Recombinant cytochrome P450 enzyme and application thereof
A technology for recombining cells and cytochromes, which is applied in applications, recombinant DNA technology, enzymes, etc., can solve the problems of low degradation effect of pure enzymes, limited range of enzyme action, single degradation strains, etc., and achieves wide applicability of pH and scope of application Wide range and high degradation efficiency
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Embodiment 1
[0047] Embodiment 1: Construction of high expression vector
[0048] (1) Obtain the coding gene of cytochrome P450 (the nucleotide sequence is shown in SEQ ID NO.1), and design primers: forward primer ATC GGATCC ATGACCCATCCGACACTCG The underlined sequence is the cutting site of restriction endonuclease BamH I; Reverse primer: ATTATA CTCGAG The underlined sequence of GGCGCGAACGGCGAC is the cut point of restriction endonuclease XhoI, using genomic DNA as a template, using RCR technology to amplify the cytochrome P450 sequence, the agarose gel electrophoresis of the PCR product is as follows figure 1 shown.
[0049] PCR mix consists of 10 μL 5× FastPfu buffer, 1 μL forward primer, 1 μL reverse primer, 1 μL template DNA, 4 μL dNTPs, 1 μL FastPfu DNA polymerase and 32 μL ddH 2 O composition. The PCR program was: denaturation at 95°C for 2 min; followed by 30 cycles with the parameters of 95°C for 20 s; 59°C for 20 s; 72°C for 30 s; and finally 72°C for 5 min.
[0050] (2) Est...
Embodiment 2
[0051] Embodiment 2: the acquisition of engineering bacteria
[0052] (1) Take 50 μL Escherichia coli BL21(DE3) competent cells (purchased from Beijing Quanshijin Company), put them on ice, and gently suspend the cells evenly after thawing completely.
[0053] (2) Take 20 μL of the plasmid pET28b(+)-cytochrome P450 obtained in Example 1, add it to the Escherichia coli BL21(DE3) competent cells, mix gently, and let stand on ice for 30 minutes.
[0054] (3) Heat shock at 42°C for 45 s, and immediately place it on ice for 2 min.
[0055] (4) Add 500 μL LB liquid medium, and culture with shaking at 37° C. and 200 r / min for 1 hour.
[0056] (5) Take 100 μL of the culture solution and spread it on an LB plate containing kanamycin (50 μg / mL) resistance, and culture it overnight at 37°C. The resulting transformant was verified by colony PCR, and it was found that there was an obvious band at about 1200 bp, After sequencing, it was found to be consistent with the sequence of cytochro...
Embodiment 3
[0057] Embodiment 3: The extraction of recombinant cytochrome P450 and the mensuration of dichloromethane dechlorination rate
[0058] 1. The recombinant Escherichia coli BL21(DE3)-pET28b(+)-cytochrome P450 obtained in Example 2 was activated in LB slant medium at 37°C and then inserted into LB liquid medium, and cultured at 37°C and 200r / min for 12h Afterwards, transfer to 50mL LB liquid culture medium with 2% (volume concentration) inoculum amount, 37 ℃, 200r / min shaking culture until OD600 reaches 0.6, add inducer isopropylthiogalactoside (IPTG) to final concentration 0.2mM, 30°C, 200r / min shaking culture for 6h, centrifuge to get the bacteria, wash with PBS buffer (pH 7), add 15mL of PBS buffer to mix, after ultrasonic crushing, centrifuge (8000rpm, 4 °C, 30 min) to collect the supernatant, and purify it through a nickel column to obtain the recombinant cytochrome P450 pure enzyme solution. The ultrasonic crushing conditions are: work for 3 seconds, pause for 2 seconds, a...
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