Organophosphate Insecticide Sensing Using Phosphotriesterase (PTE) Enzyme Derived from Sphingobium fuliginis ATCC 27551 Based-on pH-ISFET Sensor Measuremen

Abstract

Paraoxon (diethyl (4-nitrophenyl) phosphate) was chosen as a model compound of organophosphate insecticide because of their high toxicity level toward human health, animals and environment. Phosphotriesterase (PTE) as one of the potential organophosphorus hydrolase enzymes has a special ability in degrading the paraoxon in various concentration. This research aims to produce the PTE derived from Sphingobium fuliginis and evaluate its ability of PTE in hydrolyzing the paraoxon. The preparation of microbial PTE production was successfully obtained by cultivating the inoculum of Sphingobium fuliginis in LB-medium. The purified PTE was obtained by the purification process using DEAE-Toyopearl Column equilibrated by 20mM of PPB (pH=7.2). PTE enzyme assay was observed by spectrophotometric UV-vis with measured absorbance is 410nm and it was examined on two different types of the buffer which is NaHCO3 buffer 100mM (pH=8.5) and Glycine-NaOH buffer 50mM (pH=9.5). The expression of PTE was confirmed by SDS-PAGE analysis. The measurement for the time-course of pH decreased in the presence and the absence of PTE was evaluated by using the pH-ISFET sensor. The best result showed that LB-medium was chosen as the most suitable medium for Sphingobium fuliginis cultivation due to its perfect nutrients content. The molecular mass of PTE subunits protein was well-expressed in 35kDa. Moreover, the amount of paraoxon concentration and the PTE’s performance such as PTE’s specific activity, productivity, sensitivity and the higher amount of total protein of PTE (mg) was performed well in 100mM NaHCO3 buffer pH 8.5. PTE’s Km value obtained on 8.106 µM and its maximum rate velocity (Vmax) showed in 2.497 unit paraoxon mg-1 protein. Lastly, the time course of pH decreased dramatically when the PTE applied to the system. It showed a rapid detection for less than 2-5 seconds the hydrolyzation of paraoxon has been already finished. So that, PTE has been confirmed able to recognize the paraoxon in various concentration in rapid detection.