Biosensing of organophosphorus compounds using recombinant organophosphate degrading enzymes

Abstract

Agricultural advancements focusing on increasing crop production have led to excessive usage of insecticides and pesticides, resulting in leaching and accumulation of these highly toxic chemicals in soil, water, and food-chain. Organophosphorus (OP) compounds are the most commonly used insecticides and pesticides, which cause a wide range of long-lasting and life-threatening conditions. OP compounds were introduced as pesticides mainly in the 1950s and 1960s, and are widely used as insecticides, herbicides, nematicides as well as acaricides. Several derivatives of OPs are commercially marketed and they show prolonged half-life in the soil. For example, methyl parathion shows half-life in the soil for 25-130 days, whereas another OP compound, coumaphos, shows half-life in the soil for 24-1400 days (Singh and Walker 2006). Continuous or prolonged exposure of the OPs leads to deposition of these compounds at the human organ depot sites, from where they are uninterruptedly released into the blood. Subsequently, from blood, they reach the nervous system where one of their targets is the AChE enzyme. (Masson et al. 1998) Figure 1 represents the bioavailability and metabolism of OPs in humans. Once a person is exposed to OPs, these compounds may get deposited into sites like liver, kidney, muscles, adipose tissue or they may get distributed to the central nervous system via blood circulation. The amount that remains in the blood is eliminated out via urine, expired air, or in faeces (Masson et al. 1998; Paraíba et al. 2009). In addition to AChE, these compounds target some important enzymes like lipases and esterases that are crucial for proper functioning of the human body. Due to the acute toxicity and long-term side effects of OP compounds, their timely, on-the-spot, and rapid detection has gained importance for efficient healthcare management. In this respect, several OP degrading enzymes have gained the spotlight in developing enzyme-based biosensors, owing to their high activity and broad specificity. Among these enzymes, Organophosphorus acid anhydrolase (OPAA) and organophosphorus hydrolase (OPH) have emerged as a promising candidate for the detection of OP compounds, due to their ability to act on a broad range of substrates having a variety of bonds, like P-F, P-O, P-S, and P-CN, as well as chemical warfare agents and nerve agents (Kang et al. 2008). Enzymatic hydrolysis is reportedly 40-2450 times faster compared to chemical hydrolysis (Shimazu et al. 2003), and thus attracts great interest from a scientific viewpoint for the remediation as well as detection of organophosphorus compounds in the environment. The catalytic action of OPH and OPAA is shown in Figure 2. OPH and OPAA, in presence of metal ion (Vyas et al. 2010), hydrolyses methyl parathion and ethyl paraoxon into dimethyl thiophosphate and diethyl phosphoric acid, respectively, and a p-nitrophenol, a yellow-colored product, which can be detected calorimetrically (Fig. 2). This reaction also releases two protons, which eventually decreases pH and in conjunction with a pH-sensitive fluorophore or fluorescent protein like fluorescein isothiocyanate (FITC) and monomeric Teal Fluorescent Protein1 (mTFP1), respectively can be used for indirect fluorometric detection of OP substrates. There are several reports on the sensing of organophosphorus compounds using OPAA and OPH through electrochemical, optical, and other methods (Xiong et al. 2018). However, an optimal sensor that achieves good enzymatic catalytic activity and low enough detection limits to detect organophosphorus compounds in practical use cases remains elusive; primarily due to reproducibility and cost concerns. In this study, we report colorimetric, as well as fluorescence-based sensing using a known fluorescent dye FITC and fluorescent protein, mTFP1 of organophosphorus compounds, by the interaction of OPAA and OPH with OP compounds such as methyl parathion and ethyl paraoxon. Thus, the main objectives of this study are: 1. Expression and purification of recombinant organophosphorus acid anhydrolase (OPAA)-FL variant, organophosphorus hydrolase (OPH), and OPAA-FL-mTFP1 fusion protein in E. coli. 2. Development of recombinant OPAA-FL variant, OPH, and OPAA FL-mTFP1 fusion protein-based biosensors for detection of OP compounds. 3. Detection and quantification of pesticides using the recombinant OPAA-FL variant, OPH, and OPAA-FL-mTFP1 fusion protein based biosensor.