Development Of A Novel Optical Biosensor For Detection Of Organophosphorus Pesticides And Exportation Of Organochlorine Pesticides Dehydrochlorinase

In the agricultural production, cultivated crops are always influenced by agricultural destructive pests or insects, which results in a great loss in production and economy. To deal with these insects we generally use chemical reagents, such as organophosphorus, organic chloride and carbamates.Synthetic organophosphates (OPs) are a group of highly toxic chemicals widely used to control various agriculture pests, accounting for about 38% of total pesticides used globally. Over 40 million kilograms of OP pesticides are used annually in the U.S., with another 20 million kilograms produced for export. It’s easily biodegraded in a half-life period from one week to several months with a low toxicity remaining. But many high toxic organophosphorous can cause chronic poisoning. A high concentration of pesticide residue is an outstanding problem because of the large amounts abused. At present, the detection of organophosphorous residue needs complicated operation, time consuming and high cost. Organic chloride is a widely used broad-spectrum pesticide with a high fat-solubility and stable chemical properties. And it’s one of the Persistent Organic Pollutants. Though organic chloride has been banned in our country since 1983, it can be detected in soil, water and in the air. The major method for degrading the pollutants is usually biodegradation, especially aerobic degradation.In this study, we developed an optical biosensor based on methyl parathion hydrolase (MPH) immobilized by metal-chelate affinity and translocated dehydrochlorinase for organochlorine pesticides (γ-hexachlorocyclohexane) to the periplasm.1. The development of an optical biosensorWe constructed an expression vector pETM with six-histidine in the C-terminal of methyl parathion hydrolase (MPH) by inserting mpd into the original vector pET30a. MPH was expressed in the host cell Escherichia coli BL21(DE3) by T7 phage promoter. The crude protein was harvested by using Ultrasonic crushing method, and then the purified enzyme MPH was obtained using Ni-sepharose by metal-chelate affinity.We immobilized the pure enzyme on Ni-sepharose trapped in filter paper by metal-chelate affinity to make a reaction tank. The immobilized enzyme can be easily eluted by a solution with a high concentration of imidazole and regenerated when the enzyme lost its bioactivity.The production of methyl parathion after hydrolased is paranitrophenol (PNP), which has a specially OD value at 410 nm. The solution was prepared with a series concentration of methyl parathion, after hydrolased, the OD value was determined, the solution without any methyl parathion is used as a control. A standard curve between the concentration of methyl parathion and the OD value was obtained. A lower detection limit was 0.2 mg/ml methyl parathion. Hence, an optical biosensor for determination of organophosphorous residue has been successfully developed. It’s high-efficient, immediate, low-costing and maneuverable.2. The translocation of y-hexachlorocyclohexane dehydrochlorinase(LinA) fusions to the periplasm.The twin-arginine translocation (Tat) pathway was found in plant chloroplasts and prokaryotes with the conserved twin-arginine motif directly in the upstream of the protein, with a consensus sequence S/TRRxFLK. Compared with sec system, the heterologous protein can be exported to periplasm as a right folded protein by tat translocation pathway. TorA sequence is usually in the N-terminal of the protein,linA-encoded HCH dehydrochlorinase (LinA) mediates the first two steps of dehydrochlorination ofγ-HCH. Here we reported the exportation of the fusions LinA-EGFP (Enhanced Green Fluorescent Protein) to the periplasmic space by Tat pathway in Escherichia coli MC4100. A periplasmic expression vector pUTLAEG coding for TorA-LinA-EGFP was constructed. Functional periplasmic secretion of the fusions was demonstrated by cell fractionation, immunoblotting and fluorescence observation. The dehydrochlorinase activity of the periplasmic secretion, total cell lysate and whole cell was determined. It’s indicated that LinA-EGFP fusion can be exported to the periplasm of prokaryotes.