Cloning, Expression,fermentation Conditions Optimization And Characterization Of Cytochrome P450 From Bacillus Amyloliquefaciens DC-12

Cytochrome P450 is a class of heme protein superfamily end monooxygenase. CYP450 is common in human, animal, plant and microbial cells. CYP450 named because it can be combined with CO in the reduced state, and complex formation has a maximum absorption peak at 450 nm. CYP450 is the most biological catalysis and catalyst the metabolism of endogenous substances and xenobiotics. CYP450 not only can catalyze oxidation reaction but also catalytic reduction. CYP450 can transform refractory material into biodegradable substances. CYP450 in repairing and governance aspects of environmental pollutants is a hot topic in recent years.The wild-type B. amyloliquefaciens DC-12 as the starting strain. The genome CYP450 gene sequence analysis, cloning and expression of the active protein. On this basis, the fermentation conditions were optimized single factor, preliminary exploration of crude enzyme solution and enzymatic properties of CYP450 gene deletion strains.Forecast and analysis CYP450 amino acid sequence in DC-12 genome from NCBI database（GI: 410720199） Scaffold1. Successfully constructed cloning vector pMD19-T-CYP and expression vectors pET22b（+）-CYP, transformed E. coli BL21（DE3）. By Ni²⁺ affinity chromatography purified CYP450 protein with high purity. SDS-PAGE protein electrophoresis showed that about 44 KDa Department has a very strong band.By Single factor optimization experiments,we optimize the fermentation conditions.Optimum culture conditions : 0.8mmol/L IPTG, induction temperature 20℃, induced culture 16-24 h, the active expression of CYP450 enzymes reached the maximum. Expression activity amounted to 2.715706 ± 0.017657 U1 / m L, increase 18.5 times.The four specific oxidase substrate, only Nitroanisole and 7- ethoxycoumarin showed activity, while methoxyresorufin and benzphetamine did not show activity. The optimum pH was 7.6.when pH> 9.5 or <4.5, the enzyme activity is low.especially p H>7.6, the enzyme activity decreased rapidly. the optimum temperature was 30-35 ℃.when temperature> 45 ℃ or <25 ℃, CYP450 enzyme activity was low.The results of PNOD activity assay: Ca²⁺, Fe²⁺, Zn²⁺, Co²⁺ all have different degrees of inhibition to CYP450, especially when the higher ion concentration（50mmol/L） has 100% inhibition of enzyme activity. Higher concentration of Mn²⁺, Mg²⁺, NH41+ plays a weak role in promoting enzyme activity. Cationic surfactants inhibit CYP450 enzyme activity, and the greater the concentration, the stronger the inhibition. Anionic surfactant concentration of less than 0.007g/L promots enzyme activity, but as concentration was increasing, it began showing inhibition. And when the concentration reached 0.09g/L, CYP450 enzyme activity was 100% inhibition; nonionic surfactant concentration of less than 0.4g/L promots weakly CYP450 enzyme activity, but as the concentration increases, it began showing inhibition. And when the concentration reached 5g/L, CYP450 enzyme activity was 100% inhibition.The results of ECOD activity assay: Mn²⁺, NH4 +, Zn²⁺ at a concentration of 0.1mmol/L, 1mmol/L promoted weakly CYP450 enzyme activity, but with the increasing concentration, rendering the inhibition of CYP450 enzyme activity. Cationic surfactant concentration of less than 0.03g/L promoted CYP450 enzyme activity, more than 0.03 g / L inhibited enzyme activity; concentration of the anionic surface active agent in the 0.004-0.1g/L range showing inhibition; nonionic surfactants at low concentrations 0.1-0.8g/L exhibits CYP450 enzymes, but in high concentrations of 0.8-5g/L, presented the inhibition to CYP450 enzyme.By overlapping PCR method to connect the left and right arm, we get a complete homology arm fragments. The constructed gene deletion plasmid was transformed into E.coli JM110 strain demethylation. We adopted to apply electroporation transforming into wild-type B. amyloliquefaciens DC-12.