the bile salt hydrolase.

By adopting the technical scheme, the gene with the nucleotide sequence shown as SEQ ID NO. 4 is obtained by means of gene mutation; integrating the genes on an expression vector to prepare engineering bacteria containing the expression vector; inducing engineering bacteria to express target protein, purifying and separating to obtain high purity target protein.

Further, in S1, a gene having a nucleotide sequence shown in SEQ ID NO. 4 was obtained by overlap PCR.

By adopting the technical scheme, the gene containing the target mutation can be prepared and obtained through overlap PCR, and the overlap PCR technology is mature and easy to operate. The scheme optimizes and site-directed mutates gene codons of wild bile salt hydrolase, and is suitable for expression in escherichia coli.

Further, the primer combination of the overlap PCR includes: the primer with the sequence shown in SEQ ID NO. 11, the primer with the sequence shown in SEQ ID NO. 12, the primer with the sequence shown in SEQ ID NO. 15 and the primer with the sequence shown in SEQ ID NO. 16. Nucleotide fragments having the target mutations can be synthesized using the above primer combinations.

Further, in S2, the empty plasmid contains a GST tag. The GST affinity tag helps to promote solubility of the enzyme protein expressed in E.coli. In the prior art, a pET series vector is selected for expression in escherichia coli, the His affinity tag is low in expression quantity, and the expression quantity and the solubility are poor, so that the expression quantity is mostly inclusion bodies. After the label is replaced, GST affinity label is added at the front section of the protein, so that the solubility of the enzyme protein is improved, and the correct folding of the protein is facilitated, thereby improving the yield and activity of the enzyme.

Further, in S3, the escherichia coli competent cell is a BL21 DE3 competent cell. BL21 (DE 3) competent cells are conventional strains for expressing target proteins in the prior art, and are easy to obtain and realize industrialization.

Further, in S4, the engineering bacteria are induced to express proteins using IPTG. IPTG induction is a conventional induction mode for expressing target proteins by engineering bacteria in the prior art, and the technology is mature and reliable and is easy to operate.

Further, in S5, the enzymatic cells are disrupted by an ultrasonic method; the supernatant was purified using a GST affinity column. Since the GST tag is contained on the empty plasmid, the expressed enzyme can be affinity-purified using the GST tag.

Further, the application of the bile salt hydrolase with enhanced thermal stability in preparing medicines, health products or cosmetics.

By adopting the technical scheme, the bile salt hydrolase provided by the scheme has high expression quantity in the escherichia coli engineering bacteria, the expressed enzyme has good solubility, and the thermal stability of the bile salt hydrolase can be improved by a gene mutation mode, so that the industrial scale-up production is facilitated. The bile salt hydrolase obtained by the scheme can be applied to the preparation of medicines, health products, foods or cosmetics, and has wide application prospect.

Drawings

FIG. 1 is a SDS-PAGE map of the BSH (G80I) enzyme of example 2 of the present application after induced expression in BL21 (DE 3).

FIG. 2 is a SDS-PAGE map of the BSH (G80A) enzyme of example 2 of the present application after induced expression in BL21 (DE 3).

FIG. 3 is a graph showing the results of the detection of BSH (G80I) enzyme in example 2 of the present application.

FIG. 4 is a graph showing the results of the detection of BSH (G80A) enzyme in example 2 of the present application.

FIG. 5 is a SDS-PAGE of the purified BSH (G80I) enzyme (after GST tag removal) of example 3 of the present application.

FIG. 6 is a SDS-PAGE of the purified BSH (G80A) enzyme (after GST tag removal) of example 3 of the present application.

Detailed Description

Example 1: construction of expression vector containing bile salt hydrolase mutant Gene

The gene of the bile salt hydrolase of the bifidobacterium is modified according to the codon preference of the escherichia coli on the premise of unchanged amino acid sequence, so as to obtain the bile salt hydrolase gene BSH (the amino acid sequence is shown as SEQ ID NO: 5; and the nucleotide sequence is shown as SEQ ID NO: 6). The mutant gene (i.e., optimized bile salt hydrolase gene) was obtained by overlapping PCR method (overlap PCR method). The optimized bile salt hydrolase gene is BSH (G80I) (glycine at 80 th position is mutated into isoleucine), the amino acid sequence of the BSH (G80I) is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2. Another optimized bile salt hydrolase gene is BSH (G80A) (glycine at position 80 is mutated to alanine). The amino acid sequence of BSH (G80A) is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 4. Two mutant genes of BSH (G80Q) (glycine at position 80 is mutated to glutamine) and BSH (A77I) (alanine at position 77 is mutated to isoleucine) were simultaneously constructed as comparative examples. The amino acid sequence of BSH (G80Q) is shown as SEQ ID NO. 7, and the nucleotide sequence is shown as SEQ ID NO. 8; the amino acid sequence of BSH (A77I) is shown as SEQ ID NO. 9, and the nucleotide sequence is shown as SEQ ID NO. 10.

The construction of the expression vector will now be described by taking BSH (G80I) and BSH (G80A) as examples. The artificially synthesized BSH was amplified by PCR using the primer set (SEQ ID NO: 11) and (SEQ ID NO:13 or SEQ ID NO: 15) (first PCR reaction system and conditions are shown in Table 1), and then amplified by PCR using the primer set (SEQ ID NO: 12) and (SEQ ID NO:14 or SEQ ID NO: 16) (second PCR reaction system and conditions are shown in Table 2), 2 PCR products were recovered, and then cloning was performed by PCR using the 2 PCR products as templates and using the primer set (SEQ ID NO: 11) and (SEQ ID NO: 12) (third PCR reaction system and conditions are shown in Table 3). Wherein, the enzyme cutting sites of the BSH (G80I) and the BSH (G80A) are: bamHI and NotI.

The primer sequences for obtaining the BSH (G80I) gene are specifically as follows:

SEQ ID NO:11：5’-CGGGATCCCGATGTGCACCGGCGT-3’；

SEQ ID NO:12：5’-TTGCGGCCGCAATTAACGCGCCACGCT-3’；

SEQ ID NO:13：5’-AGCCCGGAAAGTTCAGGATGGCAATAGC-3’；

SEQ ID NO:14：5’-TGGCTATTGCCATCCTGAACTTTCCG-3’。

the overlapping PCR primer combinations for obtaining the BSH (G80A) gene were:

SEQ ID NO:11：5’-CGGGATCCCGATGTGCACCGGCGT-3’；

SEQ ID NO:12：5’-TTGCGGCCGCAATTAACGCGCCACGCT-3’；

SEQ ID NO:15：5’-AGCCCGGAAAGTTCAGAGCGGCAATAGC-3’；

SEQ ID NO:16：5’-TGGCTATTGCCGCTCTGAACTTTCCG-3’。

in addition to the synthetic mode of the present scheme, several mutant genes of the present scheme may be synthesized by other prior art methods, and may be delegated to biotechnology companies for synthesis.

Table 1: first PCR reaction System and conditions

Composition of the reaction System	Volume (mu L)
		PrimeSTAR Max Premix 2X	25
Primer (SEQ ID NO: 11)	2
		Primer (SEQ ID NO:13 or SEQ ID NO: 15)	2
DNA template (SEQ ID NO: 6)	1
		Sterile water	20
Total volume of	50

First PCR reaction conditions: pre-denaturation at 98 ℃ for 5 min; 35 cycles, including: denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, and extension at 72℃for 5 seconds; extension was carried out at 72℃for 10 minutes after the completion of the reaction.

Table 2: second PCR reaction System and conditions

Composition of the reaction System	Volume (mu L)
		PrimeSTAR Max Premix 2X	25
Primer (SEQ ID NO: 12)	2
		Primer (SEQ ID NO:14 or SEQ ID NO: 16)	2
DNA template (SEQ ID NO: 6)	1
		Sterile water	20
Total volume of	50

Second PCR reaction conditions: pre-denaturation at 98 ℃ for 5 min; 35 cycles, including: denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, and extension at 72℃for 5 seconds; extension was carried out at 72℃for 10 minutes after the completion of the reaction.

Table 3: third PCR reaction System and conditions

Third PCR reaction conditions: pre-denaturation at 98 ℃ for 5 min; 35 cycles, including: denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, extension at 72℃for 10 seconds; extension was carried out at 72℃for 10 minutes after the completion of the reaction.

Then, the two enzyme digestion and ligation reactions were performed, and the BSH (G80I) mutant gene and pGEX-6p-1 vector obtained by the overlap PCR were digested with BamHI and NotI restriction enzymes, respectively, and then ligated according to the system and conditions of Table 4, at 16℃overnight, to obtain pGEX-6p-1-BSH (G80I) ligation product. pGEX-6p-1-BSH (G80A) ligation products, pGEX-6p-1-BSH (G80Q) ligation products, pGEX-6p-1-BSH and pGEX-6p-1-BSH (A77I) ligation products can be obtained in the same manner.

Table 4: connection system

Composition of the reaction System	Volume (mu L)
		Mutant genes	6
pGEX-6p-1	2
		T4 Ligase	1
10×T4 Ligase buffer	1
		Total volume of	10

The ligation product was then transformed into DH 5. Alpha. Competent cells. mu.L of competent cells E.coli DH 5. Alpha. Was thawed on ice, and 2. Mu.L of ligation product pGEX-6p-1-BSH (G80I) was added to the thawed E.coli DH 5. Alpha. Competent cells and left on ice for 30 min. Heat shock at 42 ℃ for 90s and ice bath for 3 minutes. 450. Mu.L of LB medium was added, the shaking table temperature was 37℃and the shaking table was rotated at 180rpm for 45 minutes. 200. Mu.L of the Amp-coated resistant LB plate medium was aspirated and incubated overnight at 37 ℃. Single colonies were picked and inoculated into Amp+LB medium at a shaker temperature of 37℃and shaker shaking speed of 220rpm. OD600 ≡ 1.0, cells were obtained for plasmid extraction by centrifugation at 12000rpm for 5 minutes. Extracting plasmid the plasmid is extracted according to the operation of the small extraction kit instruction of the Tian Gen plasmid, and the plasmid is sent to sequencing.

Example 2: expression of the protein of interest

BL21 (DE 3) competent cells were transformed with the plasmids obtained in example 1, which were confirmed to be correct by sequencing. pGEX-6p-1-BSH (G80I) expression vector, pGEX-6p-1-BSH (G80A) expression vector, pGEX-6p-1-BSH (G80Q) expression vector, pGEX-6p-1-BSH expression vector and pGEX-6p-1-BSH (A77I) expression vector are transformed, respectively, to obtain engineering bacteria containing pGEX-6p-1-BSH (G80I) expression vector, engineering bacteria containing pGEX-6p-1-BSH (G80A) expression vector, engineering bacteria containing pGEX-6p-1-BSH (G80Q) expression vector, engineering bacteria containing pGEX-6p-1-BSH expression vector and engineering bacteria containing pGEX-6p-1-BSH (A77I) expression vector.

Taking the preparation of engineering bacteria containing pGEX-6p-1-BSH (G80I) expression vector as an example, the specific process of transforming competent cells is as follows: 50. Mu.L BL21 (DE 3) competent cells were removed at-80℃and thawed on ice, 1. Mu.L of pGEX-6p-1-BSH (G80I) plasmid was added thereto, and the mixture was left on ice for 30 minutes. Heat shock at 42 ℃ for 90 seconds and standing on ice for 3 minutes. mu.L of LB medium was added at 37℃for 45 minutes at 180 rpm. 200. Mu.L of the medium was pipetted onto an ampicillin-containing LB plate medium and incubated overnight at 37 ℃.

Then carrying out low dose expression test on the obtained engineering bacteria, wherein the specific process is as follows: 5 single clones are selected and inoculated into 5mL LB culture medium containing ampicillin, the culture is carried out at 37 ℃ and 220rpm, when the OD value is 0.6-1.0, 1mM IPTG with the final concentration is added for induction for 2 hours, SDS-PAGE (SDS-PAGE) is carried out to detect the expression quantity, and clones with high expression quantity are selected for strain preservation.

Then carrying out large-dose expression culture on the obtained engineering bacteria, wherein the specific process is as follows: 20. Mu.L of the strain was inoculated into 200mL of ampicillin-resistant LB medium, cultured overnight at 180rpm and 37℃with an OD of 2.5-4.0. Inoculating 20mL of culture solution into 1L of ampicillin-resistant LB culture medium for culturing at 120rpm and 37 ℃, cooling to 16 ℃ when the OD value is 0.8-1.2, adding 0.5mM IPTG with the final concentration for inducing overnight expression, and collecting thalli (named BSH (G80I)) at 3800rpm and 15 min; SDS-PAGE detects the expression level. SDS-PAGE images of induced expression of BSH (G80I) enzyme in BL21 (DE 3) are shown in FIG. 1 (arrow indicates the target protein band, about 58kD; lane 1 is post-induction bacterial fluid, lane 2 represents pre-induction bacterial fluid). The same method is used for protein expression by BSH (G80A) enzyme, BSH (G80Q) enzyme, BSH enzyme and BSH (A77I) enzyme. SDS-PAGE images of BSH (G80A) enzyme are shown in FIG. 2 (arrow indicates the target protein band, about 58kD; lane 1 is pre-induction broth, lane 2 represents post-induction broth).

Example 3: purification of target proteins

20G of BSH (G80I) thallus (also called enzyme thallus) is weighed, 200mL of Lysis buffer (1 XPBS) is added to resuspend the thallus, and ultrasonic bacteria breaking is carried out until bacterial liquid is clarified. The supernatant was centrifuged at 10000rpm at 4℃for 60min, and the result was shown in FIG. 3 (T represents the total bacterial liquid after disruption, N represents the pellet of the bacterial liquid after disruption, and S represents the supernatant of the bacterial liquid after disruption after centrifugation). The supernatant was passed through GST affinity column 5mL,4℃at a flow rate of 2mL/min, washed with 100mL of Lysis buffer until the UV detector showed unchanged, and digested overnight at 4℃with PreScission Protease of 5mL of PreScission Protease digestion buffer. After cleavage, the target enzyme protein was eluted with 20mL of Lysis buffer to obtain an enzyme protein solution of BSH (G80I) enzyme, which was stored at-80 ℃. After the target protein is digested (GST tag is cut off), SDS-PAGE electrophoresis detection is carried out, the molecular weight and purity of the target protein are identified, and the concentration of the purified protein is tested, as shown in figure 5, the target protein with GST cut off is about 35 kD. The same procedure was used for protein purification with BSH (G80A) enzyme, BSH (G80Q) enzyme, BSH enzyme and BSH (A77I) enzyme. A SDS-PAGE electrophoresis of BSH (G80A) enzyme is shown in FIG. 4 (T represents the total bacterial liquid after disruption, N represents the precipitate of bacterial liquid after disruption by centrifugation, S represents the supernatant of bacterial liquid after disruption by centrifugation, arrow represents the target protein), and an electrophoresis of GST-cut BSH (G80A) enzyme is shown in FIG. 6 (arrow represents the target protein).

Experimental example 1: enzyme activity assay

10. Mu.L of BSH (G80I) enzyme protein solution is diluted to 90. Mu.L by using 0.1M phosphate buffer (pH 6.0), 10. Mu.L of binding bile salt (200 mM) is added and mixed, the mixture is incubated at 37 ℃ for 30min, an equal volume of 15% (w/v) trichloroacetic acid is added to terminate the reaction, 10. Mu.L of supernatant is centrifuged and mixed with 190. Mu.L of ninhydrin reagent (comprising 50. Mu.L of 1% ninhydrin dissolved in 0.5M citric acid buffer (pH 5.5)), 120. Mu.L of glycerol and 20. Mu.L of 0.5M citric acid buffer (pH 5.5)) for 15min at 100 ℃, the absorbance is measured at 570nm after cooling, a standard curve is calculated by using glycine or taurine (glycine is used in the experiment), and the specific activity (U/mg) of the enzyme to be detected is calculated according to the detection result and the standard curve. Specific activity (U/mg) definition: under the above reaction conditions, the number of units of enzyme activity per mg of protein is contained.

Meanwhile, the enzyme activities of BSH enzyme, BSH (G80A) enzyme, BSH (G80Q) enzyme and BSH (A77I) enzyme were measured in the same manner as described above, and the enzyme activity measurement results are shown in Table 5. The results show that 4 mutants BSH (G80I), BSH (G80A), BSH (G80Q) and BSH (A77I) are all capable of hydrolyzing the bound bile salt, the specific activity of the BSH (G80I) hydrolyzed bound bile salt is 3.6 times that of the wild type BSH, and the specific activity of the BSH (G80A) hydrolyzed bound bile salt is 1.7 times that of the wild type BSH. The enzyme activities of the BSH (G80I) enzyme and the BSH (G80A) enzyme are significantly higher than the wild-type BSH, while the enzyme activities of the BSH (G80Q) enzyme and the BSH (A77I) enzyme are slightly lower than the wild-type BSH. The experimental results show that the scheme can obviously improve the enzyme activity of the enzyme by mutating the 80 th amino acid of the wild BSH into isoleucine or alanine, and the effect of improving the enzyme activity can not be obtained by mutating the amino acid of the site into other types of amino acids. In addition, the mutation of amino acid other than 80 position of wild BSH can not obtain the technical effect of improving the enzyme activity. This suggests that the selection of the mutation site and the determination of the mutated amino acid of interest are important for enhancing the activity of bile salt hydrolase.

Table 5: comparison of enzyme specific Activity of BSH with BSH (G80I), BSH (G80A), BSH (G80Q) and BSH (A77I) (GST tag)

Experimental example 2: thermal stability test

Enzyme samples (i.e., several enzyme protein solutions obtained in example 3, requiring no glycerol) were prepared according to the methods of examples 1-3, and placed in a water bath at 50℃for 20 minutes, and the residual enzyme activity was measured according to the method of measuring the enzyme activity in experimental example 1, and the residual enzyme activity was calculated in the following manner: (enzyme activity before water bath-enzyme activity after water bath)/enzyme activity before water bath multiplied by 100%. As a result, after 20 minutes of heat treatment as shown in Table 6, the enzyme activity of wild-type BSH rapidly decreased, and only about 20% of the enzyme activity was retained, while G80I still retained more than 70% of the enzyme activity, and G80A still retained more than 80% of the enzyme activity. The thermostability of the BSH (G80Q) enzyme and the BSH (A77I) enzyme were not significantly different from that of the wild-type BSH. The experiment shows that compared with wild BSH, BSH (G80I) and BSH (G80A) have better thermal stability and are more suitable for industrial application. The scheme can obviously improve the thermal stability of the enzyme by mutating the 80 th amino acid of the wild BSH into isoleucine or alanine, and can not obtain the effect of improving the thermal stability of the enzyme by mutating the amino acid of the site into other types of amino acids. In addition, the mutation of amino acid other than 80 position of wild BSH can not obtain the technical effect of improving the thermal stability of the enzyme. This suggests that the selection of the mutation site and the determination of the mutated amino acid of interest are important for improving the thermostability of the bile salt hydrolase.

Table 6: BSH was compared to BSH (G80I), BSH (G80A), BSH (G80Q) and BSH (A77I) for thermal stability (GST tag)

Experimental example 3:

the empty plasmid of examples 1-3, which had his tag, was replaced with pET28a, and the enzymatic protein solutions of BSH enzyme, BSH (G80I) enzyme and BSH (G80A) enzyme were obtained by the method of examples 1-3 (at this time, his tag was excised, but the tag affected the enzymatic activity by affecting the folding and formation of the higher structure of the protein), and the enzymatic activity measurement was performed according to the method of experimental example 1, and the experimental results are shown in Table 7. The data in Table 7 shows that the use of GST as a tag promotes the maintenance of the enzyme activity of BSH, the reduction of inclusion body formation and the promotion of the correct folding of protein, while the use of his tag does not achieve the above object.

Table 7: wild type BSH was compared with BSH (G80I) and BSH (G80A) enzyme activities (his tag)

The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims

1. A thermal stability enhanced bile salt hydrolase, characterized by: the amino acid sequence is shown as SEQ ID NO. 3.

2. A thermal stability enhanced bile salt hydrolase according to claim 1, characterized in that: the nucleotide sequence is shown as SEQ ID NO. 4.

3. A method for preparing a bile salt hydrolase with enhanced thermal stability, which is characterized in that: the method comprises the following steps of:

4. A method of preparing a thermostable enhanced bile salt hydrolase according to claim 3, characterized in that: in S1, a bile salt hydrolase gene having a nucleotide sequence shown in SEQ ID NO. 4 was obtained by overlap PCR.

5. The method for preparing the bile salt hydrolase with enhanced thermostability according to claim 4, wherein: the primer combination of the overlapping PCR comprises: the primer with the sequence shown in SEQ ID NO. 11, the primer with the sequence shown in SEQ ID NO. 12, the primer with the sequence shown in SEQ ID NO. 15 and the primer with the sequence shown in SEQ ID NO. 16.

6. The method for preparing the bile salt hydrolase with enhanced thermostability according to claim 5, wherein: in S1, the empty plasmid contains a GST tag.

7. The method for preparing the bile salt hydrolase with enhanced thermostability according to claim 6, wherein: in S2, the escherichia coli competent cell is a BL21 DE3 competent cell.

8. The method for preparing the bile salt hydrolase with enhanced thermostability according to claim 7, wherein: in S3, the engineering bacteria are induced to express proteins using IPTG.

9. The method for preparing the bile salt hydrolase with enhanced thermostability according to claim 8, wherein: in S4, the enzymatic cells are disrupted using ultrasound or high pressure; the supernatant was purified using a GST affinity column.

10. Use of a bile salt hydrolase with enhanced thermostability according to claim 1 for the preparation of a pharmaceutical, nutraceutical or cosmetic product.