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Expression of Cytochrome P450 in Prokaryotic System and Its Application in Biosensing for Endocrine Disrupting Chemical Bisphenol a

Author: YinMing
Tutor: WuYunHua
School: Central South University for Nationalities
Course: Medicinal Chemistry
Keywords: Cytochrome P450 Biosensor Environment Endocrine Disrupting Chemicals Bisphenol A
CLC: R114
Type: Master's thesis
Year: 2011
Downloads: 30
Quote: 0
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Abstract


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, thereby influencs their toxicity. They can widely be applied in biosynthesis and biological degradation.The goal of the present study is to (1) Co-expressed cytochrome P450 with human NADPH-cytochrome P450 reductase in E.coli to study the metabolism of bisphenol A; (2)Contruct an amperometric biosensor based CYP2C9 to detect bisphenol A(3)Study the electrochemical behavior of bisphenol A at nanohydroxyapatite modified carbon paste electrode and its determination. It mainly contains:(1) Using the plasmid Pcw,five forms of human cytochrome P450 co-expressed with human NADPH-cytochrome P450 reductase in E.coli. The expression level of P450 Co-expressed with reductase ranged from 3.8 to 38.7nmol/L culture. The expression level of the reductase ranged from 8.7 to 33.9U/L culture. The catalytic activity of P450 recovered from membrane was studied using bisphenol as substrate. The catalytic rates ranged from 1.8 to 65.9nmol/min/nmolP450. The Vmax of CYP2C19 was 11 times higer than that of CYP2C9 and the Km of CYP2C19 was 4 times higer than that of CYP2C9. The results suggest that bisphenol A is mainly metabolized by the CYP2C subfamily in human.(2) Direct electrochemistry of CYP2C9 in a Polyacrylamide (PAM) film on an edge-plane pyrolytic graphite electrode (EPG) has been obtained and the catalytic activity of the enzyme to bisphenol A has been demonstrated by the cyclic voltammetry. The apparent Michaelis-Menten constant for the electrocalytic activity of CYP2C9 was found to be 3.90×10-6mol/L for bisphenol A, which implies the high affinity of CYP2C9 immobilized in PAM film to its substrate.(3) A simple and high sensitive electroananlytical method for determination of bisphenol A using Nanohydroxyapatite (Nano-HAP) modified carbon paste electrode (CPE) was presented. The electrochemical oxidation behavior of bisphenol A on the modified electrode was investigated by cyclic voltammetry (CV), chronocoulometry (CC), differential pulse voltammetry (DPV), and linear sweep voltammetry (LSV) and some kinetic parameters were obtained. Bisphenol A can be adsorbed on the Nano-HAP-CPE effectively in phosphate buffer solution (PBS) and that exhibited a significant oxidation peak at 0.468 V. Under the optimal conditions, the oxidation peak current was proportional to BPA concentration in the range of 8.00×10-8～1.25×10-5 mol/L with the limit of detection 4.50×10-8 mol/L. It has been applied to determine bisphenol A in plastic product samples with satisfactory results. The recovery range was 96.10% -103.1%.

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