The potential of acetylcholinesterase from monopterus albus brain for the detection of heavy metals and insecticides
Environmental contamination of heavy metals and insecticides have been continuously increased in several areas in Malaysia because of industrial and human activity. Monitoring programmed was implemented to ensure the level of contamination can be controlled. In this study, Cholinesterase, (ChE) from...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2020
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| Online Access: | https://eprints.ums.edu.my/id/eprint/41690/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/41690/2/FULLTEXT.pdf |
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| Summary: | Environmental contamination of heavy metals and insecticides have been continuously increased in several areas in Malaysia because of industrial and human activity. Monitoring programmed was implemented to ensure the level of contamination can be controlled. In this study, Cholinesterase, (ChE) from the brain of eel, Monopterus albus was tested to determine the potential use as alternative biosensor, which is sensitive towards various insecticides and metal ions. Acetylcholinesterase (AChE) from the brain of M. albus was purified through ammonium sulphate precipitation and procainamide affinity chromatography. Enzyme recovery was obtained at 38.73% with the specific activity of 1847 U μg-1. The Michaelis constant (Km) value and maximal velocity (Vmax) were determined at 8.910 mM and 29.44 μmol min-1 mg-1, respectively, for acetylthiocholine iodide (ATC). Based on effective coefficient ratio, AChE from M. albus brain showed higher affinity to ATC compared to butyrylthiocholine iodide (BTC) and propionylthiocholine iodide (PTC) at the value of 3.304, 1.515, 2.965 Vmax.Km-1, respectively. Optimum activity of AChE was obtained at the range of 25°C to 50°C and incubated in 0.1M Tris HCl buffer pH 9.0. Purified acetylcholinesterase was exposed with a different type of metal ions, and mercury shows the highest inhibition at the percentage of 62.9% followed by chromium at 59.22% while silver, arsenic, cadmium, cobalt, copper, nickel, zinc and lead not more than 50% inhibition (around 37 to 50%). Metal ions such mercury, zinc, chromium and copper show exponential decay type inhibition curves with half-maximal inhibitory concentration; IC50 was calculated in the ascending sensitivity order of 0.005, 0.595, 0.687 and 1.329 mgL-1, respectively. Field trial works exhibited that the enzyme was applicable in sensing heavy metals pollution from the river which closes to the industrial and agricultural sites at near real-time and verified using ICP-OES. Inhibition study for insecticides resulted in this descending order of inhibition; Dimethoate> Parathion> Malathion> Diazinon> Chlorpyrifos> Bendiocarp> Methomyl> Acephate> Propoxur> Carbaryl> Trichlorfon> Carbofuran, with Dimethoate, Parathion, Malathion, Diazinon, Chlorpyrifos, Bendiocarp and Methomyl showing more than 50% inhibition at two ppm. Selected pesticides showed exponential decay type inhibition curves with calculated IC50 in the ascending Environmental contamination of heavy metals and insecticides have been continuously increased in several areas in Malaysia because of industrial and human activity. Monitoring programmed was implemented to ensure the level of contamination can be controlled. In this study, Cholinesterase, (ChE) from the brain of eel, Monopterus albus was tested to determine the potential use as alternative biosensor, which is sensitive towards various insecticides and metal ions. Acetylcholinesterase (AChE) from the brain of M. albus was purified through ammonium sulphate precipitation and procainamide affinity chromatography. Enzyme recovery was obtained at 38.73% with the specific activity of 1847 U μg-1. The Michaelis constant (Km) value and maximal velocity (Vmax) were determined at 8.910 mM and 29.44 μmol min-1 mg-1, respectively, for acetylthiocholine iodide (ATC). Based on effective coefficient ratio, AChE from M. albus brain showed higher affinity to ATC compared to butyrylthiocholine iodide (BTC) and propionylthiocholine iodide (PTC) at the value of 3.304, 1.515, 2.965 Vmax.Km-1, respectively. Optimum activity of AChE was obtained at the range of 25°C to 50°C and incubated in 0.1M Tris HCl buffer pH 9.0. Purified acetylcholinesterase was exposed with a different type of metal ions, and mercury shows the highest inhibition at the percentage of 62.9% followed by chromium at 59.22% while silver, arsenic, cadmium, cobalt, copper, nickel, zinc and lead not more than 50% inhibition (around 37 to 50%). Metal ions such mercury, zinc, chromium and copper show exponential decay type inhibition curves with half-maximal inhibitory concentration; IC50 was calculated in the ascending sensitivity order of 0.005, 0.595, 0.687 and 1.329 mgL-1, respectively. Field trial works exhibited that the enzyme was applicable in sensing heavy metals pollution from the river which closes to the industrial and agricultural sites at near real-time and verified using ICP-OES. Inhibition study for insecticides resulted in this descending order of inhibition; Dimethoate> Parathion> Malathion> Diazinon> Chlorpyrifos> Bendiocarp> Methomyl> Acephate> Propoxur> Carbaryl> Trichlorfon> Carbofuran, with Dimethoate, Parathion, Malathion, Diazinon, Chlorpyrifos, Bendiocarp and Methomyl showing more than 50% inhibition at two ppm. Selected pesticides showed exponential decay type inhibition curves with calculated IC50 in the ascending sensitivity order of Bendiocarp, Dimethoate, Malathion and Parathion at the concentration of 1.639, 1.509, 0.874 and 0.162 mgL-1, respectively. A 10-days field trial performed by testing purified ChE on C. asiatica treated with three commercial insecticides exhibited that the enzyme was applicable in sensing the presence of those compounds. This study proved that the potential use of acetylcholinesterase source from M. albus as a biomonitoring tool can be further utilized to develop a cheaper, easier and faster pesticides and heavy metals detection method as compared to conventional methods available. |
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