| Cytochrome P450 (CYP) enzymes can catalyze a lots of endogenous and exogenous compounds, including many environmental contaminants. It has been found in all living organisms such as bacteria, yeast, fungi, plants, animals, and human beings. CYP-mediated metabolism can result in either detoxification or activation of some xenobiotics and determine their validity, thereby influencing their toxicity. So they play an important role in a variety of biosynthesis and biological degradation.The goal of the present study is to (1) clone and expression of house fly cytochrome P4506A1 (CYP6A1) and rat cytochrome P4501A1 (CYP1A1) in E. coli, and then separation and purification of the two proteins primarily from membranes of E. coli; (2) construction of the biosensors based on CYP6A1 or CYP1A1 to detect aldrin, heptachlor and benz[a]pyrene, respectively; (3) over expression of CYP1A1 in tobacco and tissue localization of CYP1A1 with the fusion GFP in transformed plants.The main results are as follow:(1) Using the plasmid pCW, high-level expression of rat cytochrome p4501A1 (CYP1A1) had been achieved by making NH2-terminal translational fusions to the bacterial leader sequences ompA. The expressed 1A1 protein in bacterial membrane fractions were collected and immobilized in nano-Na-montmorillonite (nano-SWy-2) and dihexadecylphosphate (DHP) composite film. The direct electrochemistry of CYP1A1+ in a nano-SWy-2-DHP film on an edge-plane pyrolytic graphite electrode (EPG) had been obtained and the catalytic activity of the enzyme to benzo[a]pyrene had been investigated by the cyclic voltammetry.(2) A plasmid (pCW) was modified to code for the complete sequence of house fly (Musca domestica) cytochrome P450 6A1(CYP6A1) with only the second amino acid changed in the N-terminal portion and this plasmid was used to express the CYP6A1 protein in Escherichia coli cells. With the addition ofδ-aminolevulinic acid and FeCl3 to the culture, the protein was produced at a level about 0.25μmol/L (15 mg/L) of culture with approximately 50% of the P450 being associated with the membrane fraction. The CYP6A1 protein was purified with a combination of DEAE sepharose fast flow and hydroxyapatite chromatography. Direct electrochemistry of CYP6A1 in a didodecyldimethylammonium bromide (DSAB) film on an edge-plane pyrolytic graphite electrode (EPG) had been obtained, and its catalytic activity to aldrin had been demonstrated by the cyclic voltammetry. The apparent Michaelis- Menten constant for the electrocalytic activity of CYP6A1 was found to be 7.468×10-5 mol/L for aldrin and 4.316×10-5 mol/L for heptachlor, which implied the high affinity of CYP6A1 immobilized in DDAB film to its substrates. The bioelectrocatalytic products were analyzed using gas chromatography (GC) and electron ionization-mass spectrometry (EI-MS). It is confirmed that epoxidation was the main pathways for CYP6A1-mediated organochlorine pesticides oxidation. Based on these, an amperometric biosensor based on cytochrome p450 6A1 for organochlorine pesticides sensing had been developed.(3) The fusion protein expression vector for CYP1A1 and green fluorescent protein (GFP) gene was constructed by a plant expression vector pCANMBIA with CaMV 35S promoter. Through Agrobacterium-mediated plant transformation, CYP1A1 gene had been transformed into a number of tobacco seedlings. Positive transformed plants are collected after PCR test and the expression levels of CYP1A1 in the positive plants were then evaluated by real-time PCR. And CYP1A1 with GFP fusion protein was found to localize in the vein of tobacco leaves.
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