7beta-hydroxysterol dehydrogenase mutant and application of 7beta-hydroxysterol dehydrogenase mutant in ursodeoxycholic acid synthesis
A technology of hydroxysterol dehydrogenase and hydroxysterol, applied in the field of bioengineering, can solve the problems of low activity of 7β-HSDH, cumbersome application process, low substrate concentration, etc.
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Embodiment 1
[0062] Example 1 Random mutation screening of 7β-hydroxysterol dehydrogenase with improved activity and stability
[0063] Randomly mutating the 7β-hydroxysterol dehydrogenase with the amino acid sequence shown in SEQ ID No. 2 in the sequence listing by using the method of error-prone PCR.
[0064] The primers used were:
[0065] The upstream primer, as shown in the sequence table SEQ ID No.3:
[0066] 5'-CCGGAATTCATGAATCTGCGTGAAAAATAC-3', wherein the sequence shown in the underline is the restriction endonuclease EcoR I enzyme cutting site;
[0067] Downstream primers, as shown in the sequence table SEQ ID No.4:
[0068] 5'-CCGCTCGAGTTAATTGTTGCTATAGAAGC-3', wherein the underlined sequence is the restriction enzyme Xho I cutting site.
[0069] PCR system (50 μL): Taq mix 25 μl, MnCl with a final concentration of 100 μmol / L 2 , about 1ng of pET28a-7β-M0 plasmid, 2μl of upstream and downstream primers, diH 2 O to make up to 50 μl.
[0070] PCR reaction program: (1) Pre-den...
Embodiment 2
[0094] Example 2 Combined Mutation of 7β-Hydroxysterol Dehydrogenase
[0095] According to the method and primers described in Example 1, the gene DNA sequence of 7β-hydroxysterol dehydrogenase M1-M9 obtained in Example 1 is carried out to PCR amplification, the DNA fragments obtained are mixed in equal proportions, and DNase I is added for digestion , reaction system 200μl, containing 100mM Tris-HCl (pH 7.5), 0.1U DNase I, 20μg mixed DNA fragments and 10mM MnCl 2 , digested at 37°C for 3 minutes, separated by electrophoresis, and collected fragments of 50-200 bp for PCR assembly.
[0096] 50 μl of PCR assembly reaction system, containing reaction buffer, 100 ng of enzyme-digested fragments, 0.2 mM dNTP mix and 1 μl of KOD high-fidelity polymerase.
[0097] PCR reaction program: (1) Pre-denaturation at 95°C for 2min; (2) Denaturation at 94°C for 30s; (3) Annealing at 65°C for 30s; (4) Annealing at 60°C for 30s; (5) Annealing at 55°C for 30s; (6) 50°C Anneal for 30 s; (7) ann...
Embodiment 3
[0105] Example 3 Expression of recombinant E.coli BL21(DE3) / pET28a-7β-M6
[0106] The recombinant Escherichia coli E.coli BL21(DE3) / pET28a-7β-M6 of the mutant M6 obtained in Example 1 was inoculated into LB medium containing 50 μg / ml kanamycin sulfate, and cultured with shaking at 37°C until OD 600 Reach 1.2, insert in the 500ml Erlenmeyer flask that 100ml LB culture medium (containing 50 μ g / ml kanamycin sulfate) is housed by the inoculum size of 1% (v / v), place 37 ℃, 180rpm shaker shaking culture, When the OD of the culture medium 600 When it reaches 0.6, add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.2mmol / L as an inducer, and induce at 16°C for 24h. The culture solution was centrifuged at 8000×g for 10 min, the cells were collected, and washed twice with saline. Suspend the cells obtained in 100ml of culture medium in 10ml of potassium phosphate buffer (100mM, pH 8.0), sonicate, and collect the supernatant by centrifugation at 12000×g. Th...
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