Acetylcholinesterase from Osteochilus hasselti for the detection of insecticides and heavy metals

The use of acetylcholinesterase to detect insecticides such as carbamate and organophosphate is well known. However, new sources of AChE are constantly being sought since the most crucial preparation of AChE rarely is sensitive to all insecticides. At the same time, literature data shows that certain AChEs are sensitive to heavy metals and by searching new sources, AChE can be applied for the detection of heavy metals as well. In this work, an inhibitive assay for insecticide and heavy metals was developed using partially purified fraction of AChE from Osteochilus hasselti using Procainamid Sephacryl S-1000 with a purification fold of 8.1 and a recovery yield of 55.3% were obtained. The partially purified fraction showed optimal activity at pH 7.0 and optimal temperature in the range of 25oC to 30oC. Acetylthiocholine iodide (ATC) was chosen as specific substrate with higher maximal activity (Vmax) of 742.43 μmole/min and the lowest Michaelis Constant (Km) at 0.0086 mM followed by Propionylthiocholine Iodide (PTC) and Butyrylthiocholine Iodide (BTC). All of the carbamate groups such as carbaryl, bendiocarb, carbofuran, methomyl and propoxur, and several oxonated OPs such as chlorpyrifos, diazinon,malathion and parathion showed significant inhibition more than 50% of AChE activity. Partially purified AChE was not inhibited by tricholrfon, dimethoate and acephate. ANOVA analysis showed that all of the inhibition seen was significant compared to control (p<0.05) in the absence of insecticide. The carbamates; carbaryl, and the oxonated OPs; diazinon showed significantly lower IC50s in O. hasselti compared to E. electricus while parathion and diazinon showed overlapped IC50s. Chromium, copper and mercury inhibited 80%, 61.3% and 35% of AChE activity, respectively. The heavy metals exhibited exponential decay type inhibition curves with calculated IC50 for copper, and chromium at 0.198 and 0.076 mg l-1, respectively. The effect of sample matrix in detecting insecticides using tap water, sea water and waters from two pristine rivers samples spiked with carbamate showed no significant differences amongst each other (p>0.05) indicating no matrix effect. Among all the vegetable samples studied, chili shows the presence of insecticide and validated by HPLC analysis. Sungai Juru exhibited higher inhibition proved by ICP-OES high with amount of chromium and copper while other river samples showed low inhibition except for Endau Rompin national water park sample. It can be summarized that O. hasselti AChE is a new alternative source as biosensor to detect contaminant because the sensitivity of this enzyme is almost similar compare to other commercialize AChE. For further studies, it is recommended that full purification should be carried out to understand the fundamental differences between this AChE and other AChEs to further understand the mechanism of heavy metals and insecticides inhibition.

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