A method for screening enzyme mutants based on alpha-helix flexible region, mutants and application

Abstract

The application discloses a method for screening enzyme mutants based on alpha-helix flexible regions, mutants and application. Based on the multi-level computer-aided design technology of alpha-helix flexible regions, key amino acid residues affecting the thermal stability of hydroxyl steroid dehydrogenase are quickly screened, and a limited site-directed mutant library is constructed according to the key amino acid residues, and a single-point mutant with improved thermal stability or activity is efficiently screened. Compared with wild type 7alpha-HSDH, the optimal combination mutant M9 can reach a higher conversion rate in the same reaction time under the same reaction conditions, and finally realizes complete conversion (conversion rate > 99.9%) to high substrate concentration, and the yield is more than 99%. The catalytic efficiency of the mutant screened by the application is significantly improved under high substrate concentration, and has great potential for industrial application.

Description

(I) Technical Field

[0002] This invention relates to a method for screening enzyme mutants based on the α-helix flexible region, the mutants, and their applications. (II) Background Technology

[0004] In industrial biocatalysis, thermal stability is one of the important indicators for evaluating the application potential of enzymes. Higher reaction temperatures are generally beneficial for increasing reaction rates, reducing system viscosity, improving substrate mass transfer and solubility, and reducing the risk of microbial contamination. Therefore, improving the thermal stability of enzymes is of great significance for achieving industrial scale-up.

[0005] The flexible structure of proteins is a crucial factor affecting enzyme stability and catalytic efficiency. Appropriate conformational fluctuations facilitate substrate entry, product release, and catalytic cycling; however, excessive local flexibility often weakens structural stability, especially under high temperatures or extreme reaction conditions, which can easily lead to local unfolding, resulting in partial protein inactivation or decreased activity. Based on this understanding, structural modification of flexible regions has gradually become an effective strategy for improving enzyme thermal stability and catalytic performance. Identification of flexible regions typically relies on a combination of experimental and computational methods, with commonly used computational methods including molecular dynamics simulations (MD), B-FITTER, and PROFbval. Furthermore, it is noteworthy that in addition to loops and linker regions, secondary structures such as α-helices also play a vital role in maintaining the overall rigidity and thermal stability of enzymes. Studies have shown that many thermostable mutants obtained through directed evolution, rational design, or semi-rational design are located in α-helical regions.

[0006] In the biocatalytic synthesis of bile acids, hydroxysteroid dehydrogenases (HSDHs) are key catalytic enzymes. These enzymes stereoselectively catalyze the oxidation of hydroxyl groups in hydroxysteroid substrates (such as cholic acid, chenodeoxycholic acid, and taurine chenodeoxycholic acid) to synthesize bile acids. However, the catalytic activity of natural enzymes used in the synthesis of bile acids significantly decreases under high temperatures or extreme reaction conditions, affecting enzyme stability. Most natural HSDHs have short half-lives at high temperatures, making it impossible to maintain high activity for extended periods, thus limiting their industrial applications. (III) Summary of the Invention

[0008] The purpose of this invention is to provide a method for screening enzyme mutants based on the α-helix flexible region, a hydroxysteroid dehydrogenase mutant with synergistically improved thermostability and activity screened using the method, and its applications. This invention uses mutants derived from the genus *Shewanella*. Shewanella morhuaeUsing hydroxysteroid dehydrogenase (7α-HSDH) as a research example, based on the predicted structure of this hydroxysteroid dehydrogenase, and combined with computer-aided design methods such as molecular docking, molecular dynamics simulation, and folding free energy calculation, we focused on screening key sites in the α-helix flexible region and constructed mutants. We efficiently screened hydroxysteroid dehydrogenase mutants with synergistically improved thermal stability and activity, thereby improving the catalytic efficiency of hydroxysteroid dehydrogenase in the biocatalytic synthesis of bile acid compounds, laying an important foundation for the industrial production of bile acid compounds.

[0009] The technical solution adopted in this invention is:

[0010] In a first aspect, this invention provides a method for multi-level computer-aided screening of hydroxysteroid dehydrogenase mutants based on the co-evolution of thermostability and activity, using α-helical flexible regions. The method includes: constructing an enzyme-substrate-cofactor complex model based on three-dimensional structure prediction and molecular docking results of the hydroxysteroid dehydrogenase, and excluding functional core residues by combining evolutionary conservation analysis; further identifying flexible regions with significantly enhanced dynamic fluctuations under high-temperature conditions through multi-temperature molecular dynamics simulations, and focusing mutation design on highly flexible and low-conservation α-helical residues; and finally, screening for key mutation sites related to improved enzyme thermostability or catalytic activity by combining folding free energy change prediction and cross-validation. This method can synergistically converge the mutation space from multiple levels of structure-function, kinetic behavior, and thermodynamic stability, thereby efficiently guiding the construction of hydroxysteroid dehydrogenase mutant libraries.

[0011] Preferably, the method is performed according to the following steps: First, the three-dimensional structure of hydroxysteroid dehydrogenase is predicted using AlphaFold3, and molecular docking is performed using YASARA software to construct the enzyme-substrate (CDCA)-cofactor NAD. +The enzyme-substrate-cofactor complex model was developed and its quality was assessed using PROCHECK, with Ramachandran plot analysis as the primary evaluation criterion. A model was deemed qualified if over 90% of the amino acid residues were located in the most favorable region. Pymol was used to visualize the active site region, identifying key functional residues that directly interact with the substrate and cofactor. ConSurf analysis was employed for evolutionary conservation, eliminating highly conserved sites with a conservation score ≥7 to prevent interference with the catalytic core region from the outset. After excluding core functional residues, molecular dynamics (MD) simulations were performed on the enzyme-substrate-cofactor complex model at 303.15 K and 323.15 K, respectively, to calculate the root mean square fluctuation (RMSF) distribution of each residue. ΔRMSF was defined as the difference in RMSF between the low-temperature (303.15 K) and high-temperature (323.15 K) conditions. Statistical analysis of the overall ΔRMSF distribution showed that ΔRMSF < -0.2 was considered acceptable. Using Å as the threshold, residues exhibiting significantly enhanced dynamic fluctuations under high-temperature conditions were screened as candidate flexible sites. Based on the above analysis results, the mutation design focused on α-helix residues located in highly flexible regions with low conservation, aiming to optimize the overall conformational stability of the protein by regulating the local rigidity of the α-helix. For the selected candidate sites, Pythia was used to perform virtual saturation mutation calculations, and the change in folding free energy (ΔΔG) for all amino acid substitutions at each site was predicted. ΔΔG < 0 was used as the criterion for potential stabilizing mutations, screening favorable mutation sites from a thermodynamic perspective. Furthermore, PROSS was used for cross-validation to screen key mutation sites while considering both evolutionary rationality and structural stability.

[0012] Preferably, the method is derived from the genus *Shewanella* as shown in SEQ ID NO.1. Shewanella morhuae Based on the hydroxysteroid dehydrogenase (WP_076500293.1), the key mutation sites screened were D12K, V15I, V50E, I56R, D66A, T76R, A126I, A132H, S158A, S161A, A203P and H208Q.

[0013] Secondly, the present invention provides a hydroxysteroid dehydrogenase mutant with synergistically improved thermostability and activity screened by the method described above. The mutant is obtained by performing single-site or multi-site combination mutations on positions 12, 15, 50, 56, 66, 76, 126, 132, 158, 161, 203, or 208 of the amino acid sequence shown in SEQ ID NO.1.

[0014] Preferably, the hydroxysteroid dehydrogenase mutant is formed by mutating one of the following amino acid sequences shown in SEQ ID NO.1: (1) mutating aspartic acid at position 12 to lysine (D12K); (2) mutating valine at position 15 to isoleucine (V15I); (3) mutating valine at position 50 to glutamic acid (V50E); (4) mutating isoleucine at position 56 to arginine (I56R); (5) mutating aspartic acid at position 66 to alanine (D66A); (6) mutating threonine at position 76 to arginine (T76R); (7) mutating aspartic acid at position 12 to arginine (D15I); (8) mutating valine at position 15 to glutamic acid (V15I); (9) mutating valine at position 16 to glutamic acid (V50E); (10) mutating valine at position 16 to arginine (I56R); (11) mutating valine at position 16 to glutamic acid (V50E); (12) mutating valine at position 16 to arginine (I56R); (13) mutating valine at position 16 to glutamic acid (V50E); (14) mutating valine at position 16 to arginine (I56R); (15) mutating valine at position 16 to glutamic acid (I56R); (16) mutating valine at position 16 to glutamic acid (I56R); (17) mutating valine at position 16 to glutamic acid (I56R); (18) mutating valine at position 16 to glutamic acid (I56R); (19) (26) Alanine is mutated to isoleucine (A126I); (8) Alanine is mutated to histidine (A132H) at position 132; (9) Serine is mutated to alanine (S158A) at position 158; (10) Serine is mutated to alanine (S161A) at position 161; (11) Alanine is mutated to proline (A203P) at position 203; (12) Histidine is mutated to glutamine (H208Q) at position 208; and multiple combinations of the above single mutations.

[0015] Preferably, the amino acid sequence of the hydroxysteroid dehydrogenase mutant is shown in one of SEQ ID NO.3-SEQ ID NO.11, more preferably in one of SEQ ID NO.8-SEQ ID NO.11.

[0016] Thirdly, the present invention provides a gene encoding the hydroxysteroid dehydrogenase mutant.

[0017] Fourthly, the present invention provides a recombinant expression vector containing the gene encoding the hydroxysteroid dehydrogenase mutant.

[0018] Furthermore, the recombinant expression vector was constructed using pET 28a(+) as the base plasmid.

[0019] Fifthly, 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. E. coli BL21(DE3).

[0020] 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 host cells. E. coli BL21(DE3) was used to obtain recombinant genetically engineered bacteria.

[0021] 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 (CDCA).

[0022] Furthermore, the application involves using wet bacterial cells obtained through induced culture of recombinant genetically engineered bacteria containing a hydroxysteroid dehydrogenase mutant encoding gene, the supernatant obtained by ultrasonic disruption and centrifugation of wet bacterial cells, or the extracted pure enzyme solution as a catalyst, with CDCA as a substrate, and adding NAD... + Sodium pyruvate and lactate dehydrogenase (LDH) were used to form a conversion system with a pH 6-9 buffer (preferably pH 8.0, 100 mM KH2PO4-K2HPO4 buffer) as the reaction medium. The conversion reaction was carried out at 25-50 °C (preferably 40 °C) and 600 rpm to obtain a conversion solution containing 7-KLCA.

[0023] Furthermore, in the conversion system, the substrate concentration is 125-375 mM (preferably 300 mM); NAD + The concentration of added sodium pyruvate is 0.1-1 mM (preferably 0.4 mM); the concentration of added sodium pyruvate is 150-750 mM (preferably 300 mM); the amount of catalyst added is 5-15 g / L (preferably 5 g / L) based on the wet cell volume before crushing, and 5-15 mg / L (preferably 10 mg / L) based on the protein content.

[0024] Furthermore, the lactate dehydrogenase is added in the form of the supernatant obtained by ultrasonic disruption and centrifugation of wet bacterial cells obtained from recombinant Escherichia coli containing the lactate dehydrogenase gene through induced culture, with an addition amount of 1-10 g / L (preferably 2.5 g / L) based on the wet bacterial cells before disruption. The nucleotide sequence of the lactate dehydrogenase gene is shown in SEQ ID NO.12.

[0025] Further, the wet bacterial cells 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 ℃ and 180 rpm for 10 h to obtain a seed culture; the seed culture was then diluted with water at a volume concentration of 1.0% (…). v / v The inoculum was inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin and incubated at 37 ℃ and 180 rpm until OD500. 600 =0.6~0.8, then add isopropyl thiogalactoside (IPTG) to the culture medium to a final concentration of 0.2 mM, incubate at 28 ℃ for 12 h, and centrifuge at 4 ℃ and 8000 rpm for 10 min to obtain wet bacterial cells containing the 7α-HSDH mutant.

[0026] The inoculation, transfer, induction, and cell recovery of the recombinant engineered bacteria described in this invention are preferably performed using 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.

[0027] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0028] This invention utilizes multi-level computer-aided design technology based on the α-helix flexible region to rapidly screen key amino acid residues affecting thermal stability, and constructs a limited site-directed mutant library accordingly. This allows for efficient screening of single-point mutations that significantly enhance thermal stability or catalytic activity. Further screening yields combined mutants D12K / V15I (M1), V50E / I56R / D66A / T76R (M2), A126I / A132H (M3), S158A / S161A (M4), A203P / H208Q (M5), and D12K / V15I / V50... E / I56R / D66A / T76R (M6), D12K / V15I / V50E / I56R / D66A / T76R / A126I / A132H (M7), D12K / V15I / V50E / I56R / D66A / T76R / A126I / A132H / A203P / H208Q (M8), D12K / V15I / V50E / I56R / D66A / T76RA126I / A132H / S158A / S161A / A203P / H208Q (M9), half-inactivation temperature ( Compared with wild-type 7α-HSDH, the increases were 2.1℃, 6.5℃, 8.5℃, 2.9℃, 3.2℃, 3.4℃, 7.4℃, 8.2℃, and 11.4℃, respectively. t 1 / 2 The time intervals were extended to 30.4 h, 33.2 h, 46.2 h, 37.7 h, 40.1 h, 47.5 h, 59.2 h, 69.3 h, and 75.3 h, respectively, for each mutant. T m Compared to the wild type, the catalytic efficiency of each mutant was increased by 0.7℃, 1.2℃, 2.8℃, 1.1℃, 1.7℃, 4.4℃, 6.1℃, 6.2℃, and 10.5℃, respectively. cat / K m The percentages for the wild type were 156.46%, 407.37%, 84.27%, 157.81%, 193.53%, 331.18%, 215.03%, 264.41%, and 414.13%, respectively.

[0029] Compared to wild-type 7α-HSDH, the optimal mutant M9 of this invention achieves a higher conversion rate within the same reaction time under the same reaction conditions, ultimately achieving complete conversion of high substrate concentrations (300 mM) (conversion rate > 99.9%), with a yield exceeding 99%. The mutants screened in this invention exhibit significantly improved catalytic efficiency at high substrate concentrations, demonstrating great potential for industrial application. (iv) Description of the attached drawings

[0031] Figure 1 A schematic diagram of the reaction for the two-step coupling catalysis of 7α-HSDH and 7β-HSDH to prepare UDCA (ursodeoxycholic acid) from CDCA.

[0032] Figure 2 The NADH concentration and OD in Example 3 340nm The standard curve.

[0033] Figure 3 The optimal temperature curves for the catalytic reaction of wild-type 7α-HSDH and its optimal combination mutant M9 in Example 4 are shown.

[0034] Figure 4 The optimal pH curve for the catalytic reaction of wild-type 7α-HSDH and its optimal combination mutant M9 in Example 4 is shown.

[0035] Figure 5 The half-inactivation temperatures of wild-type 7α-HSDH and its combined mutants M1, M2, M3, M4, M5, M6, M7, M8 and M9 in Example 4 are ( (See graph for measurement results)

[0036] Figure 6 The half-lives of wild-type 7α-HSDH and its combined mutants M1, M2, M3, M4, M5, M6, M7, M8 and M9 in Example 4 are ( t 1 / 2 (See graph for measurement results)

[0037] Figure 7 The melting temperatures of wild-type 7α-HSDH and its combined mutants M1, M2, M3, M4, M5, M6, M7, M8 and M9 in Example 4 are ( T m (See graph for measurement results)

[0038] Figure 8 The optimal cofactor concentration for the catalytic reaction of wild-type 7α-HSDH and its optimal combination mutant M9 in Example 5.

[0039] Figure 9 This represents the optimal ratio of substrate to co-substrate for the catalytic reaction of wild-type 7α-HSDH and its optimal combination mutant M9 in Example 5.

[0040] Figure 10 This represents the optimal ratio of α-HSDH to LDH concentrations for the catalytic reaction of the wild-type and its optimal combination mutant M9 in Example 5.

[0041] Figure 11 This is the standard curve of 7-KLCA concentration versus peak area in Example 6.

[0042] Figure 12 This is a flowchart of the reaction process for the synthesis of 7-KLCA catalyzed by wild-type 7α-HSDH and its optimal combination mutant M9 in Example 6. (V) Detailed Implementation Methods

[0044] 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:

[0045] Example 1: Construction, induction, and protein purification of wild-type recombinant strains

[0046] 1. Construction and induced expression of wild-type 7α-HSDH recombinant strain

[0047] According to the NCBI database, it originates from the genus Shewanella. Shewanella morhuae The 7α-hydroxysteroid dehydrogenase (7α-HSDH, Accession No: WP_076500293.1) was synthesized using a 7α-HSDH gene fragment (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2), denoted as […]. sm7α-hsdh And insert it into the carrier pET28a(+) Nco I and Xho Between the I restriction sites, construct the recombinant expression plasmid pET28a- sm7α-hsdh After confirming that the DNA sequencing was correct, the recombinant plasmid was transferred into the host bacteria. E. coli BL21(DE3) was used to obtain a recombinant strain of wild-type 7α-HSDH.

[0048] 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 to obtain seed culture. The seed culture was then 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. IPTG was then added to the culture medium to a final concentration of 0.2 mM, and the culture was incubated at 28°C for 12 h. The culture was then 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.

[0049] SEQ ID NO.1

[0050] MYNPKDFTLNGDVAVITGAGAGIGRAIAETFAAAGAAVMVSDLKQETANVVAQAIIAQGGKAVAIDCDITQEDDLTLVAQTISEFGKLTILVSNAGGGGPKPFDMPMADFRRAFDLNVFSLFRLAQI AAPAMEKAGGGSILGITSMAGENKNEHMASYASSKAATNHLIRNIAFDLGPKNIRVNGIAPGATRTTALESVLTAEIEQHMLKKTPIHRLGEPQDMANAALFLSSPAASWISGQILTVSGGGVQELE.

[0051] SEQ ID NO.2

[0052] ATGTATAACCCGAAAGACTTCACCCTGAATGGCGACGTTGCAGTTATTACCGGTGCAGGTGCAGGTATTGGTCGTGCAATTGCAGAAACCTTTGCAGCAGCAGGTGCAGCAGTTATGGTTAGCGATCTGAAACAGGAAACCGCAAATGTTGTTGCACAGGCAATTATTGCACAGGGTGGTAAAGCAGTTGCAATTGATTGTGATATTACCCAGGAAGACGATCTGACCCGTCTGGTTGCACAGACCATTAGCGAATTTGGTAAACTGACCATTCTGGTGAGCAATGCCGGTGGTGGTGGTCCGAAACCGTTTGATATGCCGATGGCAGATTTTCGTCGTGCATTTGATCTGAATGTTTTCAGCCTGTTTCGCCTGGCACAGATTGCAGCACCGGCAATGGAAAAAGCAGGTGGTGGTAGCATTCTGGGTATTACCAGCATGGCAGGTGAAAATAAAAATGAGCATATGGCAAGCTACGCCAGCAGCAAAGCAGCAACCAATCATCTGATTCGTAATATCGCATTTGACCTGGGCCCGAAAAATATTCGCGTTAATGGTATTGCACCGGGCGCAACCCGTACCACCGCATTAGAAAGTGTTCTGACCGCAGAAATTGAACAGCATATGCTGAAAAAAACCCCGATTCATCGTCTGGGTGAACCGCAGGATATGGCAAATGCAGCACTGTTTCTGAGCAGCCCGGCAGCAAGTTGGATTAGCGGTCAGATTCTGACCGTTAGCGGTGGTGGTGTTCAGGAACTGGAA.

[0053] 2. Construction and induced expression of recombinant strain of wild-type lactate dehydrogenase (LDH)

[0054] According to the sequence derived from Escherichia coli in the NCBI database ( E. coliD-lactate dehydrogenase (GenBank accession number: MCV5771625.1) was synthesized, and a lactate dehydrogenase gene fragment (amino acid sequence shown in SEQ ID NO.12, nucleotide sequence shown in SEQ ID NO.13) was artificially synthesized. This gene fragment was then directionally inserted into the multiple cloning site of the pETDuet vector, successfully constructing the recombinant expression vector pETDuet- esldh And this recombinant expression vector was transferred into E. coli BL21(DE3) was obtained through antibiotic screening and sequencing verification. E. coli BL21(DE3) / pETDuet- esldh 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.

[0055] SEQ ID NO.12

[0056] MKLAVYSTKQYDKKYLDDVNESFGFELEFFDFLLTEKTAKTANGCEAVCIFVNDDGSRPVLEELKKHGVKYIALRCAGFNNVDLDAAKELGLKVVRVPAYDPEAVAEHAIGMMMMTLNRRIHRATQRTRDANFSLEGLTGFTMYGKTAGVIGTGKIGVAMLRILKG FGMRLLAFDPYPSAAALELGVEYVDLPTLFSESDVISLHCPLTPENYHLLNEAAFEQMKNGVMIVNTSRGALIDSQAAIEALKNQKIGSLGMDVYENERDLFFEDKSNDVIQDDVFRRLSACHNVLFTGHQAFLTAEALTSISQTTLQNLSNLEKGETCPNELVW.

[0057] SEQ ID NO.13 (underlined is histidine tag)

[0058] ATGAAACTGGCAGTTTACTCTACCAAACAGTACGACAAGAAATACCTGCAGCAGGTTAACGAAAGCTTCGGTTTCGAACTGGAATTTTTCGACTTTCTGCTGACCGAGAAGACCGCGAAGACTGCGAACGGCTGCGAAGCTGTTTGCATCTTCGTTAACGACGACGGTTCTCGTCCAGTTCTGGAAGAACTGAAGAAACACGGTGTGAAGTACATCGCGCTGCGTTGTGCGGGTTTCAACAACGTGGACCTGGACGCAGCGAAAGAACTGGGTCTGAAAGTTGTACGTGTACCGGCGTATGATCCGGAAGCGGTTGCTGAACACGCGATCGGTATGATGATGACCTTGAACCGTCGTATCCACCGTGCGTACCAGCGTACTCGTGATGCAAACTTCTCTCTGGAAGGTCTGACCGGTTTCACCATGTACGGTAAGACCGCGGGCGTTATCGGTACCGGTAAGATCGGTGTTGCGATGCTGCGTATTCTGAAAGGTTTCGGTATGCGTCTGCTGGCGTTCGATCCATATCCGTCCGCTGCGGCACTGGAACTGGGTGTTGAATACGTTGACCTGCCAACTCTGTTCTCCGAATCCGACGTTATCTCTCTGCACTGTCCGCTGACTCCGGAGAACTACCACCTGCTGAACGAAGCAGCGTTCGAACAGATGAAGAACGGCGTAATGATCGTGAACACCAGCCGTGGTGCACTGATCGATTCTCAGGCAGCTATCGAAGCTCTGAAGAACCAGAAGATCGGTTCTCTGGGTATGGACGTTTACGAAAACGAACGTGACCTGTTCTTCGAAGACAAGAGCAACGACGTTATCCAAGACGACGTATTCCGTCGTCTGTCTGCTTGCCACAACGTGCTGTTCACCGGTCACCAGGCGTTCTTGACCGCTGAAGCGCTGACCTCTATCAGCCAGACCACTCTGCAGAACCTGTCTAACTTGGAGAAAGGCGAAACCTGTCCGAACGAACTGGTT CATCATCATCATCATCATTAA.

[0059] 3. Protein purification

[0060] The purified enzyme solution was prepared as follows: the crude enzyme solution was centrifuged at 8000 rpm and 4℃ for 10 min, the precipitate was discarded, and the supernatant was collected. The supernatant was microfiltered (0.22 μm filter membrane) for protein purification. Ni-NTA resin was packed into a chromatography column, and the column was washed with 5 column volumes of buffer B (Elution Buffer, pH 7.5, 500 mM imidazole, 300 mM NaCl, 10 mM Tris HCl buffer), followed by rinsing with buffer A (Bindind Buffer, pH 7.5, 10 mM imidazole, 10 mM Tris HCl buffer). The microfiltered supernatant was loaded onto the sample and rinsed with 5-10 column volumes of buffer A to remove unbound and contaminating proteins. To accurately determine whether contaminating proteins had been sufficiently removed, approximately 1 mL of eluent was collected after rinsing to the 8th column volume, and the protein concentration was determined using a BCA protein concentration assay kit. If the protein concentration in the effluent is <0.01 mg / mL, it indicates that unbound contaminating proteins have been completely removed. If the protein concentration is higher than this threshold, elution continues until the measured value meets the requirements. Eluent is then used to elute with buffer B, and the eluent is collected in four tubes, 1 mL per tube. The collected target protein eluent is dialyzed in pH 7.0 potassium phosphate buffer (20 mM) for 16–20 h. The eluent after dialyzing is the purified enzyme solution of the target protein; wild-type 7α-HSDH is denoted as WT. The purity of the target enzyme protein is analyzed by protein gel electrophoresis.

[0061] Example 2: Construction and screening of 7α-HSDH mutants

[0062] 1. Design of mutation sites

[0063] Based on the structural information of wild-type 7α-HSDH, and combined with a multi-level computer-aided design strategy, key amino acid sites affecting the thermal stability and catalytic activity of the enzyme molecule were systematically screened, and a site-directed mutant library was constructed. The specific methods are as follows:

[0064] First, use AlphaFold3 (https: / / alphafoldserver.com) to... Sm The three-dimensional structure of the 7α-HSDH protein (amino acid sequence as shown in SEQ ID NO.1) was predicted, and molecular docking was performed using YASARA software to construct the enzyme-substrate (CDCA)-cofactor NAD. +The complex model was developed and its quality was assessed using PROCHECK (https: / / saves.mbi.ucla.edu), with Ramachandran plot analysis as the primary evaluation criterion. The model was deemed qualified if 92.2% of the amino acid residues were located in the most favorable region (over 90%). Pymol was used to visualize the active site region, identifying key functional residues that directly interact with the substrate and cofactors. ConSurf was employed for evolutionary conservation analysis, and highly conserved sites were removed with a conservation score ≥7 to prevent interference with the catalytic core region from the outset.

[0065] After excluding functional core residues, 7α-HSDH-CDCA-NAD + The complex was subjected to MD simulations at 303.15 K and 323.15 K to calculate the root mean square fluctuation (RMSF) distribution of each residue, and ΔRMSF was defined as the difference in RMSF between the low-temperature (303.15 K) and high-temperature (323.15 K) conditions. Statistical analysis of the overall ΔRMSF distribution was performed, and residues showing significantly enhanced dynamic fluctuations at high temperatures were selected as candidate flexible sites using ΔRMSF < -0.2 Å as the threshold. The results showed that the flexible regions were mainly concentrated in Zone 1 (N10-G20), Zone 2 (Q45-A80), Zone 3 (P101-A136), Zone 4 (N151-G178), and Zone 5 (A203-K211). Further structural analysis revealed that these regions were mainly distributed within α-helices or their adjacent structural units.

[0066] Based on the above analysis, the mutation design focused on α-helical residues located in highly flexible regions with low conservation, aiming to optimize the overall conformational stability of the protein by regulating the local rigidity of the α-helix. For the selected candidate sites, Pythia was used to perform virtual saturation mutation calculations. The folding free energy change (ΔΔG) of all amino acid substitutions at each site was predicted, and ΔΔG < 0 was used as the criterion for potential stabilizing mutations, screening favorable mutation sites from a thermodynamic perspective. Further cross-validation using PROSS was conducted to screen key mutation sites while considering both evolutionary rationality and structural stability. Ultimately, 12 candidate residues were obtained: D12K, V15I, V50E, I56R, D66A, T76R, A126I, A132H, S158A, S161A, A203P, and H208Q.

[0067] 2. Site-directed mutation

[0068] Based on the candidate residues in step 1, corresponding specific amplification primers were designed to amplify the recombinant plasmid pET-28a(+)- constructed in Example 1. sm7α-hsdh Using the upstream and downstream primers in Table 1 as templates, site-directed mutagenesis PCR amplification was performed.

[0069] Table 1. Primer design for 7α-HSDH site-directed mutagenesis

[0070]

[0071] 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.

[0072] The PCR amplification program was as follows: pre-denaturation at 95℃ for 5 min, followed by 32 cycles (denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 6 min), 72℃ for 5 min, and storage at 4℃.

[0073] 3. Construction of mutant strains

[0074] After PCR, 2 μL of the amplification product was taken for nucleic acid gel electrophoresis analysis. The PCR product with a clear target band was added to 1 μL of the gel electrophoresis solution. Dpn Endonuclease was used for overnight digestion at 37°C. After purification of the digested PCR product, 10 μL was added to a 1.5 mL centrifuge tube containing 100 μL of competent cells. The tube was incubated on ice for 30 min, heat-shocked at 42°C for 1.5 min, and then rapidly transferred to ice for 2 min. 600 μL of antibiotic-free, sterile LB broth was added to the tube, and the cells were cultured at 37°C and 200 rpm for 1 h to revive them. The cells were then centrifuged at 4°C and 6000 rpm for 1 min, and after discarding 650 μL of supernatant, the cells were resuspended and plated on LB agar plates containing 50 μg / mL kanamycin. The plates were incubated at 37°C for approximately 12 h to obtain transformants containing the 7α-HSDH recombinant plasmid. Sequencing identification yielded a series of 7α-HSDH mutant strains. Wet cells of the mutant strains were prepared using the method described in Example 1.

[0075] 4. Screening of dominant mutants

[0076] The obtained positive transformants were screened for dominant mutants. The final concentration composition and conditions of the screening system were as follows: 25 mM CDCA, 1 mM NAD. +A 10 mL reaction system was prepared using 50 mM sodium pyruvate, 5 g / L wild-type 7α-HSDH and its mutant wet cells (wet weight), and 2.5 g / L LDH wet cells (wet weight), in a pH 8.0 potassium phosphate buffer (100 mM). After reacting in a water bath at 30℃ and 600 rpm for 5 min, parallel samples were taken. The reaction was terminated with 3 M HCl, and the samples were extracted three times with equal volumes 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. The dominant strains with high catalytic efficiency were screened using relative enzyme activity as the evaluation index. The results are shown in Table 2.

[0077] HPLC detection method for 7-KLCA: Ultimate 3000 HPLC system, C18 column (50×2.1 mm), mobile phase: 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 ℃, and the detection wavelength was 210 nm.

[0078] The definition of enzyme activity is the same as in Example 3.

[0079] Table 2. Relative enzyme activities of single-point mutant strains in 7-KLCA synthesis

[0080]

[0081] 5. Construction and screening of multi-site combined mutants

[0082] Based on step 4, any two or more single mutants were combined, and the relative enzyme activity was detected using the method and conditions in step 4. The results are shown in Table 3.

[0083] Table 3. Relative enzyme activities of 7-KLCA synthesized by the combined mutant strains

[0084]

[0085] Screening for dominant strains with high catalytic efficiency E. coli BL21(DE3)-7α-HSDH-D12K / V15I (amino acid sequence as shown in SEQ ID NO.3) is denoted as M1. E. coli BL21(DE3)-7α-HSDH-V50E / I56R / D66A / T76R (amino acid sequence as shown in SEQ ID NO.4) is denoted as M2. E. coli BL21(DE3)-7α-HSDH-A126I / A132H (amino acid sequence as shown in SEQ ID NO.5) is designated as M3. E. coliBL21(DE3)-7α-HSDH-S158A / S161A (amino acid sequence as shown in SEQ ID NO. 6) is designated as M4. E. coli BL21(DE3)-7α-HSDH-A203P / H208Q (amino acid sequence as shown in SEQ ID NO.7) is designated as M5. E. coli BL21(DE3)-7α-HSDH-D12K / V15I / V50E / I56R / D66A / T76R (amino acid sequence as shown in SEQ ID NO.8) is denoted as M6. E. coli BL21(DE3)-7α-HSDH-D12K / V15I / V50E / I56R / D66A / T76R / A126I / A132H (amino acid sequence as shown in SEQ ID NO.9) is denoted as M7. E. coli BL21(DE3)-7α-HSDH-D12K / V15I / V50E / I56R / D66A / T76R / A126I / A132H / A203P / H208Q (amino acid sequence as shown in SEQ ID NO.10) is denoted as M8 and E. coli BL21(DE3)-7α-HSDH-D12K / V15I / V50E / I56R / D66A / T76R / A126I / A132H / S158A / S161A / A203P / H208Q (amino acid sequence as shown in SEQ ID NO.11) is denoted as M9.

[0086] Example 3: Determination of enzyme activity and kinetic parameters

[0087] 1. Determination of enzyme activity

[0088] Preparation of NADH standard curve: NADH standard solutions of 1.5 mM, 1 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, and 0.1 mM were prepared using 100 mM KH₂PO₄-K₂HPO₄ buffer (pH 8.0). The OD values ​​of each standard solution were then measured 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 The NADH standard curve is plotted with the ordinate as the vertical axis, and the result is as follows: Figure 2 As shown. Therefore, the standard curve equation for NADH is: y = 2.9320x + 0.162, R... 2 =0.9997.

[0089] 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 pure enzyme solution prepared according to the method of Example 1 (concentration of 0.01 mg / mL based on protein content), and make up to 200 μL with pH 8.0, 100 mM KH2PO4-K2HPO4 buffer. Incubate at 30°C with shaking for 1 min, and measure the absorbance at 340 nm every 10 s. Calculate NAD based on the NADH standard curve. + Consumption rate.

[0090] 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. Specific enzyme activity is defined as the number of enzyme activity units per milligram of enzyme protein, expressed in U / mg.

[0091] The enzyme activities of the pure enzyme solutions of wild-type 7α-HSDH and mutants M1-M9 screened by the method in Example 2 were 601.35 U / mg, 791.22 U / mg, 1468.71 U / mg, 595.13 U / mg, 782.32 U / mg, 1017.54 U / mg, 710.74 U / mg, 1033.67 U / mg, 1072.46 U / mg, and 1368.68 U / mg, respectively.

[0092] 2. Measurement of dynamic parameters

[0093] The purified enzyme solutions of wild-type 7α-HSDH and mutants M1-M9 screened by the method in Example 2 were adjusted to a protein concentration of 1 mg / mL using pH 8.0 and 100 mM KH2PO4-K2HPO4 buffer, respectively. 10 μL of the 1 mg / mL enzyme solution was added to the wells of an ELISA plate pre-allocated with 190 μL of reaction mixture. The final concentration of the reaction mixture consisted of: 100 mM KH2PO4-K2HPO4 buffer (pH 8.0), 1 mM NAD... + The substrates, CDCA, were prepared at concentrations ranging from 20 µM to 1000 µM, with 13 concentration gradients: 20 µM, 40 µM, 80 µM, 100 µM, 200 µM, 300 µM, 400 µM, 500 µM, 600 µM, 700 µM, 800 µM, 900 µM, and 1000 µM. The reaction was carried out at 30 °C with shaking for 1 min, and the absorbance at 340 nm was measured every 10 s. The initial rate of the enzymatic reaction was calculated based on the slope of the absorbance increase during the initial linear phase of the reaction, and converted to specific enzyme activity according to the definition of enzyme activity. The reaction rate versus substrate concentration curve was fitted using the Michaelis-Menten equation from Origin to calculate the corresponding kinetic parameters.

[0094] Table 4. Summary of kinetic parameters of wild-type 7α-HSDH and its mutants on the substrate CDCA

[0095]

[0096] Table 4 shows that, under the same reaction conditions, the catalytic efficiencies of each mutant, M1, M2, M3, M4, M5, M6, M7, M8, and M9, are (…). k cat / K m The percentages for the wild type were 156.46%, 407.37%, 84.27%, 157.81%, 193.53%, 331.18%, 215.03%, 264.41%, and 414.13%, respectively.

[0097] Example 4: Enzymatic characterization of pure enzyme solutions of wild-type 7α-HSDH and its mutants

[0098] The enzymatic properties of WT and mutant M9 were systematically characterized (including optimal catalytic temperature and optimal catalytic pH), and the thermal stability parameters of WT and mutant M1-M9 were characterized (determination of half-inactivation temperature, half-life, and melting temperature).

[0099] 1. Enzymatic properties

[0100] (1) Optimal catalytic temperature

[0101] Using the enzyme activity detection standard conditions of Example 3, the effect of temperature on enzyme activity was evaluated at different temperatures ranging from 25℃ to 50℃. The highest enzyme activity under each temperature condition was taken as 100%, and the relative enzyme activity was calculated. Results are shown below. Figure 3 Through optimization, its optimal catalytic temperature was determined to be 40℃.

[0102] (2) Optimal catalytic pH

[0103] The enzyme activity assay was performed using the standard conditions of Example 3, with the temperature changed to 40℃. Different pH buffer systems (pH 4.0-6.0 sodium acetate buffer; pH 6.0-8.0 phosphate buffer; pH 8.0-10.0 Tris-HCl buffer) were used for the assay. The highest enzyme activity under each pH condition was taken as 100%, and the relative enzyme activity was calculated. Results are shown below. Figure 4 Through optimization, its optimal catalytic pH value was determined to be 9.0.

[0104] 2. Determination of thermal stability parameters

[0105] The pure enzyme solutions of wild-type 7α-HSDH and mutants M1-M9 screened by the method in Example 2 were adjusted to a protein concentration of 1 mg / mL using pH 8.0 and 100 mM KH2PO4-K2HPO4 buffer, respectively.

[0106] Half-deactivation temperature ( The residual enzyme activity (REV) refers to the temperature required for the enzyme activity to decrease to 50% of its original activity after incubation for 15 minutes within a certain temperature gradient range. Pure enzyme solutions of 1 mg / mL wild-type 7α-HSDH and its mutants (M1-M9) 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 minutes. Immediately after incubation, the REV was cooled on ice. The residual enzyme activity after each temperature treatment was determined using the enzyme activity detection standard conditions of Example 3. The enzyme activity of the untreated sample was taken as 100%, and the relative residual enzyme activity after each temperature treatment was calculated. The half-inactivation temperature was obtained by fitting a nonlinear curve using Origin 2022. The result is as follows Figure 5 As shown in Table 5.

[0107] half life( t 1 / 2 : This refers to the incubation time corresponding to when the enzyme activity decreases to 50% of its initial value. Pure enzyme solutions of 1 mg / mL wild-type 7α-HSDH and its mutants (M1-M9) were incubated in a 40°C constant temperature metal bath. Samples were taken at regular intervals, and residual enzyme activity was determined using the enzyme activity detection standard conditions of Example 3. A first-order inactivation kinetic model was used (…). Fit the experimental data, by The half-life was calculated, and the result is as follows: Figure 6 As shown in Table 5.

[0108] melting temperature ( T m : refers to the temperature at which a protein, during heating, has 50% of its native conformation (folded state) and 50% of its denatured conformation (unfolded state). T m It is typically used to characterize the thermal stability of proteins. T m A higher value indicates a more stable protein structure and stronger heat resistance. Pure enzyme solutions of wild-type 7α-HSDH (1 mg / mL) and its mutants (M1-M9) were diluted with deionized water to a concentration of 0.1 mg / mL and transferred to 10 mm quartz cuvettes, taking care to avoid bubble formation during the process. Data were recorded and the protein's thermal stability was analyzed by scanning the wavelength range of 180 nm-260 nm. The results are shown below. Figure 7 As shown in Table 5.

[0109] Table 5. Half-inactivation temperatures of 7α-HSDH and its mutants

[0110]

[0111] Table 5 shows that, under the same reaction conditions, the combined mutants M1, M2, M3, M4, M5, M6, M7, M8, and M9 exhibit different characteristics. Compared to the wild type, the increases were 2.1℃, 6.5℃, 8.5℃, 2.9℃, 3.2℃, 3.4℃, 7.4℃, 8.2℃, and 11.4℃, respectively. t 1 / 2 The durations were extended to 30.4 h, 33.2 h, 46.2 h, 37.7 h, 40.1 h, 47.5 h, 59.2 h, 69.3 h, and 75.3 h, respectively, while the combined mutants... T m Compared to the wild type, the temperature was increased by 0.7℃, 1.2℃, 2.8℃, 1.1℃, 1.7℃, 4.4℃, 6.1℃, 6.2℃ and 10.5℃ respectively.

[0112] Example 5: Optimization of Whole-Cell Catalytic Reaction Conditions

[0113] Based on the optimal temperature and pH determined in Example 4, using wet cells of WT and M9 as catalysts, key reaction parameters affecting the synthesis efficiency of 7-KLCA were systematically optimized, including the cofactor NAD. + Concentration, molar ratio of substrate CDCA to cosubstrate sodium pyruvate, and enzyme ratio of 7α-HSDH to LDH.

[0114] (1) Determination of optimal cofactor concentration

[0115] The final concentration composition of the 10 mL reaction system was: 0.1–1.0 mM (0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM) of NAD. + 200 mM CDCA, 400 mM sodium pyruvate, 5 g / L 7α-HSDH wet cells, and 2.5 g / L LDH wet cells were added to a final volume of 10 mL with pH 8.0, 100 mM KH₂PO₄-K₂HPO₄ buffer. The mixture was reacted at 40℃ and 600 rpm for 5 min with shaking. 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 7-KLCA content of the product was determined by liquid chromatography, with relative enzyme activity as the evaluation index, and the highest enzyme activity recorded as 100%. The results are shown in [Figure number missing]. Figure 8 The results showed that M9 could maintain high catalytic activity at a low cofactor concentration (0.4 mM), while the wild type was more sensitive to the cofactor concentration, and its catalytic activity decreased significantly at a cofactor concentration of 0.4 mM.

[0116] (2) Optimal ratio of substrate (CDCA) to co-substrate (sodium pyruvate)

[0117] Final concentration composition of 10 mL reaction system: 0.4 mM NAD + Add 200 mM of substrate CDCA, along with co-substrate (sodium pyruvate) at different molar ratios to CDCA (substrate to co-substrate molar ratios of 1:0.5, 1:1.0, 1:1.5, 1:2.0, and 1:2.5), 5 g / L of 7α-HSDH wet cells, and 2.5 g / L of LDH wet cells. Make up to 10 mL with pH 8.0, 100 mM KH2PO4-K2HPO4 buffer. React at 40℃ and 600 rpm for 5 min. Take parallel samples, process them in the same way as in step (1), and use relative enzyme activity as the evaluation index. Results are shown in […]. Figure 9 The results showed that the optimal molar ratio of substrate to co-substrate was 1:1.

[0118] (3) The optimal ratio of 7α-HSDH to LDH wet cells

[0119] Final concentration composition of 10 mL reaction system: 0.4 mM NAD + 200 mM of substrate CDCA, 200 mM of co-substrate sodium pyruvate, 5 g / L of 7α-HSDH wet cells, and then added LDH wet cells prepared by the method in Example 1. The mass ratios of 7α-HSDH to LDH wet cells were 1:0.25, 1:0.5, 1:0.75, 1:1.0, 1:1.25, and 1:1.5, respectively. The mixture was brought to a final volume of 10 mL with pH 8.0, 100 mM KH2PO4-K2HPO4 buffer. The mixture was shaken at 40℃ and 600 rpm for 5 min. Parallel samples were then taken and processed in the same manner as in step (1). The relative enzyme activity was used as the evaluation index. The results are shown in […]. Figure 10 The results showed that the optimal ratio of 7α-HSDH to LDH enzyme was 1:0.5.

[0120] Example 6: Synthesis of 7-KLCA catalyzed by wild-type and optimal combination mutant M9

[0121] Under the optimized reaction conditions of Example 5, the efficiency of wild-type WT and the optimal combination mutant M9 in catalytic synthesis of 7-KLCA at high substrate concentrations was compared.

[0122] 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. The peak areas were detected using the liquid chromatograph described in Example 2. 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 11 As shown. The 7-KLCA standard curve equation obtained by linear regression fitting is: y = 6.8247x - 2.0744, R0 2 =0.9991.

[0123] Crude enzyme solutions of wild-type 7α-HSDH and mutants M9 and LDH were prepared according to the method described in Example 1. The final concentrations of each component in a 10 mL reaction system were as follows: 300 mM CDCA, 0.4 mM NAD. + 300 mM sodium pyruvate, 5 g / L crude enzyme solution (based on wet cell weight before lysis) prepared by wild-type 7α-HSDH or M9 according to the method of Example 1, 2.5 g / L crude enzyme solution of LDH (based on wet cell weight before lysis), and 100 mM KH2PO4-K2HPO4 buffer (pH 8.0) were added to a final volume of 10 mL. The reaction was carried out at 40°C and 600 rpm in a water bath (30, 60, 90, 120, 150, 180, 210, 240, 270 min). Parallel samples were taken, and the reaction was terminated by adding an appropriate amount of 3 M HCl. The samples were 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 product was reconstituted with methanol and appropriately diluted. The peak area was detected using high-performance liquid chromatography as described in Example 2. The content and yield of 7-KLCA were calculated based on the 7-KLCA standard curve. The reaction progress curve is shown below. Figure 12 As shown in the figure. The results showed that M9 could completely convert 300 mM CDCA within 3.5 h (conversion rate > 99.9%), with a yield of 99.5%, while the wild-type 7α-HSDH had a yield of only 64% in the same time, and the final yield was only close to 80%, which could not achieve complete conversion of high substrate concentration.

Claims

1. A method for screening hydroxysteroid dehydrogenase mutants based on the co-evolution of thermostability and activity using α-helical flexible regions, characterized in that, The method includes: constructing an enzyme-substrate-cofactor complex model based on the three-dimensional structure prediction and molecular docking results of hydroxysteroid dehydrogenase, and excluding functional core residues by combining evolutionary conservation analysis; further identifying flexible regions with significantly enhanced dynamic fluctuations under high temperature conditions through multi-temperature molecular dynamics simulation, and focusing the mutation design on highly flexible and low-conserved α-helical residues; and then screening out key mutation sites related to the improvement of enzyme thermal stability or catalytic activity by combining folding free energy change prediction and cross-validation.

2. The method as described in claim 1, characterized in that, The method is performed as follows: First, the three-dimensional structure of hydroxysteroid dehydrogenase is predicted using AlphaFold3; then, molecular docking is performed using YASARA software to construct the enzyme-substrate CDCA-cofactor NAD. + The enzyme-substrate-cofactor complex model was developed and its quality was assessed using PROCHECK, with Ramachandranplot analysis as the primary evaluation criterion. A model was deemed qualified if over 90% of the amino acid residues were located in the most favorable region. Pymol was used to visualize the active site region, identifying key functional residues that directly interact with the substrate and cofactor. ConSurf analysis was employed for evolutionary conservation, eliminating highly conserved sites with a conservation score ≥7 to prevent interference with the catalytic core region from the outset. After excluding core functional residues, MD simulations were performed on the enzyme-substrate-cofactor complex model at 303.15 K and 323.15 K, respectively. The root mean square fluctuation (RMSF) distribution of each residue was calculated, and ΔRMSF was defined as the difference in RMSF between the low-temperature (303.15 K) and high-temperature (323.15 K) conditions. Statistical analysis of the overall ΔRMSF distribution showed that ΔRMSF < -0.2 was considered acceptable. Using Å as the threshold, residues exhibiting significantly enhanced dynamic fluctuations under high-temperature conditions were screened as candidate flexible sites. Based on the above analysis results, the mutation design focused on α-helix residues located in highly flexible regions with low conservation, aiming to optimize the overall conformational stability of the protein by regulating the local rigidity of the α-helix. For the selected candidate sites, Pythia was used to perform virtual saturation mutation calculations, and the folding free energy change ΔΔG was predicted for all amino acid substitutions at each site. ΔΔG < 0 was used as the criterion for potential stabilizing mutations, screening favorable mutation sites from a thermodynamic perspective. Furthermore, PROSS was used for cross-validation to screen key mutation sites while considering both evolutionary rationality and structural stability.

3. A hydroxysteroid dehydrogenase mutant with synergistically enhanced thermostability and activity, screened using the method of claim 1, characterized in that... The mutant is obtained by performing single-site or multi-site combination mutations on positions 12, 15, 50, 56, 66, 76, 126, 132, 158, 161, 203, or 208 of the amino acid sequence shown in SEQ ID NO.

1.

4. The mutant as described in claim 3, characterized in that, The amino acid sequence of the hydroxysteroid dehydrogenase mutant is shown in one of SEQ ID NO.3-SEQ ID NO.

11.

5. A recombinant genetically engineered bacterium containing the encoding gene of the hydroxysteroid dehydrogenase mutant of claim 1.

6. The use of the hydroxysteroid dehydrogenase mutant of claim 3 in the catalytic synthesis of 7-ketolithocholic acid from chenodeoxycholic acid.

7. The application as described in claim 6, characterized in that, The application is: using the recombinant genetically engineered bacteria containing hydroxyl steroid dehydrogenase mutant gene as the catalyst, using chenodeoxycholic acid as the substrate, adding NAD + , sodium pyruvate and lactate dehydrogenase, using the buffer solution with pH 6-9 as the reaction medium to form a conversion system, and performing the conversion reaction under the conditions of 25-50 DEG C and 600 rpm to obtain the conversion liquid containing 7-ketolithocholic acid.

8. The application as described in claim 7, characterized in that, In the conversion system, the substrate concentration was added at 125-375 mM; NAD + The concentration of added sodium pyruvate is 0.1-1 mM; the concentration of added sodium pyruvate is 150-750 mM; the amount of catalyst added is 5-15 g / L based on the wet cell volume before crushing, and 5-15 mg / L based on the protein content.

9. The application as described in claim 7, characterized in that, The lactate dehydrogenase was added in the form of the supernatant obtained by ultrasonic disruption and centrifugation of recombinant Escherichia coli containing the lactate dehydrogenase gene after induced culture. The amount added was 1-10 g / L based on the wet bacterial cells before disruption. The nucleotide sequence of the lactate dehydrogenase gene is shown in SEQ ID NO.

12.

10. The application as described in claim 7, characterized in that, The wet bacterial cells 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 ℃ 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 centrifuge at 4 ℃ and 8000 rpm for 10 min to obtain wet bacterial cells containing the 7α-HSDH mutant.

Images