The Development of Bacterial Carboxylesterase Biological Recognition Elements for Cocaine Detection.

Enzyme recognition element-based biosensors are very attractive for biosensor application due to a variety of measurable reaction products arising from a catalytic process. In this study, biosensor recognition elements have been developed via engineer bacterial enzymes (carboxylesterases (CEs)) which will used for narcotic detection because of their role in narcotics metabolism. The modification (insertion of cys-tag) allows the enzyme to bind into a transducer surface of a biosensor which will translate the reaction product into the detection system. The results demonstrate the successful isolation, cloning, expression, and purification of recombinant (pnbA1 and pnbA2), and engineered (pnbA1-cys and pnbA2-cys) bacterial carboxylesterases. Enzyme capability to hydrolyse cocaine into benzoylecgonine and methanol was quantified using HPLC. Both enzymes showed broad maximal activity between pH (8.0, 8.5, and 9.0), PnbA1 temperature stability ranging between (25 and 45 °C); however, PnbA2 stability range was (25-40 °C). Insertion of cys-tag at the N-terminal of the enzyme did not limit entrance to the active site which is located at the base of a cavity with dimensions 20 by 13 by 18 Å, and did not prevent substrate hydrolysis. Bacterial carboxylesterases pnbA1 and pnbA2 mimic hCE1 and not hCE2 in its reaction pathways hydrolysing cocaine into benzoylecgonine and methanol rather than ecgonine methyl ester and benzoic acid. These results are the first experimental evidence confirming the capability of bacterial carboxylesterase to hydrolyse cocaine into its main metabolites, therefore opening up the possibility to use these enzymes in numerous biotechnological applications in addition to a cocaine biosensor.