A hydroxysteroid dehydrogenase mutant with improved thermal stability, screening method and application thereof

Abstract

The application discloses a hydroxyl steroid dehydrogenase mutant with improved thermal stability, a screening method and application. Based on a hydroxyl steroid dehydrogenase structure prediction and PROSS and other computer-aided design technologies, key hot amino acid residues related to thermal stability are rapidly identified, so that a limited site-directed mutant library is constructed, and an advantage mutant is further screened. The half-inactivation temperature (T5 1 0 5 ) of the obtained mutant is improved by 1.0-7.7 DEG C relative to that before mutation, wherein the half-inactivation temperature (T5 1 0 5 ) of the mutants A126I and A132H is improved by 7.7 DEG C and 3.7 DEG C respectively. The relative enzyme activity of the mutant reaches 99.86-136.73% of that before mutation. The application provides a new method for rapidly improving the thermal stability of an enzyme, and lays a foundation for the application of the hydroxyl steroid dehydrogenase in industry.

Description

(I) Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to a hydroxysteroid dehydrogenase mutant with improved thermal stability, a screening method, and its application. (II) Background Technology

[0002] Natural enzymes often fail to meet the demands of industrial production in terms of activity, stability, and substrate selectivity, thus necessitating enzyme molecule modification to enhance their catalytic properties. Enzyme molecule modification techniques primarily include directed evolution, rational design, semi-rational design, and AI-aided design. Currently, breakthroughs in artificial intelligence technology and the emergence of innovative advanced algorithms are driving computer-aided protein design to the forefront of this field. By constructing a multi-scale molecular simulation platform and combining deep learning algorithms with quantum chemical calculations, researchers have demonstrated unique advantages in key areas such as complex enzyme structure prediction, stability enhancement path planning, selective regulation mechanism analysis, and catalytic active site reshaping. This data-driven design paradigm not only overcomes the limitations of traditional trial-and-error methods but also opens up a new dimension of enzyme molecule design based on first principles, providing new solutions for developing high-performance industrial enzyme preparations.

[0003] Thermal stability is a fundamental requirement for enzymes used in the bioindustry. Operating at higher temperatures offers significant advantages because: (1) the rate of chemical reactions approximately doubles for every 10°C increase; (2) substrate diffusion is greater due to reduced medium viscosity; (3) molecular solubility increases; and (4) the risk of potential contamination is lower. Stabilization strategies encompass a wide range of methods based on protein free energy calculations, including the use of statistical, empirical, or physical energy functions, bioinformatics methods based on consensus design or ancestor sequence reconstruction, and contemporary machine learning (ML) algorithms. Each strategy has its advantages and disadvantages. Currently, the PROSS strategy, developed using automated algorithms based on atomic Rosetta modeling and phylogenetic sequence information, is widely used. It can design mutants with large-area stabilizing effects without modifying protein function. For example, in CN 118599800 A, Li et al. addressed the problem of poor stability of microbial glycosyltransferases by obtaining a series of single-point mutant strains of glycosyltransferases with significantly improved thermostability through comprehensive calculation (FireProt and PROSS) and site-directed mutagenesis. Based on this, they obtained a series of combined mutants of glycosyltransferases with improved thermostability through combined mutagenesis, which significantly increased the yield of rhodioloside.

[0004] This invention uses a hydroxysteroid dehydrogenase derived from *Shewanella morhuae* as a research example. This enzyme stereoselectively catalyzes the hydroxyl oxidation of hydroxysteroids (cholic acid, chenodeoxycholic acid, and taurine chenodeoxycholic acid, etc.) to synthesize bile acid compounds. Based on the predicted structure of this hydroxysteroid dehydrogenase, this invention uses computer-aided design techniques such as PROSS to analyze relevant amino acid residue sites, constructs a defined point mutant library, rapidly identifies key hotspot residues affecting thermostability, and improves the thermostability of the hydroxysteroid dehydrogenase. By enhancing the thermostability of this enzyme through mutation, the reaction time required for the enzyme to catalyze the conversion of chenodeoxycholic acid to 7-ketolithocholic acid (7-KLCA) with a yield of 99% is effectively shortened. This invention provides a new method for modifying the thermostability of hydroxysteroid dehydrogenases, which helps lay the foundation for the industrial production of bile acid compounds such as ursodeoxycholic acid. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a thermo-stable hydroxysteroid dehydrogenase mutant, a screening method, and its application. This invention utilizes computer-aided design to efficiently screen thermo-stable hydroxysteroid dehydrogenase mutants, effectively improving screening efficiency. At the same time, it can effectively improve the stability of hydroxysteroid dehydrogenase in the biocatalytic synthesis of steroid compounds, solving the problem of poor stability of existing hydroxysteroid dehydrogenases.

[0006] The technical solution adopted in this invention is:

[0007] In a first aspect, the present invention provides a hydroxysteroid dehydrogenase mutant with improved thermal stability, wherein the mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 at positions 50, 126, 132, 173 or 203 at one or more sites.

[0008] The amino acid sequence shown in SEQ ID NO.1 of this invention is a hydroxysteroid dehydrogenase (WP_076500293.1) derived from the genus Shewanella morhuae.

[0009] Preferably, the hydroxysteroid dehydrogenase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 by one of the following: (1) valine at position 50 is mutated to alanine (V50A), and the amino acid sequence is shown in SEQ ID NO.3; (2) valine at position 50 is mutated to glutamic acid (V50E), and the amino acid sequence is shown in SEQ ID NO.4; (3) alanine at position 126 is mutated to isoleucine (A126I), and the amino acid sequence is shown in SEQ ID NO.5; (4) alanine at position 132 is mutated to histidine (A132H), and the amino acid sequence is shown in SEQ ID NO.6; (5) isoleucine at position 173 is mutated to leucine (I173L), and the amino acid sequence is shown in SEQ ID NO.7; (6) alanine at position 203 is mutated to proline (A203P), and the amino acid sequence is shown in SEQ ID NO.8.

[0010] Secondly, the present invention provides a gene encoding the hydroxysteroid dehydrogenase mutant.

[0011] Thirdly, the present invention provides a recombinant expression vector containing the gene encoding the hydroxysteroid dehydrogenase mutant.

[0012] Furthermore, the recombinant expression vector was constructed using pET 28a(+) as the base plasmid.

[0013] Fourthly, the present invention provides a recombinant genetically engineered bacterium containing the gene encoding the hydroxysteroid dehydrogenase mutant. The recombinant genetically engineered bacterium is obtained by expressing the recombinant expression vector in a host bacterium. The host bacterium can be any conventional host cell in the art, as long as the recombinant expression vector can stably replicate spontaneously and can effectively express the target protein after induction with an inducer, including E. coli BL21(DE3).

[0014] Furthermore, the recombinant genetically engineered bacteria are constructed according to the following steps: the gene encoding the hydroxysteroid dehydrogenase mutant is cloned into pET 28a(+) to construct a recombinant expression vector; the obtained recombinant expression vector is transformed into the host cell E.coli BL21(DE3) to obtain the recombinant genetically engineered bacteria E.coli BL21(DE3)(pET 28a-sm7α-hsdh).

[0015] Fifthly, the present invention provides a method for screening the hydroxysteroid dehydrogenase mutant, the method comprising:

[0016] First, AlphaFold3 (https: / / alphafoldserver.com) was used to predict the structure of Sm7α-HSDH (amino acid sequence as shown in SEQ ID NO.1). Molecular docking simulations were performed using YASARA software to construct an enzyme-substrate (chenodeoxycholic acid) complex model. The model's reliability was assessed using PROCHECK (https: / / saves.mbi.ucla.edu). Pymol was used for structural visualization analysis to clarify the spatial arrangement of active sites and the intermolecular interaction network. Then, based on this three-dimensional structural model, PROSS (https: / / pross.weizmann.ac.il / step / pross-terms / ) was used for analysis, and the model with the highest prediction accuracy was selected to construct a site-directed mutation model. PROSS provides a visualization interface for displaying mutation sites, supporting batch generation and evaluation of different mutation schemes. Based on this, ConSurf (https: / / consurf.tau.ac.il / consurf_index.php) is integrated for conservation assessment. ConSurf assigns a conservation score (1-9) to each amino acid site, with 1-3 points (low conservation) prioritized for engineering targets; 4-6 points (moderate conservation) may involve structure or function and require careful selection; 7-9 points (high conservation) are highly conserved and usually involve key functions (such as catalytic sites or binding sites) and are retained. Mutation combinations with excessively high conservation (7-9 points) or that do not conform to evolutionary trends are removed. DeepDDG (http: / / protein.org.cn / ddg.html) is used to calculate the change in mutation free energy to assess the impact of mutation on protein folding free energy, excluding those with ΔΔG values ​​< 0.5 kcal·mol⁻¹. -1 Unfavorable mutations were identified, and flexible amino acid sites were determined using B-FITTER software (http: / / www.kofo.mpg.de / en / research / biocatalysis), while rigid sites with B-factor values ​​≤0.72 were excluded. The following mutants with significant impact on thermal stability were ultimately selected: V50A, V50E, A126I, A132H, I173L, and A203P.

[0017] In a sixth aspect, the present invention provides the application of the hydroxysteroid dehydrogenase mutant in the catalytic synthesis of 7-ketolithocholic acid (7-KLCA) from chenodeoxycholic acid.

[0018] Furthermore, the application involves using the supernatant obtained by ultrasonically disrupting and centrifuging wet bacterial cells obtained from the induced culture of recombinant genetically engineered bacteria containing a hydroxysteroid dehydrogenase mutant encoding gene as a catalyst, and adding NAD+ as a substrate.+ Sodium pyruvate and lactate dehydrogenase (LDH) were used to form a conversion system with a pH 6-9 buffer (preferably pH 8.0, 100mM KH2PO4-K2HPO4 buffer) as the reaction medium. The conversion reaction was carried out at 20-40℃ (preferably 30℃) and 600rpm to obtain a conversion solution containing 7-KLCA.

[0019] Furthermore, in the conversion system, the substrate concentration is 50-150 g / L (preferably 80 g / L); NAD + The concentration of added catalyst is 1-5 mM (preferably 1 mM); the concentration of added sodium pyruvate is 100-500 mM (preferably 400 mM); the amount of added catalyst is 5-15 g / L (preferably 10 g / L) based on the wet cell volume before crushing, and 5-15 mg / L (preferably 10 mg / L) based on the protein content.

[0020] Further, the supernatant obtained by ultrasonic disruption and centrifugation of recombinant Escherichia coli containing the lactate dehydrogenase gene obtained through induced culture is added, at a concentration of 1-10 g / L (preferably 5 g / L) based on the amount of wet bacterial cells before disruption. The nucleotide sequence of the lactate dehydrogenase gene is shown in SEQ ID NO.10.

[0021] Further, the wet bacterial cells are prepared as follows: The recombinant genetically engineered bacteria encoding the hydroxysteroid mutant gene are inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 10 h to obtain a seed culture; the seed culture is then inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at an inoculation rate of 1.0% (v / v) and cultured at 37°C and 180 rpm until OD... 600 =0.6~0.8, then add isopropyl thiogalactoside (IPTG) to the culture medium to a final concentration of 0.2mM, incubate at 28℃ for 12h, and centrifuge at 4℃ and 8000rpm for 10min to obtain wet bacterial cells containing the 7α-HSDH mutant.

[0022] This invention relates to the inoculation, transfer, induction, and cell recovery of hydroxysteroid mutants. The preferred culture medium is LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in distilled water and adjusted to pH 7.0. There are no special limitations on the culture method and conditions.

[0023] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0024] This invention rapidly identifies key hotspot amino acid residues related to thermal stability based on hydroxysteroid dehydrogenase structure prediction analysis and computer-aided design techniques such as PROSS, thereby constructing a limited site-directed mutant library and efficiently screening mutants with significantly improved thermal stability.

[0025] The half-inactivation temperature of the obtained mutant The relative pre-mutation temperature increased by 1.0-7.7℃, with the half-inactivation temperature of mutants A126I and A132H being the highest. The temperature was increased by 7.7℃ and 3.7℃, respectively. The relative enzyme activity of the mutants reached 99.86-136.73% of the pre-mutation level, with mutants V50E and V50A achieving 136.73% and 127.84% of the wild-type activity, respectively. The mutants of this invention can shorten the reaction time (60-90 min) in high substrate concentration (80 g / L) reaction systems, while wild-type hydroxysteroid dehydrogenase requires at least 120 min to reach a yield of over 99%, thus shortening the reaction time by 30-60 min. Mutant V50A can achieve a 7-ketolithocholic acid (7-KLCA) yield of over 99% within 60 min, while mutants A132H, V50E, I173L, A126I, and A203P can all achieve a yield of over 99% within 90 min, significantly shortening the reaction time. In summary, this invention provides a new method for modifying the thermostability of hydroxysteroid dehydrogenases, laying a solid foundation for the industrial production of ursodeoxycholic acid. (iv) Description of the attached drawings

[0026] Figure 1 A schematic diagram of the reaction for the two-step coupling catalysis of 7α-HSDH and 7β-HSDH to prepare UDCA from CDCA.

[0027] Figure 2 This is the standard curve for NADH in Example 3.

[0028] Figure 3 The half-inactivation temperatures of wild-type 7α-HSDH and its mutant enzymes A132H, V50E, V50A, I173L, A126I, and A203P in Example 4 are shown. Measurement results diagram.

[0029] Figure 4 This is the standard curve for 7-KLCA in Example 5.

[0030] Figure 5 The reaction process diagram for the synthesis of 7-KLCA catalyzed by wild-type 7α-HSDH and its mutant enzymes A132H, V50E, V50A, I173L, A126I, and A203P in Example 5 is shown. (V) Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0032] Example 1: Construction and Induction of Recombinant Strains

[0033] 1. Construction and induced expression of wild-type 7α-HSDH recombinant strain

[0034] Based on the 7α-HSDH gene fragment (WP_076500293.1) from the genus *Shewanella* morhuae in the NCBI database, a 7α-HSDH gene fragment (nucleotide sequence as shown in SEQ ID NO.2, amino acid sequence as shown in SEQ ID NO.1), denoted as sm7α-hsdh, was artificially synthesized and inserted between the NcoⅠ and Xho I restriction sites of the vector pET28a(+) to construct the recombinant expression plasmid pET 28a-sm7α-hsdh. After confirming correct DNA sequencing, the recombinant plasmid was transformed into the host bacterium *E. coli* BL21(DE3) to obtain a recombinant strain of wild-type 7α-HSDH.

[0035] The recombinant strain of wild-type 7α-HSDH was inoculated into test tubes containing LB liquid medium with a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 8 h. Then, it was inoculated at a volume concentration of 1% into 100 mL of fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 2-3 h. Next, 0.2 mM IPTG was added to the culture medium, and the culture was incubated at 28°C for 12 h. Afterward, the culture was centrifuged at 4°C and 8000 rpm for 10 min to obtain wet cells. The wet cells were then resuspended at a concentration of 10 g / L in 100 mM phosphate buffer (pH 8.0). The suspension was ultrasonically disrupted for 10 min on an ice-water mixture under the following conditions: 200 W power, 2 s disruption, 1 s pause. The disrupted mixture was collected to obtain the corresponding crude enzyme solution.

[0036] SEQ ID NO.1

[0037] MYNPKDFTLNGDVAVITGAGAGIGRAIAETFAAAGAAVMVSDLKQETANVVAQAIIAQGGKAVAIDCDITQEDDLTRLVAQTISEFGKLTILVSNAGGGGPKPFDMPMADFRRAFDLNVFSLFRLA QIAAPAMEKAGGGSILGITSMAGENKNEHMASYASSKAATNHLIRNIAFDLGPKNIRVNGIAPGATRTTALESVLTAEIEQHMLKKTPIHRLGEPQDMANAALFLSSPAASWISGQILTVSGGGVQELE.

[0038] SEQ ID NO.2

[0039] ATGTATAACCCGAAAGACTTCACCCTGAATGGCGACGTTGCAGTTATTACCGGTGCAGGTGCAGGTATTGGTCGTGCAATTGCAGAAACCTTTGCAGCAGCAGGTGCAGCAGTTATGGTTAGCGATCTGAAACAGGAAACCGCAAATGTTGTTGCACAGGCAATTATTGCACAGGGTGGTAAAGCAGTTGCAATTGATTGTGATATTACCCAGGAAGACGATCTGACCCGTCTGGTTGCACAGACCATTAGCGAATTTGGTAAACTGACCATTCTGGTGAGCAATGCCGGTGGTGGTGGTCCGAAACCGTTTGATATGCCGATGGCAGATTTTCGTCGTGCATTTGATCTGAATGTTTTCAGCCTGTTTCGCCTGGCACAGATTGCAGCACCGGCAATGGAAAAAGCAGGTGGTGGTAGCATTCTGGGTATTACCAGCATGGCAGGTGAAAATAAAAATGAGCATATGGCAAGCTACGCCAGCAGCAAAGCAGCAACCAATCATCTGATTCGTAATATCGCATTTGACCTGGGCCCGAAAAATATTCGCGTTAATGGTATTGCACCGGGCGCAACCCGTACCACCGCATTAGAAAGTGTTCTGACCGCAGAAATTGAACAGCATATGCTGAAAAAAACCCCGATTCATCGTCTGGGTGAACCGCAGGATATGGCAAATGCAGCACTGTTTCTGAGCAGCCCGGCAGCAAGTTGGATTAGCGGTCAGATTCTGACCGTTAGCGGTGGTGGTGTTCAGGAACTGGAA。

[0040] 2. Construction and Induced Expression of Recombinant Bacteria of Lactate Dehydrogenase (LDH)

[0041] Based on the D-lactate dehydrogenase (GenBank accession number MCV5771625.1) derived from *E. coli* in the NCBI database, a lactate dehydrogenase gene fragment (nucleotide sequence shown in SEQ ID NO.10, amino acid sequence shown in SEQ ID NO.9) was artificially synthesized. This gene fragment was directionally inserted into the multiple cloning site of the pETDuet vector, successfully constructing the recombinant expression vector pETDuet-esldh. This recombinant expression vector was then transformed into *E. coli* BL21(DE3), and *E. coli* BL21(DE3) / pETDuet-esldh was obtained after antibiotic screening and sequencing verification. LDH crude enzyme solution was prepared using the same conditions and methods as those used to prepare crude enzyme solution from wild-type 7α-HSDH recombinant strains.

[0042] SEQ ID NO.9

[0043] MKLAVYSTKQYDKKYLDDVNESFGFELEFFDFLLTEKTAKTANGCEAVCIFVNDDGSRPVLEELKKHGVKYIALRCAGFNNVDLDAAKELGLKVVRVPAYDPEAVAEHAIGMMMMTLNRRIHRATQRTRDANFSLEGLTGFTMYGKTAGVIGTGKIGVAMLRILKG FGMRLLAFDPYPSAAALELGVEYVDLPTLFSESDVISLHCPLTPENYHLLNEAAFEQMKNGVMIVNTSRGALIDSQAAIEALKNQKIGSLGMDVYENERDLFFEDKSNDVIQDDVFRRLSACHNVLFTGHQAFLTAEALTSISQTTLQNLSNLEKGETCPNELVW.

[0044] SEQ ID NO.10 (underlined is histidine tag)

[0045] ATGAAACTGGCAGTTTACTCTACCAAACAGTACGACAAGAAATACCTGCAGCAGGTTAACGAAAGCTTCGGTTTCGAACTGGAATTTTTCGACTTTCTGCTGACCGAGAAGACCGCGAAGACTGCGAACGGCTGCGAAGCTGTTTGCATCTTCGTTAACGACGACGGTTCTCGTCCAGTTCTGGAAGAACTGAAGAAACACGGTGTGAAGTACATCGCGCTGCGTTGTGCGGGTTTCAACAA CGTGGACCTGGACGCAGCGAAAGAACTGGGTCTGAAAGTTGTACGTGTACCGGCGTATGATCCGGAAGCGGTTGCTGAACACGCGATCGGTATGATGATGACCTTGAACCGTCGTATCCACCGTGCGTACCAGCGTACTCGTGATGCAAACTTCTCTCTGGAAGGTCTGACCGGTTTCACCATGTACGGTAAGACCGCGGGCGTTATCGGTACCGGTAAGATCGGTGTTGCGATGCTGCGTATTCTGAAAGGTTTCGGTATGCGTCTGCTGGCGTTCGATCCATATCCGTCCGCTGCGGCACTGGAACTGGGTGTTGAATACGTTGACCTGCCAACTCTGTTCTCCGAATCCGACGTTATCTCTCTGCACTGTCCGCTGACTCCGGAGAACTACCACCTGCTGAACGAAGCAGCGTTCGAACAGATGAAGAACGGCGTAATGATCGTGAACACCAGCCGTGGTGCACTGATCGATTCTCAGGCAGCTATCGAAGCTCTGAAGAACCAGAAGATCGGTTCTCTGGGTATGGACGTTTACGAAAACGAACGTGACCTGTTCTTCGAAGACAAGAGCAACGACGTTATCCAAGACGACGTATTCCGTCGTCTGTCTGCTTGCCACAACGTGCTGTTCACCGGTCACCAGGCGTTCTTGACCGCTGAAGCGCTGACCTCTATCAGCCAGACCACTCTGCAGAACCTGTCTAACTTGGAGAAAGGCGAAACCTGTCCGAACGAACTGGTT CATCATCATCATCATCATTAA.

[0046] Example 2: Construction and screening of 7α-HSDH mutants

[0047] This invention uses 7α-HSDH structural simulation analysis and computer-aided design techniques such as PROSS to guide the construction of site-directed mutant libraries, thereby screening out key residue sites related to thermal stability and activity. The specific method is as follows:

[0048] First, AlphaFold3 (https: / / alphafoldserver.com) was used to predict the structure of Sm7α-HSDH (amino acid sequence as shown in SEQ ID NO.1). Molecular docking simulations were then performed using YASARA software to construct an enzyme-substrate (chenodeoxycholic acid) complex model. The model's reliability was assessed using PROCHECK (https: / / saves.mbi.ucla.edu). Pymol was used for structural visualization analysis to clarify the spatial arrangement of active sites and the intermolecular interaction network. Finally, based on this three-dimensional structural model, PROSS (https: / / pross.weizmann.ac.il / step / pross-terms / ) was used for analysis. The model with the highest prediction accuracy was selected to construct a site-directed mutation library. PROSS provides a visualization interface to display mutation sites and supports batch generation and evaluation of different mutation schemes. Based on this, ConSurf (https: / / consurf.tau.ac.il / consurf_index.php) was integrated for conservation assessment. ConSurf assigns a conservation score (1-9) to each amino acid site, with 1-3 points (low conservation) prioritized for engineering targets; 4-6 points (moderate conservation) may involve structure or function and require careful selection; 7-9 points (high conservation) are highly conserved and usually involve key functions (such as catalytic sites and binding sites) and are retained. Mutation combinations with excessively high conservation (7-9 points) or that do not conform to evolutionary trends were removed. DeepDDG (http: / / protein.org.cn / ddg.html) was used to calculate the change in mutational free energy to assess the impact of mutation on protein folding free energy, excluding mutations with ΔΔG values ​​< 0.5 kcal·mol⁻¹. -1Unfavorable mutations were identified, and flexible amino acid sites were analyzed using B-FITTER software (http: / / www.kofo.mpg.de / en / research / biocatalysis), while rigid sites with B-factor values ​​≤0.72 were excluded. The following mutants with significant impact on thermal stability were ultimately selected: V50A, V50E, A126I, A132H, I173L, and A203P. Compared to traditional modification methods, this approach prioritizes factors such as the energy balance between local and overall protein structure, the coordinated distribution of surface charge characteristics and hydrophobic regions, and intramolecular interaction networks.

[0049] Based on the optimized mutation scheme, specific amplification primers were designed, and site-directed PCR amplification was performed at each site. The amplified products were transformed into E. coli BL21(DE3) competent cells. After resistance selection, expression was induced in E. coli cells according to the method in Example 1, and crude enzyme solution was prepared as a catalyst to catalyze the synthesis of 7-KLCA from CDCA. Dominant mutants were screened based on product yield. The screening process for dominant mutants is as follows:

[0050] Using the recombinant plasmid pET-28a(+)-sm7α-hsdh obtained in Example 1 as a template, site-directed mutagenesis PCR amplification was performed using the upstream and downstream primers in Table 1.

[0051] Table 1. Primer design for 7α-HSDH site-directed mutagenesis

[0052]

[0053] The PCR (50 μL) amplification system is as follows: 25 μL of 2×PCR buffer, 1 μL each of forward and reverse primers, 1 μL of template plasmid, 1 μL of dNTP, 1 μL of high-fidelity enzyme, and ddH2O added to make up to 50 μL.

[0054] The PCR amplification program was as follows: pre-denaturation at 95℃ for 5 min, followed by 32 cycles (denaturation at 95℃, annealing at 60℃ for 30 s, extension at 72℃ for 6 min), 72℃ for 5 min, and storage at 4℃.

[0055] After PCR, 2 μL of the amplification product was analyzed by nucleic acid gel electrophoresis. The PCR product with a clear target band was added to 1 μL of DpnI restriction enzyme and incubated overnight at 37°C for digestion. 10 μL of the digested PCR product was added to a 1.5 mL centrifuge tube containing 100 μL of E. coli BL21(DE3) competent cells and incubated on ice for 30 min. The tube was then heat-shocked at 42°C for 1.5 min and rapidly transferred to ice for 2 min. 600 μL of antibiotic-free, sterile LB liquid medium was added, and the cells were incubated at 37°C and 200 rpm for 1 h to revive the cells. The cells were then centrifuged at 4°C and 6000 rpm for 1 min, and 650 μL of supernatant was discarded. The cells were resuspended in the centrifuge tube and plated on LB solid medium containing 50 μg / mL kanamycin. The cells were incubated at 37°C for approximately 12 h, and positive transformants were picked to prepare crude enzyme solution according to the method in Example 1.

[0056] The obtained positive transformants were screened for dominant mutants under the following conditions: 10 g / L CDCA, 1 mM NAD. + 50 mM sodium pyruvate, 5 g / L wild-type 7α-HSDH and its mutant crude enzyme solution, and 2.5 g / L LDH crude enzyme solution were reacted in a water bath at 30℃ and 600 rpm for 5 min. Parallel samples were then taken, and the reaction was terminated with 3 M HCl. The mixture was then extracted three times with an equal volume of ethyl acetate. The organic phases were combined, concentrated under reduced pressure to remove the organic solvent, and the product was collected. The content of 7-KLCA in the product was determined by liquid chromatography. Using the amount of 7-KLCA produced as an indicator, the dominant strains with high catalytic efficiency were screened: *E. coli* BL21(DE3)-7α-HSDH-V50A (amino acid sequence as shown in SEQ ID NO.3), *E. coli* BL21(DE3)-7α-HSDH-V50E (amino acid sequence as shown in SEQ ID NO.4), *E. coli* BL21(DE3)-7α-HSDH-A126I (amino acid sequence as shown in SEQ ID NO.5), and *E. coli* BL21(DE3)-7α-HSDH-A126I (amino acid sequence as shown in SEQ ID NO.5). BL21(DE3)-7α-HSDH-A132H (amino acid sequence as shown in SEQ ID NO.6), E. coli BL21(DE3)-7α-HSDH-I173L (amino acid sequence as shown in SEQ ID NO.7), E. coli BL21(DE3)-7α-HSDH-A203P (amino acid sequence as shown in SEQ ID NO.8).

[0057] 7-KLCA HPLC detection method: Ultimate 3000 HPLC system, C18 column (50×2.1mm), mobile phase 1mM KH2PO4 buffer (pH adjusted to 3.0 with phosphoric acid): acetonitrile = 40:60 (v / v), flow rate 1.0mL / min, column temperature 30℃, detection wavelength 210nm.

[0058] Example 3: Enzyme activity assay of 7α-HSDH wild type and its mutants

[0059] Preparation of NADH standard curve: Prepare NADH standard solutions of 2mM, 1.5mM, 1mM, 0.5mM, 0.4mM, 0.3mM, 0.2mM, and 0.1mM using 100mM KH2PO4-K2HPO4 buffer (pH 8.0). Detect the standard solutions at OD values ​​using a microplate reader. 340nm The absorbance values ​​at the specified points are plotted on the x-axis, with different concentrations of NADH as the corresponding OD values. 340nm Plot the NADH standard curve using absorbance values ​​as the ordinate, and the results are as follows: Figure 2 As shown. Therefore, the standard curve equation for NADH is: y = 2.9319x + 0.162, R... 2 =0.9994.

[0060] Enzyme activity assay standard conditions: The reaction system volume is 200 μL, and NAD+ is added to a final concentration of 2 mM. + Add 2 mM CDCA and an appropriate amount of crude enzyme solution (0.01 mg / mL based on protein concentration), and bring the volume to 200 μL with pH 8.0, 100 mM KH₂PO₄-K₂HPO₄ buffer. Incubate at 30°C with shaking for 1 min. Measure the absorbance at 340 nm every 10 s. Calculate NAD based on the NADH standard curve. + Consumption rate. The unit cell enzyme activity of 7α-HSDH and its mutants is shown in Table 2.

[0061] The enzyme activity (U) of 7α-HSDH is defined as the amount of enzyme required to generate 1 μmol of NADH per minute under standard enzyme activity detection conditions.

[0062] Unit bacterial cell enzyme activity (U g) -1 wcw ): Enzyme activity of wet bacterial cells containing the 7α-HSDH gene per gram.

[0063] Table 2. Unit cell enzyme activity of 7α-HSDH and its mutants

[0064] Enzyme <![CDATA[Activity(U g -1 wcw )]]> Wild-type 13507.42 V50A 17267.89 V50E 18468.70 A126I 13519.58 A132H 13488.51 I173L 13699.23 A203P 14336.78

[0065] The specific results obtained from Table 2 are as follows: Under the same reaction conditions, the enzyme activities of the mutants A132H, V50E, V50A, I173L, A126I, and A203P were 99.86%, 136.73%, 127.84%, 101.42%, 100.09%, and 106.14% of the wild-type enzyme activity, respectively.

[0066] Example 4: Determination of the thermal stability of 7α-HSDH wild type and its mutants

[0067] Half-deactivation temperature This refers to the temperature required to reduce enzyme activity to 50% of its original activity after incubation for 15 minutes within a certain temperature gradient range.

[0068] The crude enzyme solutions of wild-type 7α-HSDH and its mutants prepared by the method in Example 1 were incubated at different temperatures (30.0℃, 31.1℃, 32.8℃, 35.2℃, 38.2℃, 41.1℃, 43.7℃, 47.1℃, 50.0℃, 52.3℃, 54.0℃, 55℃) for 15 min each. Immediately after incubation, they were placed on ice to cool. The residual enzyme activity was measured at each temperature using the enzyme activity detection standard conditions of Example 3, thus obtaining... The half-deactivation temperature was obtained using Origin 2022 nonlinear curve fitting. The results are shown in Table 3 below.

[0069] Table 3. Half-inactivation temperatures of 7α-HSDH and its mutants

[0070]

[0071] As shown in Table 3, under the same reaction conditions, the mutants A132H, V50E, V50A, I173L, A126I, and A203P showed different results. The temperatures increased by 3.7℃, 1.0℃, 2.0℃, 2.1℃, 7.7℃, and 2.6℃, respectively.

[0072] Example 5: Synthesis of 7-KLCA catalyzed by wild-type 7α-HSDH and its mutants

[0073] 7-Ketolithocholic acid was prepared using the wild-type and mutant 7α-HSDH obtained in Example 1 and crude lactate dehydrogenase. The specific procedure is as follows: 10 mL of the reaction system was supplemented with 80 g / L CDCA and 1 mM NAD. +400 mM sodium pyruvate, wild-type 7α-HSDH or its mutant, crude enzyme solution prepared according to the method of Example 1 (added in an amount of 10 g / L based on the weight of wet cells before lysis), and LDH crude enzyme solution prepared according to the method of Example 1 (added in an amount of 5 g / L based on the weight of wet cells before lysis) were added to a final volume of 10 mL with pH 8.0 and 100 mM KH2PO4-K2HPO4 buffer. The mixture was reacted in a water bath at 30 °C and 600 rpm for 10, 20, 30, 45, 60, 90, 120, and 150 min. Parallel samples were taken, and the reaction was terminated with 3 M HCl. The mixture was then extracted three times with an equal volume of ethyl acetate. The organic phases were combined, concentrated under reduced pressure to remove the organic solvent, and the product was collected. The peak area was detected by liquid chromatography, and the 7-KLCA content of the product was calculated based on the 7-KLCA standard curve. The specific reaction process curve is shown in Figure 1. Figure 5 As shown in the figure. The results showed that under the same reaction conditions, wild-type 7α-HSDH required at least 120 min to achieve a yield of over 99%, while mutant V50A achieved a yield of >99% after 60 min of reaction, and mutants A132H, V50E, I173L, A126I, and A203P achieved a yield of >99% after 90 min of reaction, which shortened the reaction time by 30-60 min compared to wild-type.

[0074] Preparation of the 7-KLCA standard curve: 7-KLCA standard solutions of 5 g / L, 4 g / L, 3 g / L, 2 g / L, 1 g / L, 0.5 g / L, and 0.25 g / L were prepared using pure methanol, and the peak areas were detected by liquid chromatography. A 7-KLCA standard curve was plotted with 7-KLCA concentration on the x-axis and the corresponding peak area on the y-axis. The results are shown below. Figure 4 As shown. Therefore, the equation for the 7-KLCA standard curve is: y = 6.8247x - 2.0744, R... 2 =0.9991.

Claims

1. A hydroxysteroid dehydrogenase mutant with improved thermal stability, characterized in that, The hydroxysteroid dehydrogenase mutant is formed by making one of the following point mutations to the amino acid sequence shown in SEQ ID NO.1: (1) valine at position 50 is mutated to alanine; (2) valine at position 50 is mutated to glutamic acid; (3) alanine at position 126 is mutated to isoleucine; (4) alanine at position 132 is mutated to histidine; (5) isoleucine at position 173 is mutated to leucine; (6) alanine at position 203 is mutated to proline.

2. The encoding gene of the hydroxysteroid dehydrogenase mutant as described in claim 1.

3. A recombinant expression vector containing the encoding gene of the hydroxysteroid dehydrogenase mutant as described in claim 2.

4. A recombinant genetically engineered bacterium containing the encoding gene of the hydroxysteroid dehydrogenase mutant of claim 2.

5. The use of the hydroxysteroid dehydrogenase mutant of claim 1 in the catalytic synthesis of 7-ketolithocholic acid from chenodeoxycholic acid.

6. The application as described in claim 5, characterized in that, The application involves using the supernatant obtained by ultrasonically disrupting and centrifuging wet bacterial cells obtained from the induced culture of recombinant genetically engineered bacteria containing a gene encoding a hydroxysteroid dehydrogenase mutant as a catalyst, and chenodeoxycholic acid as a substrate, with the addition of NAD+. + Sodium pyruvate and lactate dehydrogenase were used to form a conversion system with a buffer solution of pH 6-9 as the reaction medium. The conversion reaction was carried out at 20-40℃ and 600 rpm to obtain a conversion solution containing 7-ketolithocholic acid.

7. The application as described in claim 6, characterized in that, In the conversion system, the substrate concentration is 50-150 g / L; NAD + The concentration of added components is 1-5 mM; the concentration of added sodium pyruvate is 100-500 mM; the amount of added catalyst is 5-15 g / L based on the wet bacterial cells before lysis, or 5-15 mg / L based on the protein content; the lactate dehydrogenase is added in the form of the supernatant obtained by ultrasonic disruption and centrifugation of wet bacterial cells obtained by induced culture of recombinant Escherichia coli containing the lactate dehydrogenase gene, and the amount added is 1-10 g / L based on the wet bacterial cells before lysis; the nucleotide sequence of the lactate dehydrogenase gene is shown in SEQ ID NO.

10.

8. The application as described in claim 6, characterized in that, The wet bacterial cells were prepared as follows: the recombinant genetically engineered bacteria containing the gene encoding the hydroxysteroid dehydrogenase mutant were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37 ℃ and 180 rpm for 10 h to obtain the seed culture; The seed culture was inoculated at a volume concentration of 1.0% into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37 ℃ and 180 rpm until OD. 600 =0.6~0.8, then add isopropyl thiogalactoside to the culture medium to a final concentration of 0.2 mM, incubate at 28 ℃ for 12 h, and then centrifuge at 4 ℃ and 8000 rpm for 10 min to obtain the wet bacterial cells.

Images