7beta-hsdh mutant with alkali resistance and application thereof

Abstract

The application relates to the technical field of enzyme engineering, and discloses a 7beta-HSDH mutant with alkali resistance and application thereof. In order to improve the alkali resistance of 7beta-HSDH, the 7beta-HSDH from Collinsella aerofaciens is subjected to site-directed mutation modification, and a plurality of 7beta-HSDH mutants with alkali resistance are obtained. The 7beta-HSDH from Collinsella aerofaciens is used as a wild type, and the 7beta-HSDH mutant provided by the application is a single-point mutant or a multi-point combination mutant of E26K, P104S, N113Y and M144T. The optimum pH of the wild type enzyme is 5.5, the optimum pH of the 7beta-HSDH mutant provided above is improved from 5.5 to 7.0 after mutation, and the enzyme activity of the 7beta-HSDH mutant is much higher than that of the wild type enzyme under the condition of pH 7.0; under the alkaline condition of pH 8.0-8.5, the 7beta-HSDH mutant still has relatively high enzyme activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme engineering, and in particular to a 7β-HSDH mutant with alkali resistance and application thereof. BACKGROUND

[0002] Ursodeoxycholic acid (UDCA), chemical name 3α, 7β-dihydroxy-5β-cholestane-24-acid, is one of the main active components of precious Chinese medicinal material bear bile powder, and is clinically used for treating liver diseases such as gallstones and primary biliary cirrhosis (PBC).

[0003] The synthesis methods of UDCA mainly include chemical synthesis and biological synthesis. The chemical synthesis method is usually to synthesize UDCA from bovine, sheep, pig bile acid and chenodeoxycholic acid as raw materials through multi-step chemical reactions. The reaction conditions are severe, the cost is high, the safety performance is poor, and there may be toxic chemical reagent residues. The method of using biological synthesis to prepare UDCA is an economical and green preparation method.

[0004] In the biological synthesis process of UDCA, 7β-hydroxysteroid dehydrogenase (7β-HSDH) is often used to catalyze the reduction of chemical drug intermediate 7-keto-lithocholic acid (7K-LCA) to synthesize UDCA. Wild-type 7β-HSDH is widely available. At present, researchers have screened a large number of 7β-HSDH-producing microorganisms and cloned their encoding genes, such as Ruminococcus gnavus, Ruminococcus torques ATCC 35915, Collinsella aerofaciens, Clostridium absonum, etc. The 7β-HSDH from Collinsella aerofaciens has relatively good enzyme activity and enzyme activity stability, and thus has great application prospects.

[0005] In the prior art, a 7β-hydroxysteroid dehydrogenase mutant, a coding sequence, a recombinant expression vector, a genetically engineered bacterium and an application are provided in Chinese Patent Publication No. CN109182284A. The patent mutates and modifies the 7β-HSDH from Collinsella aerofaciens to obtain a mutant Ca7β 2, the mutant Ca7β 2, the mutant Ca7β

[0006] Since the substrate 7K-LCA of 7β-HSDH has high solubility in a slightly alkaline solution, the catalytic reaction of 7β-HSDH in a slightly alkaline solution is beneficial to the improvement of the yield of UDCA. Therefore, the improvement of the alkali resistance of 7β-HSDH is beneficial to the catalytic reaction of 7K-LCA as the substrate. Therefore, more 7β-HSDH mutants need to be researched and discovered. SUMMARY

[0007] In order to solve the technical problem of low enzyme activity of 7β-HSDH in alkaline conditions, the present application provides a 7β-HSDH mutant with alkali resistance and application thereof.

[0008] The specific technical scheme of the present application is as follows:

[0009] As a first aspect of the present application, the present application provides a 7β-HSDH mutant with alkali resistance, which is obtained by any one of single-point mutation, double-site combination mutation, triple-site combination mutation or four-site combination mutation of the amino acid sequence shown in SEQ ID NO. 1 at the following sites:

[0010] The single-point mutation is:

[0011] the 26th amino acid is mutated from glutamic acid (E) to lysine (K);

[0012] the 104th amino acid is mutated from proline (P) to serine (S);

[0013] the 144th amino acid is mutated from methionine (M) to threonine (T);

[0014] The double-site combination mutation is:

[0015] the 104th amino acid is mutated from proline (P) to serine (S), and the 144th amino acid is mutated from methionine (M) to threonine (T);

[0016] the 113th amino acid is mutated from asparagine (N) to tyrosine (Y), and the 144th amino acid is mutated from methionine (M) to threonine (T);

[0017] The triple-site combination mutation is:

[0018] the 26th amino acid is mutated from glutamic acid (E) to lysine (K), the 113th amino acid is mutated from asparagine (N) to tyrosine (Y), and the 144th amino acid is mutated from methionine (M) to threonine (T);

[0019] the 104th amino acid is mutated from proline (P) to serine (S), the 113th amino acid is mutated from asparagine (N) to tyrosine (Y), and the 144th amino acid is mutated from methionine (M) to threonine (T);

[0020] the four-site combination mutation is:

[0021] the 26th amino acid is mutated from glutamic acid (E) to lysine (K), the 104th amino acid is mutated from proline (P) to serine (S), the 113th amino acid is mutated from asparagine (N) to tyrosine (Y), and the 144th amino acid is mutated from methionine to threonine.

[0022] In order to improve the alkali resistance of 7β-HSDH, the 7β-HSDH derived from Collinsella aerofaciens is subjected to site-directed mutation modification to obtain a plurality of 7β-HSDH mutants with alkali resistance. Among them, the 7β-HSDH derived from Collinsella aerofaciens has an amino acid sequence as shown in SEQ ID NO. 1. The 7β-HSDH mutant provided by the present application is a single-point mutation or a multi-point combination mutation of E26K, P104S, N113Y and M144T. The optimum pH of the wild-type 7β-HSDH enzyme is 5.5, and after mutation, the optimum pH of the 7β-HSDH mutant provided above is improved from 5.5 to 7.0, and under the condition that the substrate 7K-LCA has good solubility at pH 8.0-8.5, the 7β-HSDH mutant provided above still has high enzyme activity.

[0023] As a second aspect of the present application, the present application provides a coding gene of the above-mentioned 7β-HSDH mutant.

[0024] As a third aspect of the present application, the present application provides an expression vector comprising the above-mentioned coding gene.

[0025] As preferred, the expression vector is a plasmid, a bacteriophage or a viral vector.

[0026] As a fourth aspect of the present application, the present application provides a host cell comprising the above-mentioned 7β-HSDH mutant or the above-mentioned coding gene.

[0027] As preferred, the host cell is Escherichia coli.

[0028] As a fifth aspect of the present application, the present application provides a use of the 7β-HSDH mutant in the preparation of ursodeoxycholic acid.

[0029] The method of application includes the following steps: using 7-carbonyllithocholic acid as a substrate and the above-mentioned alkali-resistant 7β-HSDH enzyme mutant as a catalyst to carry out a catalytic reaction.

[0030] Preferably, the catalytic reaction is carried out in a system with a pH of 5.5 to 9.0.

[0031] Under pH conditions of 5.5 to 9.5, the 7β-HSDH mutants provided by this invention all exhibit high enzyme activity.

[0032] Further preferably, the catalytic reaction is carried out in a system with a pH of 7.5 to 8.5.

[0033] Under conditions where the substrate 7K-LCA has good solubility at pH 7.5–8.5, the 7β-HSDH mutant provided above still exhibits high enzyme activity.

[0034] Compared with the prior art, the present invention has the following technical effects:

[0035] To improve the alkali tolerance of 7β-HSDH, this invention involves site-directed mutagenesis of 7β-HSDH derived from Collinsella aerofaciens, resulting in several alkali-tolerant 7β-HSDH mutants. The amino acid sequence of the 7β-HSDH derived from Collinsella aerofaciens is shown in SEQ ID NO.1. The 7β-HSDH mutants provided by this invention are single-point or multi-point combination mutations of E26K, P104S, N113Y, and M144T. The optimal pH for wild-type 7β-HSDH enzyme is 5.5. After mutation, the optimal pH of the provided 7β-HSDH mutants is increased from 5.5 to 7.0. Furthermore, at pH 5.5, the enzyme activity of the 7β-HSDH mutants is significantly higher than that of the wild-type enzyme; under alkaline conditions of pH 8.0–8.5, the 7β-HSDH mutants still exhibit high enzyme activity. Attached Figure Description

[0036] Figure 1 The enzyme activity assay results for the four-point combination mutants of E26K, P104S, N113Y, and M144T under different pH conditions are shown.

[0037] Figure 2 The results show the enzyme activity assays of different mutants at pH 7.0. Detailed Implementation

[0038] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0039] In the following examples, 7β-HSDH from Collinsella aerofaciens was mutated as a wild-type enzyme. The amino acid sequence of wild-type 7β-HSDH is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. The nucleotide sequence of the 7β-HSDH gene was synthesized by Jinan Boshan Biotechnology Co., Ltd. using whole-genome synthesis technology and ligated into the pET-28a plasmid vector to construct the pET-28a-7β-HSDH recombinant expression vector. The pET-28a plasmid vector is a conventional plasmid in the field, widely used in genetic engineering, and commercially available.

[0040] In the following examples, the primer names and sequence information of the primers involved are shown in Table 1.

[0041] Table 1 Primer Information

[0042]

[0043] Example 1

[0044] This embodiment uses the 7β-HSDH sequence from Collinsella aerofaciens as the original sequence. The 7β-HSDH gene was randomly mutated using error-prone PCR and expressed in E. coli BL21(DE3). The crude enzyme of the randomly mutated 7β-HSDH recombinant protein was obtained through fermentation and isolation. High-yield alkali-tolerant 7β-hydroxysteroid dehydrogenase strains were screened by measuring enzyme activity under alkaline conditions. The specific steps are as follows:

[0045] (1) Construction of randomly mutated recombinant strains

[0046] Using the pET-28a-7β-HSDH recombinant expression vector as a template, and the random mutation forward and reverse primers listed in Table 1 as primers, error-prone PCR products were obtained through error-prone PCR. The error-prone PCR reaction system is shown in Table 2.

[0047] Table 2 Commonly Misunderstood PCR Reaction Systems

[0048]

[0049] Error-prone PCR reaction conditions: First, pre-denaturate at 94℃ for 3 min; then denature at 94℃ for 30 s, anneal at 55℃ for 30 s, and anneal at 72℃ for 1 min, for a total of 30 cycles; finally, extend at 72℃ for 10 min.

[0050] The error-prone PCR products obtained above were subjected to electrophoresis and gel extraction for purification. The prokaryotic expression vector pET-28a(+) was double-digested with BamHI and HindIII at 37℃ for 1.5 h and then gel-extracted. The double digestion reaction system is shown in Table 3.

[0051] Table 3. Double enzyme digestion reaction system

[0052]

[0053] The purified error-prone PCR product and the linearized plasmid obtained by double digestion of pET-28a(+) were ligated at 50℃ for 15 min according to the reaction system in Table 4. 10 μL of the ligated plasmid was added to E. coli BL21(DE3) competent cells, placed on ice for 30 min, heat-shocked at 42℃ for 90 s, placed on ice for another 2 min, and then 500 μL of LB medium was added. The cells were cultured at 37℃ and 220 rpm for 1 h. 100 μL of the culture was then spread on plates containing Kana antibiotic and cultured overnight at 37℃ to obtain random mutant colonies.

[0054] Table 4 Connection Reaction System

[0055]

[0056] (2) Screening of random mutant strains

[0057] Single colonies obtained from step (1) were inoculated into 96-well cell culture dishes containing 200 μL of LB liquid medium and cultured overnight at 37°C with shaking. Then, 4% (v / v) of the colonies were inoculated into test tubes containing 200 μL of LB liquid medium and cultured at 37°C until the OD600 reached 0.4–0.6. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mmol / L for induction, and the temperature was lowered to 16°C for overnight further culture. The cells were centrifuged at 4000 r / min for 20 min, collected, and resuspended in 20 μL of BugBuster protein extraction reagent. After incubation for 10 min, the cells were centrifuged again, and the supernatant was used for later use.

[0058] Prepare 0.5 mmol / L NADPH and 5 mmol / L 7-KLCA using 50 mmol / L Tris-HCl (pH 9.0). Add 150 μL of 0.5 mmol / L NADPH and 45 μL of 5 mmol / L 7-KLCA to each well of a 96-well cell culture dish, followed by 5 μL of protein extraction supernatant. Incubate at 30°C for 15 minutes and measure the absorbance change at 340 nm. Lower absorbance values ​​indicate greater NADPH consumption and higher enzyme activity, thus allowing for screening of mutant proteins for enzyme activity under alkaline conditions.

[0059] This step screened approximately 10,000 clones, and selected 59 strains with significant variations in absorbance values. These were then subjected to a second round of screening and verification using the same method described above, resulting in the strain with the highest enzyme activity under alkaline conditions.

[0060] (3) Fermentation, protein isolation and purification and enzyme activity determination of alkali-tolerant mutant strains

[0061] The bacterial strain was inoculated into 100 mL of LB liquid medium (containing 35 μg / mL Kana) at a volume ratio of 1:1000 and cultured overnight at 37°C and 220 rpm. The fermented bacterial solution was then transferred to TB liquid medium at a 2% inoculation rate and cultured at 37°C and 220 rpm until the OD600 reached 0.4–0.6. IPTG was added to a final concentration of 0.5 mmol / L for induction, and the temperature was lowered to 16°C for further overnight culture. The bacterial cells were collected by centrifugation at 8000 rpm for 10 min, sonicated, and then centrifuged at 12000 rpm for 20 min. The supernatant was collected as the crude enzyme solution. 100 μL of the crude enzyme solution was sampled for protein SDS-PAGE electrophoresis detection.

[0062] The protease was purified using a Ni column. The Ni column was equilibrated with Lysis buffer, impurities were washed with wash buffer, and the target protein was eluted with elution buffer. The protein was then concentrated and dialyzed to remove salt, yielding the 7β-HSDH recombinant protein. Protein concentration was determined using a Bradford assay kit (Soluble).

[0063] The enzyme activity of the purified 7β-HSDH recombinant protein was measured under different pH conditions, and the results are shown in Table 5. Figure 1 Enzyme activity unit (U): Under appropriate conditions, the amount of enzyme required to convert 1 μmol of NADPH substrate per minute is defined as one enzyme activity unit. Specific activity of an enzyme is the number of active units per milligram of enzyme protein, expressed in U / mg.

[0064] The enzyme activity assay was performed as follows: Different pH gradient buffers were prepared: sodium phosphate buffer (pH 5.0–8.0, 50 mmol / L); Tris-HCl buffer (pH 8.0–9.0, 50 mmol / L); and glycine (Gly)-NaOH buffer (pH 9.0–11.0, 50 mmol / L), with each gradient separated by 0.5 pH values. The enzyme activity of 7β-HSDH recombinant protein in buffers at pH 5.0–11.0 was measured to determine the optimal pH for 7β-HSDH (using the original enzyme as a control).

[0065] Table 5

[0066]

[0067] From Table 5 and Figure 1 It is evident that, compared to the original strain, the optimal pH of the mutant strain increased from 5.5 to 7.0, and its enzyme activity was higher across the entire pH range of 5.0–10.0. Specifically, at pH 7.0, the enzyme activity of the mutant strain was 3.6 times that of the original strain. Colony sequencing revealed the following mutation sites for this alkali-tolerant mutant strain: E26K, P104S, N113Y, and M144T.

[0068] Example 2

[0069] Based on the four mutation sites E26K, P104S, N113Y, and M144T obtained in Example 1, this example conducts a detailed study of site-directed mutagenesis at these mutation sites. The steps are as follows: Site-directed mutagenesis primers as shown in Table 1 were designed based on the base sequences of the four mutation sites. Then, using the pET-28a-7β-HSDH recombinant expression vector as a template, site-directed mutagenesis PCR was performed using a site-directed mutagenesis kit to obtain the site-directed mutagenesis expression plasmid. The plasmid was then introduced into E. coli BL21(DE3) competent cells and cultured to form single colonies of recombinant bacteria. Whole-genome sequencing was then performed to obtain single-site mutants and multi-site combined mutants related to the four sites E26K, P104S, N113Y, and M144T. The mutants were inoculated into 100 mL of LB liquid medium (containing 35 μg / mL Kana) at a volume ratio of 1:1000 and cultured overnight at 37°C and 220 rpm. The fermented bacterial culture was inoculated at a 2% (v / v) in medium containing TB transfer. The culture was incubated at 37°C and 220 rpm until the OD600 reached 0.4–0.6. IPTG was added to a final concentration of 0.5 mmol / L for induction, and the temperature was lowered to 16°C for overnight incubation. The bacterial cells were collected by centrifugation at 8000 r / min for 10 min, sonicated, and then centrifuged at 12000 rpm for 20 min. The supernatant was collected to obtain the crude enzyme solution, which yielded the crude enzyme solutions corresponding to the single-point mutants and multi-site combination mutants related to the four sites E26K, P104S, N113Y, and M144T as shown in Table 6.

[0070] Following the reaction system described in Example 1, the enzyme activity of the obtained mutant crude enzyme solution was verified and measured under the optimal pH condition (pH 7.0). The results are shown in Table 6. Figure 2 . Figure 2 In the diagram, "0" represents the absence of a mutation site, indicating the wild type. Enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 μmol of NADPH substrate per minute under the given conditions. Enzyme activity is the number of active units per milliliter of enzyme protein, expressed in U / mL.

[0071] Table 6

[0072]

[0073] From Table 6 and Figure 2As can be seen, through site-directed mutagenesis and combined mutagenesis, several mutant strains with high activity were obtained, including single-site mutants E26K, P104S, and M144T, as well as two-site mutants P104S / M144T, two-site mutants N113Y / M144T, three-site mutants E26K / N113Y / M144T, three-site mutants P104S / N113Y / M144T, and four-site mutants E26K / P104S / N113Y / M144T.

[0074] Unless otherwise specified, the above embodiments were performed under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0075] Unless otherwise specified, the raw materials and equipment used in the above embodiments are all commonly used raw materials and equipment in the field.

[0076] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A 7β-HSDH mutant with alkali tolerance, characterized in that: Obtained from the amino acid sequence shown in SEQ ID NO.1 by any one of the following mutations: single-site mutation, two-site combination mutation, three-site combination mutation, or four-site combination mutation: The single-point mutation is: The 26th amino acid was changed from glutamic acid to lysine. The amino acid at position 104 is mutated from proline to serine; The 144th amino acid was mutated from methionine to threonine; The two-site combined mutation is: The 104th amino acid was mutated from proline to serine, and the 144th amino acid was mutated from methionine to threonine. The 113th amino acid was mutated from asparagine to tyrosine, and the 144th amino acid was mutated from methionine to threonine. The three-point combination mutation is: The 26th amino acid was mutated from glutamic acid to lysine, the 113th amino acid was mutated from asparagine to tyrosine, and the 144th amino acid was mutated from methionine to threonine. The amino acid at position 104 was mutated from proline to serine, the amino acid at position 113 was mutated from asparagine to tyrosine, and the amino acid at position 144 was mutated from methionine to threonine. The four-site combination mutation is: The 26th amino acid was mutated from glutamic acid to lysine, the 104th amino acid from proline to serine, the 113th amino acid from asparagine to tyrosine, and the 144th amino acid from methionine to threonine.

2. The encoding gene of the 7β-HSDH mutant as described in claim 1.

3. An expression vector comprising the encoding gene of claim 2.

4. The expression vector as described in claim 3, characterized in that: The expression vector is a plasmid, bacteriophage, or viral vector.

5. A host cell comprising the 7β-HSDH mutant of claim 1 or the encoding gene of claim 2.

6. The host cell as described in claim 5, characterized in that: The host cell is Escherichia coli.

7. The application of the 7β-HSDH mutant as described in claim 1 in the preparation of ursodeoxycholic acid.

8. The application as described in claim 7, characterized in that: The method of application includes the following steps: using 7-carbonyllithocholic acid as a substrate and the 7β-HSDH mutant of claim 1 as a catalyst to carry out a catalytic reaction.

9. The application as described in claim 7, characterized in that: The catalytic reaction is carried out in a system with a pH of 5.5 to 9.

5.

10. The application as described in claim 7, characterized in that: The catalytic reaction was carried out in a system with a pH of 7.5 to 8.5.

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