An amperometric biosensor based on rat cytochrome p450 1A1 for benzo[a]pyrene determination.
Using the plasmid pCW, high-level expression of rat cytochrome p4501A1 (CYP1A1) has been achieved by making NH(2)-terminal translational fusions to bacterial leader sequences ompA (ompA-1A1/pCW). The construct ompA-1A1 was compared with an expression construct in which the Ala codon GCT was placed in the second position and 5'-terminal codons were maximized for A T content (1A1/pCW). Both constructs produced spectrally active, functional protein. However, the ompA-1A1 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with the construct 1A1/pCW. The expressed 1A1 from the construct ompA-1A1/pCW 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) has been obtained and the catalytic activity of the enzyme to benzo[a]pyrene has been investigated by the cyclic voltammetry. The immobilized CYP1A1 displayed a pair of redox peaks with a formal potential of -0.36 mV in pH 7.0 O(2)-free phosphate buffers at scan rate of 1 V s(-1). The CYP1A1 in the nano-SWy-2-DHP film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon the addition of its substrate benzo[a]pyrene (B[a]P) to the air-saturated solution, the reduction peak current of dissolved oxygen increased, which indicates the catalytic behavior of CYP1A1 to B[a]P. By amperometry a calibration linear range for B[a]P was obtained to be 3.31-16.56 μM with a sensitivity of 58.57 μA mM(-1). And the apparent Michaelis-Menten constant for the electrocatalytic activity of CYP1A1 was estimated to be 46.27 μM for B[a]P.
Key Lab for Biotechnology of National Commission for Nationalities, College of Life Science, South Central University for Nationalities, Wuhan 430074, PR China. yunhuawu@yahoo.com.cn
Yunhua
Y
Wu
Xuequn
X
Liu
Li
L
Zhang
Chuntai
C
Wang
D000818:Animals; D001564:Benzo(a)pyrene / Q000032:analysis* / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation*; D003217:Conductometry / Q000295:instrumentation*; D019363:Cytochrome P-450 CYP1A1 / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D051381:Rats
D001564:Benzo(a)pyrene; D019363:Cytochrome P-450 CYP1A1