Application of cytochrome P450 monooxygenase in catalyzing lithocholic acid to produce ursodesoxycholic acid
A technology for ursodeoxycholic acid and monooxygenase, which is applied in the field of enzyme engineering, can solve the problems of complicated steps, insufficient product concentration, low enzyme activity, etc., and achieves good selectivity, easy industrial amplification, and high substrate concentration. Effect
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Embodiment 1
[0025] Example 1: Cloning of Cytochrome P450 Monooxygenase Gene
[0026] According to the genome of the Allokutzneria albata bacteria (gene accession number: SDN52915.1) recorded in Genbank and predicted to be a monooxygenase, the PCR primers were designed as follows (SEQ ID NO.3-4):
[0027] Upstream primer: 5'-gtgccgcgcggcagc catatg ATGACCGCCGTCGATCCC-3'
[0028] Downstream primer: 5'-gtggtggtggtggtg ctcgag TCACCAGCTCACCGGAAGG-3';
[0029] Wherein, the underlined part of the upstream primer is the NdeI restriction site, and the downstream primer part is the XhoI restriction site.
[0030] The genomic DNA of Allokutzneria albata was used as a template for PCR amplification. The PCR system is: 10 μL of 2×Taq PCR MasterMix, 1 μL (0.3 μmol / L) of each upstream primer and downstream primer, 1 μL (0.1 μg) of DNA template and ddH 2 O 7.0 μL. The PCR amplification program is: (1) 95°C, pre-denaturation for 3 minutes; (2) 94°C, denaturation for 30 seconds; (3) 55°C annealing f...
Embodiment 2
[0031] Example 2: Preparation of Cytochrome P450 Monooxygenase Recombinant Plasmid and Recombinant Expression Transformant
[0032] The cytochrome P450 monooxygenase gene DNA fragment obtained in Example 1 and the pET28a empty plasmid were digested with restriction endonucleases NdeI and XhoI at 37°C for 2 h, purified by agarose gel electrophoresis, and purified by agarose gel electrophoresis. DNA Recovery Kit recovers target fragments. Put the target fragment at T 4 Under the action of DNA ligase, the recombinant expression plasmid pET28a-CYP was obtained by ligation overnight at 4°C.
[0033] The above recombinant expression plasmids were transformed into Escherichia coli E.coli DH5α competent cells, positive recombinants were screened on a resistance plate containing kanamycin, single clones were picked, and positive clones were verified by colony PCR. Cultivate the recombinant bacteria, extract the plasmid after the amplification of the plasmid, and retransform into E. ...
Embodiment 3
[0034] Example 3: Expression of Cytochrome P450 Monooxygenase
[0035]The recombinant Escherichia coli obtained in Example 2 was inoculated into LB medium containing kanamycin (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH7.0), and cultured overnight at 37°C with shaking, According to the inoculation amount of 1% (v / v), insert into the 500mL Erlenmeyer flask that 100mLLB culture medium is housed, place 37 ℃, 180rpm shaker shaking culture, when the OD of culture solution 600 When it reaches 0.6, add IPTG with a final concentration of 0.2mmol / L as an inducer, and after induction at 25°C for 12 hours, centrifuge the culture medium, collect the cells, and wash twice with normal saline to obtain resting cells, which can be obtained by freeze-drying for 24 hours Cells were freeze-dried and stored at 4°C after collection. The obtained quiescent cells can also be suspended in a pH 7.0 buffer, ultrasonically disrupted in an ice bath, and centrifuged to collect the supernatant, whi...
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