Hydroxysteroid dehydrogenase mutant with improved thermal stability, screening method and application

Abstract

The invention discloses a hydroxysteroid dehydrogenase mutant with improved thermal stability, a screening method and application, key hotspot amino acid residues related to the thermal stability are rapidly identified based on computer-aided design technologies such as hydroxysteroid dehydrogenase structure prediction, PROSS and the like, so that a limited site-directed mutant library is constructed; and further screening to obtain a dominant mutant. The semi-inactivation temperature (T5105) of the obtained mutant is increased by 1.0-7.7 DEG C compared with that before mutation, wherein the semi-inactivation temperatures (T5105) of the mutants A126I and A132H are increased by 7.7 DEG C and 3.7 DEG C respectively. And the relative enzyme activity of the mutant reaches 99.86-136.73% of the relative enzyme activity before mutation. The invention provides a new method for rapidly improving the thermal stability of the enzyme, and lays a foundation for the industrial application of the hydroxysteroid dehydrogenase.

Description

(1) Technical field

[0001] The invention belongs to the field of genetic engineering and relates to a hydroxysteroid dehydrogenase mutant with improved thermal stability, a screening method and an application. (2) Background technology

[0002] Natural enzymes often fail to meet the demands of industrial production in terms of activity, stability, and substrate selectivity, so enzyme molecules need to be modified to improve their catalytic properties. Enzyme molecule modification technologies mainly include directed evolution, rational design, semi-rational design, and artificial intelligence-assisted design. Currently, breakthroughs in artificial intelligence technology and the emergence of innovative advanced algorithms are driving computer-aided protein design to become a cutting-edge direction in this field. By building a multi-scale molecular simulation platform and combining deep learning algorithms with quantum chemical calculations, researchers have demonstrated unique advantages in key issues such as complex enzyme structure prediction, stability enhancement path planning, selective regulation mechanism analysis, and catalytic active site remodeling. This data-driven design paradigm not only breaks through 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 the development of 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 doubles approximately every 10°C, (2) substrate diffusion is higher due to reduced medium viscosity, (3) the solubility of molecules is increased, and (4) the potential risk of contamination is lower. Stabilization strategies encompass a wide range of approaches based on protein free energy calculations, including the use of statistical, empirical, or physical energy functions, bioinformatics methods based on consensus design or ancestral sequence reconstruction, and contemporary machine learning (ML) algorithms. Each strategy has its strengths and weaknesses. Currently, the PROSS strategy, developed based on automated algorithms based on atomistic Rosetta modeling and phylogenetic sequence information, has been widely used and can design mutants with large-scale stabilizing effects without modifying protein function. For example, in CN 118599800 A, researchers such as Li et al. addressed the problem of poor stability of microbial glycosyltransferases by using comprehensive calculations (FireProt and PROSS) and site-directed mutagenesis to obtain a series of glycosyltransferase single-point mutants with significantly improved thermal stability. On this basis, through combined mutagenesis, they obtained a series of glycosyltransferase combination mutants with improved thermal stability, significantly increasing the production of salidroside.

[0004] This invention uses a hydroxysteroid dehydrogenase derived from Shewanella morhuae as a research example. This enzyme can stereoselectively catalyze the oxidation of the hydroxyl groups of hydroxysteroids (cholic acid, chenodeoxycholic acid, and taurochenodeoxycholic acid, among others) to synthesize bile acid compounds. Based on the predicted structure of this hydroxysteroid dehydrogenase, the invention uses computer-aided design techniques such as PROSS to analyze relevant amino acid residues and construct a library of defined point mutants. This allows for the rapid identification of key hotspot residues affecting thermal stability, thereby improving the thermal stability of the hydroxysteroid dehydrogenase. By enhancing the enzyme's thermal stability through mutations, the reaction time required for the enzyme to catalyze the production of 7-ketolithocholic acid (7-KLCA) from chenodeoxycholic acid reaches a 99% yield. This invention provides a new method for modifying the thermal stability of hydroxysteroid dehydrogenase, which will help lay the foundation for the industrial production of bile acid compounds such as ursodeoxycholic acid. (3) Summary of the invention

[0005] The present invention aims to provide a hydroxysteroid dehydrogenase mutant with improved thermal stability, a screening method and an application thereof. The present invention utilizes computer-aided design to efficiently screen hydroxysteroid dehydrogenase mutants with improved thermal stability, thereby effectively improving the screening efficiency and effectively improving the stability of hydroxysteroid dehydrogenase in the biocatalytic synthesis of steroid compounds, thereby solving the problem of poor stability of existing hydroxysteroid dehydrogenases.

[0006] The technical solution adopted in the present 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 performing single-site or multi-site mutation at position 50, 126, 132, 173 or 203 of the amino acid sequence shown in SEQ ID NO.1.

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

[0009] Preferably, the hydroxysteroid dehydrogenase mutant is a mutant of the amino acid sequence shown in SEQ ID NO.1, wherein one of the following mutations is performed: (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] In a second aspect, the present invention provides a gene encoding the hydroxysteroid dehydrogenase mutant.

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

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

[0013] In a fourth aspect, 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 on its own and effectively express the target protein after induction with an inducer, including E. coli BL21 (DE3).

[0014] Furthermore, the recombinant genetically engineered bacteria were constructed according to the following steps: the gene encoding the hydroxysteroid dehydrogenase mutant was cloned into pET 28a(+) to construct a recombinant expression vector; the obtained recombinant expression vector was 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] In a fifth aspect, the present invention provides a method for screening the hydroxysteroid dehydrogenase mutant, the method comprising:

[0016] First, the structure of Sm7α-HSDH (amino acid sequence such as SEQ ID NO.1) was predicted using AlphaFold3 (https: / / alphafoldserver.com), and molecular docking simulation was performed using YASARA software to construct an enzyme-substrate (chenodeoxycholic acid) complex model. The quality assessment was performed using PROCHECK (https: / / saves.mbi.ucla.edu) to ensure the reliability of the model. Structural visualization analysis was performed using Pymol to clarify the spatial arrangement of the active site 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 analysis. For the mutation library, PROSS provides a visual interface to display mutation sites and supports batch generation and evaluation of different mutation schemes. On this basis, ConSurf (https: / / consurf.tau.ac.il / consurf_index.php) is integrated for conservation assessment. ConSurf scores each amino acid site for conservation (1-9 points), of which 1-3 points (low conservation) are prioritized as engineering targets; 4-6 points (moderate conservation) may be involved in structure or function and need to be selected with caution; 7-9 points (high conservation) are highly conserved and usually involve key functions (such as catalytic sites and binding sites) and should be retained. Mutation combinations that are too conserved (7-9 points) or do not conform to evolutionary trends are removed. At the same time, DeepDDG (http: / / protein.org.cn / ddg.html) is used to calculate the change in mutation free energy to evaluate the impact of mutations on protein folding free energy, excluding those with ΔΔG values < 0.5 kcal·mol -1 Unfavorable mutations were identified using B-FITTER software (http: / / www.kofo.mpg.de / en / research / biocatalysis). Flexible amino acid sites were identified, while rigid sites with B-factor values ≤ 0.72 were excluded. The following mutants with the greatest impact on thermal stability were selected: V50A, V50E, A126I, A132H, I173L, and A203P.

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

[0018] Furthermore, the application is: using the supernatant of wet bacteria obtained by ultrasonic disruption and centrifugation of recombinant genetically engineered bacteria containing a hydroxysteroid dehydrogenase mutant encoding gene as a catalyst, using chenodeoxycholic acid (CDCA) as a substrate, adding NAD+ , sodium pyruvate and lactate dehydrogenase (LDH), a conversion system is formed with a pH 6-9 buffer (preferably pH 8.0, 100mM KH2PO4-K2HPO4 buffer) as the reaction medium, and the conversion reaction is carried out at 20-40°C (preferably 30°C) and 600 rpm to obtain a conversion solution containing 7-KLCA.

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

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

[0021] Furthermore, the wet bacteria were prepared as follows: the recombinant genetically engineered bacteria encoding the hydroxysteroid mutant gene were 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 solution; the seed solution was inoculated into a fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at an inoculum volume concentration of 1.0% (v / v), and cultured at 37°C and 180 rpm until the OD 600 =0.6-0.8, and then add isopropylthiogalactoside (IPTG) to the culture medium with a final concentration of 0.2 mM. After culturing at 28°C for 12 h, the culture medium is centrifuged at 4°C and 8000 rpm for 10 min to obtain wet cells containing the 7α-HSDH mutant.

[0022] The culture medium for inoculation, transfer, induction, and cell recovery of the hydroxysteroid mutant of the present invention is preferably 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 particular restrictions on the culture method and culture conditions.

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

[0024] The present invention is based on hydroxysteroid dehydrogenase structure prediction analysis and computer-aided design technologies such as PROSS to rapidly identify key hot spot amino acid residues related to thermal stability, thereby constructing a limited site-directed mutant library and efficiently screening to obtain mutants with significantly improved thermal stability.

[0025] Half-inactivation temperature of the obtained mutant The half-inactivation temperature of mutants A126I and A132H increased by 1.0-7.7℃ compared to before mutation. The results show that the enzyme activity of the mutants reached 99.86-136.73% of that before mutation, of which the enzyme activities of mutants V50E and V50A reached 136.73% and 127.84% of that of the wild type, respectively. The mutants of the present invention can shorten the reaction time (60-90 min) in a reaction system with a high substrate concentration (80 g / L), while the wild-type hydroxysteroid dehydrogenase requires at least 120 min to achieve a yield of more than 99%, and the reaction time is shortened by 30-60 min. Among them, the mutant V50A can achieve a 7-ketolithocholic acid (7-KLCA) yield of more than 99% within 60 min, while the mutants A132H, V50E, I173L, A126I, and A203P can all achieve a yield of more than 99% within 90 min, significantly shortening the reaction time. In summary, the present invention provides a new method for modifying the thermal stability of hydroxysteroid dehydrogenase, laying a solid foundation for the industrial production of ursodeoxycholic acid. (IV) Description of the accompanying drawings

[0026] Figure 1 Schematic diagram of the reaction of preparing UDCA from CDCA catalyzed by 7α-HSDH and 7β-HSDH in two steps.

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

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

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

[0030] Figure 5 This is a reaction process diagram of the synthesis of 7-KLCA catalyzed by the wild-type 7α-HSDH and its mutant enzymes A132H, V50E, V50A, I173L, A126I, and A203P in Example 5. (V) Specific implementation methods

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

[0032] Example 1: Construction of recombinant strain and induced expression

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

[0034] Based on the 7α-HSDH gene fragment from Shewanella morhuae (WP_076500293.1) listed in the NCBI database, a synthetic 7α-HSDH gene fragment (nucleotide sequence shown in SEQ ID NO. 2, amino acid sequence shown in SEQ ID NO. 1) was synthesized and designated sm7α-hsdh. This fragment was then inserted into the pET28a(+) vector between the Nco I and Xho I restriction sites to construct the recombinant expression plasmid pET 28a-sm7α-hsdh. After DNA sequencing confirmed the identity of the recombinant plasmid, it was transformed into the host strain E. coli BL21(DE3) to obtain a recombinant strain expressing the wild-type 7α-HSDH.

[0035] The wild-type 7α-HSDH recombinant strain was inoculated into a test tube containing LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 8 hours. The inoculum was then inoculated into 100 mL of fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 1% and cultured at 37°C and 180 rpm for 2-3 hours. IPTG was then added to the culture at a final concentration of 0.2 mM and cultured at 28°C for 12 hours. The culture was then centrifuged at 8000 rpm for 10 minutes at 4°C to obtain wet cells. The wet cells were then resuspended in 100 mM phosphate buffer, pH 8.0, at a concentration of 10 g / L. The suspension was then ultrasonically disrupted in an ice-water mixture for 10 minutes using the following ultrasonication conditions: 200 W power, 2 seconds of disruption, and a 1-second pause. The resulting suspension was then 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 from Escherichia coli (E. coli) in the NCBI database (GenBank accession number MCV5771625.1), 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). After antibiotic screening and sequencing verification, E. coli BL21(DE3) / pETDuet-esldh was obtained. Crude LDH enzyme solution was prepared using the same conditions and methods as those used to prepare crude enzyme solution from the wild-type 7α-HSDH recombinant strain.

[0042] SEQ ID NO.9

[0043] MKLAVYSTKQYDKKYLDDVNESFGFELEFFDFLLTEKTAKTANGCEAVCIFVNDDGSRPVLEELKKHGVKYIALRCAGFNNVDLDAAKELGLKVVRVPAYDPEAVAEHAIGMMMMTLNRRIHRATQRTRDANFSLEGLTGFTMYGKTAGVIGTGKIGVAMLRILKG FGMRLLAFDPYPSAAALELGVEYVDLPTLFSESDVISLHCPLTPENYHLLNEAAFEQMKNGVMIVNTSRGALIDSQAAIEALKNQKIGSLGMDVYENERDLFFEDKSNDVIQDDVFRRLSACHNVLFTGHQAFLTAEALTSISQTTLQNLSNLEKGETCPNELVW.

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

[0045] ATGAAACTGGCAGTTTACTCTACCAAACAGTACGACAAGAAATACCTGCAGCAGGTTAACGAAAGCTTCGGTTTCGAACTGGAATTTTTCGACTTTCTGCTGACCGAGAAGACCGCGAAGACTGCGAACGGCTGCGAAGCTGTTTGCATCTTCGTTAACGACGACGGTTCTCGTCCAGTTCTGGAAGAACTGAAGAAACACGGTGTGAAGTACATCGCGCTGCGTTGTGCGGGTTTCAACAA CGTGGACCTGGACGCAGCGAAAGAACTGGGTCTGAAAGTTGTACGTGTACCGGCGTATGATCCGGAAGCGGTTGCTGAACACGCGATCGGTATGATGATGACCTTGAACCGTCGTATCCACCGTGCGTACCAGCGTACTCGTGATGCAAACTTCTCTCTGGAAGGTCTGACCGGTTTCACCATGTACGGTAAGACCGCGGGCGTTATCGGTACCGGTAAGATCGGTGTTGCGATGCTGCGTATTCTGAAAGGTTTCGGTATGCGTCTGCTGGCGTTCGATCCATATCCGTCCGCTGCGGCACTGGAACTGGGTGTTGAATACGTTGACCTGCCAACTCTGTTCTCCGAATCCGACGTTATCTCTCTGCACTGTCCGCTGACTCCGGAGAACTACCACCTGCTGAACGAAGCAGCGTTCGAACAGATGAAGAACGGCGTAATGATCGTGAACACCAGCCGTGGTGCACTGATCGATTCTCAGGCAGCTATCGAAGCTCTGAAGAACCAGAAGATCGGTTCTCTGGGTATGGACGTTTACGAAAACGAACGTGACCTGTTCTTCGAAGACAAGAGCAACGACGTTATCCAAGACGACGTATTCCGTCGTCTGTCTGCTTGCCACAACGTGCTGTTCACCGGTCACCAGGCGTTCTTGACCGCTGAAGCGCTGACCTCTATCAGCCAGACCACTCTGCAGAACCTGTCTAACTTGGAGAAAGGCGAAACCTGTCCGAACGAACTGGTT CATCATCATCATCATCATTAA.

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

[0047] The present invention is based on 7α-HSDH structural simulation analysis and computer-aided design technologies such as PROSS to guide the construction of a site-directed mutant library, thereby screening key residue sites related to thermal stability and activity. The specific method is as follows:

[0048] First, the structure of Sm7α-HSDH (amino acid sequence shown in SEQ ID NO. 1) was predicted using AlphaFold3 (https: / / alphafoldserver.com). Molecular docking simulations were performed using YASARA software to construct a model of the enzyme-substrate (chenodeoxycholic acid) complex. PROCHECK (https: / / saves.mbi.ucla.edu) was used for quality assessment to ensure model reliability. Pymol was used for structural visualization to clarify the spatial arrangement of the active site and the intermolecular interaction network. This three-dimensional structural model was then analyzed using PROSS (https: / / pross.weizmann.ac.il / step / pross-terms / ). The model with the highest prediction accuracy was selected to construct a site-directed mutagenesis library. PROSS provides a visual interface for displaying mutation sites and supports the batch generation and evaluation of different mutation schemes. On this basis, ConSurf (https: / / consurf.tau.ac.il / consurf_index.php) was integrated for conservation assessment. ConSurf assigns a conservation score (1-9 points) to each amino acid site. Score 1-3 (low conservation) prioritizes them as engineering targets; 4-6 (moderate conservation) may be involved in structure or function and require careful selection; 7-9 (high conservation) is highly conserved and usually involves key functions (such as catalytic sites and binding sites) and should be retained. Mutation combinations with 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 mutation free energy to assess the impact of mutations on protein folding free energy. ΔΔG values < 0.5 kcal·mol were excluded. -1Unfavorable mutations were identified using B-FITTER software (http: / / www.kofo.mpg.de / en / research / biocatalysis). Flexible amino acid sites were identified, while rigid sites with B-factors ≤ 0.72 were excluded. The following mutants with significant thermal stability effects were ultimately selected: V50A, V50E, A126I, A132H, I173L, and A203P. Compared to traditional modification methods, this protocol prioritizes factors such as the energy balance between local and global protein structure, the coordinated distribution of surface charge and hydrophobic regions, and the intramolecular interaction network.

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

[0050] The recombinant plasmid pET-28a(+)-sm7α-hsdh obtained in Example 1 was used as a template and the upstream and downstream primers in Table 1 were used to perform site-directed mutagenesis PCR amplification.

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

[0052]

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

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

[0055] After PCR, 2 μL amplified products were taken for nucleic acid gel electrophoresis analysis, and the PCR product with clear target bands was added with 1 μL DpnI endonuclease, and cultured overnight in a 37°C incubator for enzyme digestion. 10 μL digested PCR products were added to a 1.5 mL centrifuge tube containing 100 μL of E. coli BL21 (DE3) competent cells, and allowed to stand on ice for 30 min, heat-shocked at 42°C for 1.5 min, and quickly transferred to an ice bath on ice for 2 min. 600 μL of non-antibiotic, sterile LB liquid medium was then added to the tube and cultured at 37°C, 200 rpm for 1 h to resuscitate the cells. The cells were subsequently centrifuged at 4°C, 6000 rpm for 1 min, and 650 μL of supernatant was removed and resuspended in the centrifuge tube. The cells were then plated on LB solid medium containing 50 μg / mL kanamycin, cultured at 37°C for about 12 h, and the positive transformants were prepared by the method of Example 1 using a crude enzyme solution.

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

[0057] Liquid chromatography for 7-KLCA was performed on an Ultimate 3000 liquid chromatograph using a C18 column (50 × 2.1 mm) and a mobile phase consisting of 1 mM KH2PO4 buffer (adjusted to pH 3.0 with phosphoric acid): acetonitrile (40:60 v / v). The flow rate was 1.0 mL / min, the column temperature was 30°C, and the detection wavelength was 210 nm.

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

[0059] Preparation of NADH standard curve: 100mM KH2PO4-K2HPO4 buffer (pH 8.0) was used to prepare 2mM, 1.5mM, 1mM, 0.5mM, 0.4mM, 0.3mM, 0.2mM, and 0.1mM NADH standard solutions, and the standard solutions were detected by microplate reader at OD 340nm The absorbance value at different concentrations of NADH is the horizontal axis, and the corresponding OD 340nm The absorbance value is the vertical axis, and the NADH standard curve is drawn. The results are as follows Figure 2 As shown. The standard curve equation of NADH is: y = 2.9319x + 0.162, R 2 =0.9994.

[0060] Standard conditions for enzyme activity detection: the reaction volume is 200 μL, and NAD is added to a final concentration of 2 mM. + Add 2mM CDCA and an appropriate amount of crude enzyme solution (the concentration is 0.01mg / mL based on protein concentration) and add pH 8.0, 100mM KH2PO4-K2HPO4 buffer to 200μL. Oscillate the reaction at 30℃ for 1min. Measure the absorbance at 340nm every 10s and calculate NAD based on the NADH standard curve. + The unit cell enzyme activities of 7α-HSDH and its mutants are shown in Table 2.

[0061] Definition of 7α-HSDH enzyme activity (U): Under standard enzyme activity detection conditions, the amount of enzyme required to generate 1 μmol NADH per minute is one enzyme activity unit.

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

[0063] Table 2. Unit bacterial 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: Under the same reaction conditions, the enzyme activities of the A132H, V50E, V50A, I173L, A126I, and A203P mutants 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-inactivation temperature It refers to the temperature required for the enzyme activity to drop to 50% of the original activity after incubation for 15 minutes within a certain temperature gradient range.

[0068] The crude enzyme solutions of the wild-type 7α-HSDH and its mutants prepared by the method of Example 1 were placed at different temperatures (30.0°C, 31.1°C, 32.8°C, 35.2°C, 38.2°C, 41.1°C, 43.7°C, 47.1°C, 50.0°C, 52.3°C, 54.0°C, 55°C) and incubated for 15 min. After the incubation, they were immediately placed on ice to cool down. The enzyme activity detection standard conditions of Example 3 were used for detection, and the residual enzyme activity at each temperature was measured to obtain The half-inactivation temperature was obtained using Origin2022 nonlinear curve fitting. The results are shown in Table 3 below.

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

[0070]

[0071] It can be seen from Table 3 that under the same reaction conditions, the mutants A132H, V50E, V50A, I173L, A126I, and A203P 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 7α-HSDH and its mutants and the crude lactate dehydrogenase enzyme solution obtained by the method of Example 1. The specific process is as follows: 10 mL of the reaction system was added with the following concentrations: 80 g / L CDCA, 1 mM NAD +, 400 mM sodium pyruvate, crude enzyme solution of wild-type 7α-HSDH or its mutant prepared according to the method of Example 1 (the amount added is 10 g / L based on the weight of wet cells before crushing) and crude LDH enzyme solution prepared according to the method of Example 1 (the amount added is 5 g / L based on the weight of wet cells before crushing), pH 8.0, 100 mM KH2PO4-K2HPO4 buffer is made up to 10 mL, and the mixture is reacted in a water bath at 30°C and 600 rpm for 10, 20, 30, 45, 60, 90, 120, and 150 min. After that, parallel samples are taken. After terminating the reaction with 3M HCl, the mixture is extracted three times with an equal volume of ethyl acetate, and the organic phases are combined and concentrated under reduced pressure to remove the organic solvent, and the product is collected. The peak area is detected by liquid chromatography, and the content of the product 7-KLCA is calculated according to the 7-KLCA standard curve. The specific reaction progress curve is shown in FIG. Figure 5 The results showed that under the same reaction conditions, the wild-type 7α-HSDH required at least 120 minutes to achieve a yield of more than 99%, while the mutant V50A achieved a yield of >99% after 60 minutes of reaction, and the mutants A132H, V50E, I173L, A126I, and A203P achieved yields of >99% after 90 minutes of reaction, shortening the reaction time by 30-60 minutes compared to the wild-type.

[0074] Preparation of 7-KLCA standard curve: Use pure methanol to prepare 5g / L, 4g / L, 3g / L, 2g / L, 1g / L, 0.5g / L, and 0.25g / L 7-KLCA standard solutions, and use liquid chromatography to detect the peak area. Use 7-KLCA concentration as the horizontal axis and the corresponding peak area as the vertical axis to draw the 7-KLCA standard curve. The results are as follows: Figure 4 As shown. The equation of 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 mutant is obtained by performing single-site or multi-site mutation on position 50, position 126, position 132, position 173 or position 203 of the amino acid sequence shown in SEQ ID NO.

1.

2. The hydroxysteroid dehydrogenase mutant according to claim 1, wherein The hydroxysteroid dehydrogenase mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO.1 to one of the following mutations: (1) mutation of valine at position 50 to alanine; (2) mutation of valine at position 50 to glutamic acid; (3) mutation of alanine at position 126 to isoleucine; (4) mutation of alanine at position 132 to histidine; (5) mutation of isoleucine at position 173 to leucine; and (6) mutation of alanine at position 203 to proline.

3. A gene encoding the hydroxysteroid dehydrogenase mutant according to claim 1.

4. A recombinant expression vector containing the gene encoding the hydroxysteroid dehydrogenase mutant according to claim 3.

5. A recombinant genetically engineered bacterium containing the gene encoding the hydroxysteroid dehydrogenase mutant according to claim 3.

6. A method for screening hydroxysteroid dehydrogenase mutants according to claim 1, characterized in that: The method comprises the following steps: First, AlphaFold3 was used to predict the amino acid sequence structure shown in SEQ ID NO.

1. Molecular docking simulation was performed using YASARA software to construct an enzyme-substrate chenodeoxycholic acid complex model, and quality assessment was performed using PROCHECK to ensure model reliability. Pymol was used for structural visualization analysis to clarify the spatial arrangement of the active site and the intermolecular interaction network. Then, based on this three-dimensional structural model, PROSS was used for analysis, and the model with the highest prediction accuracy was selected to construct a site-directed mutation library. On this basis, ConSurf was integrated to score the conservation of each amino acid site, where 1-3 was low conservation, 4-6 was moderate conservation, and 7-9 was high conservation. Highly conserved regions with a score of 7-9 or mutation combinations that did not conform to evolutionary trends were removed. DeepDDG was used to calculate the change in mutation free energy to evaluate the impact of the mutation on the protein folding free energy, and to exclude mutations with a ΔΔG value of < 0.5 kcal·mol -1 Unfavorable mutations were detected, and flexible amino acid sites were identified by B-FITTER software analysis, while rigid sites with B-factor values ≤ 0.72 were excluded; finally, mutants with greater impact on thermal stability were selected.

7. Use of the hydroxysteroid dehydrogenase mutant according to claim 1 in catalyzing the synthesis of 7-ketolithocholic acid from chenodeoxycholic acid.

8. The use according to claim 7, characterized in that The application is: using the supernatant of wet bacteria obtained by ultrasonic disruption and centrifugation of recombinant genetically engineered bacteria containing a hydroxysteroid dehydrogenase mutant encoding gene as a catalyst, using chenodeoxycholic acid as a substrate, adding NAD + , sodium pyruvate and lactate dehydrogenase, a conversion system is formed with a pH 6-9 buffer as a reaction medium, and the conversion reaction is carried out at 20-40° C. and 600 rpm to obtain a conversion liquid containing 7-ketolithocholic acid.

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

10.

10. The use according to claim 8, characterized in that The wet bacteria were prepared as follows: the recombinant genetically engineered bacteria encoding the hydroxysteroid dehydrogenase mutant gene were inoculated into LB liquid culture medium containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37° C. and 180 rpm for 10 hours to obtain seed liquid; The seed solution was inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at an inoculum concentration of 1.0% by volume, and cultured at 37°C and 180 rpm until the OD 600 =0.6-0.8, and then adding isopropylthiogalactoside with a final concentration of 0.2 mM to the culture solution. After culturing at 28°C for 12 hours, centrifuging at 4°C and 8000 rpm for 10 minutes to obtain the wet bacteria.

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