STUDIES ON BIOREMEDIATION POTENTIAL OF HYDROLASE FAMILY ENZYMES

Abstract

Bioremediation, particularly enzyme-mediated remediation, is the most sensitive, eco-friendly, and cost-effective approach to eliminate toxic pollutants from the environment. Among the various enzyme classes, 'Hydrolase' emerges as a crucial group catalyzing the hydrolysis of toxic contaminants. Hydrolases further fall into sub-classes like phosphatases, esterases, proteases, lipases, dehalogenases, and nucleases, offering a versatile range of solutions for different classes of pollutant remediation. Hydrolases from different sources, including microorganisms, plants, and insects, have the ability to break down pesticide residues into harmless byproducts through various biochemical reactions. Pesticides, which were a boon, have now turned into a curse to mankind and the environment. Strategic and scientifically guided pesticide application proved advantageous, acting as a blessing. Conversely, the widespread and imprudent use has resulted in the gradual deterioration of the ecosystem. The concerns have extended to the trophic levels and have resulted in a global imbalance of resources and life forms. The urgent demand of the situation is to adopt green technology to detect and detoxify long-standing pesticide accumulations. Engineering these enzymes into enzyme-based tools such as biosensors enables swift detection with uncomplicated instrumentation and high efficacy. Additionally, the customization of enzymes into nanostructures enhances stability and catalytic efficiency through the immobilization of enzymes on biodegradable nanofibers. The first hydrolase enzyme considered in our study is carboxylesterase (CarEs, EC 3.1.1.1), belonging to the sub-class esterase. Insects utilize CarEs as phase-I detoxifying agents, proficiently breaking down a range of xenobiotic molecules, including insecticide, insect and plant volatiles, insect growth hormones and pheromones. However, high genomic plasticity and the intense selection pressure from the overuse of insecticides have contributed to the emergence of insecticide resistance in pests such as Helicoverpa armigera. Consequently, an investigation of the functional expression and an in-depth characterization signifying CarE-mediated insecticide detoxification has been conducted. Additionally, the enzyme was immobilized on nanofibers to enhance its stability. This research not only provided insights into a pivotal enzyme but also played a significant role in the fabrication of enzyme-mediated biosensor. Consequently, it laid the foundation for future research on the development and practical applications of insecticide-resistant enzymes, addressing a diverse range of chemical remediation.The second analysis conducted adds to the study of another hydrolase enzyme associated with the haloacid dehalogenase superfamily (HADSF), sourced from the bacterium. The HADSF is one of the largest superfamilies, with 479,051 sequences deposited in databases (InterPro IPR023214). Notably, these ubiquitous enzymes are reported in the genomes of 33 different families encoding essential proteins having diversified functions. The acid hydrolase group of HADSF is well-known to catalyze phosphate hydrolysis and phosphoryl transfer reactions; it includes P-type ATPase, phosphoserine phosphatase, phosphomannomutase, and L-2-haloacid dehalogenase. Several studies have underscored the potential of haloacid dehalogenases (HAD) in bioremediation. Consequently, one such enzyme from Staphylococcus lugdunensis was identified and characterized to assess its functional and structural attributes. Furthermore, site-directed mutagenesis was performed to obtain better insights into the activity and explore the unique properties of the bacterial HAD enzyme. The present research focuses on two hydrolase proteins, specifically carboxyl/cholinesterase (Ha006a) and haloacid dehalogenase (SLHAD1) enzymes. Helicoverpa armigera, a worldwide pest, is the source of the first hydrolase enzyme, Ha006a. The second enzyme, SLHAD1, is sourced from Staphylococcus lugdunensis, an opportunistic human pathogen. The goal of investigating the potential of these enzymes for pesticide bioremediation was accomplished through their effective cloning, over-expression, and purification. The enzymes were subjected to biochemical, biophysical, bioinformatics, and structural investigations using a systematic approach. Furthermore, the enzyme was immobilized on nanofibers, thereby modifying the enzyme's stability and catalytic effectiveness. Additionally, the electroanalytical biosensor for pesticide detoxification was designed using the enzyme Ha006a. Moreover, a screening process using in silico approach was implemented to identify potent inhibitors of Ha006a protein which could be employed as synergists with organophosphate (OP) insecticides. Following the characterization of the phytochemical inhibitors, biochemical and biophysical methods were used to confirm the interaction. In the case of SLHAD1, the crystal structure was obtained at a resolution of 1.7 Å. The investigations also unveiled unique characteristics, such as the time-dependent self-cleaving property of SLHAD1, suggesting potential regulatory implications. Moreover, the sequence and functional analysis showed SLHAD1, belonging to HAD subfamily II, to be a magnesium-dependent acid phosphatase with a broad substrate specificity and a possible role in thiamine metabolism. Thus, the thesis is organized into five chapters, encompassing the various aspects of the study.CHAPTER 1 includes a detailed explanation of the essential role played by enzymes, specifically hydrolases, in bioremediation. An elaborate discussion on enzyme-mediated bioremediation, mainly highlighting the mechanism of action of esterases and haloacid dehalogenases in the remediation of toxic compounds, is mentioned. Additionally, a comprehensive literature review to investigate pesticide consumption worldwide, further revealing the recent data on pesticide consumption in India, is elaborated. Moreover, pesticide classification has been explained in detail. Subsequently, the adverse consequences associated with their usage are also discussed. CHAPTER 2 contains the cloning, over-expression and purification of the carboxyl/cholinesterase gene (Ha006a) from an economically important pest, Helicoverpa armigera. The protein was purified using several chromatographic techniques and the pure protein exhibited a monomeric conformation of ~60kDa upon analysis by SDS-PAGE. The protein was meticulously characterized, employing several biochemical and biophysical methods. The esterase activity was monitored against a natural substrate, α-naphthyl acetate (α-NA). The optimum temperature, thermostability, optimum pH, and pH stability were evaluated using this substrate. The protein underwent structural analysis using Circular Dichroism spectroscopy. A total of six insecticides belonging to organophosphates (OP) and synthetic pyrethroid (SP) classes exhibited inhibitory responses towards the enzyme, thus showing its potential of remediating insecticides. Furthermore, pyrethroid insecticides were hydrolyzed efficiently in the presence of the enzyme, and their products were analyzed using the gas chromatography-mass spectrometry (GC-MS) technique. The Isothermal Titration Calorimetry was employed to evaluate the interaction studies of Ha006a with several OP insecticides to gain thermodynamic insights. Overall, this chapter provides an overview of the characterization of Ha006a, highlighting its capacity to sequester organophosphate pesticides and hydrolyze synthetic pyrethroids. CHAPTER 3 encompasses the practical application of the Ha006a enzyme for the detection of pesticides through the creation of an enzyme-mediated technique. The bioremediation approaches, i.e., biosensor and immobilization, were employed independently to detoxify the pesticides in a sustainable manner. To enhance esterase catalytic efficiency over a broader range of temperature and pH conditions, Ha006a was immobilized using the electrospinning technique. A systematic approach was employed to optimize electrospinning parameters, enzyme concentration, and nanofiber diameter for immobilization. Characterization of thenanofibers involved various techniques such as Field Emission Scanning Electron Microscopy, Fourier Transform Infrared Spectroscopy, Thermogravimetric Analyzer and X-ray Diffraction. Furthermore, a Ha006a-based biosensor was fabricated to detoxify OP insecticides from solution. The Ha006a-based electroanalytical biosensor exhibited significant sensitivity towards paraoxon ethyl, profenofos, and chlorpyrifos, effectively sequestering organophosphates. The results indicated an enhanced response in enzyme activity and stability after immobilization, highlighting its potential as a reliable tool for bioremediation applications. Thus, this chapter explains strategies for the development of insect enzyme-immobilized nanofibers and enzyme-mediated biosensors, unlocking their potential for diverse applications, including pesticide remediation and environmental protection. CHAPTER 4 describes the development of another effective intervention for pest management. It includes the screening of the FooDB library to identify potent inhibitory compounds targeting carboxylesterase, Ha006a. The compounds with the highest scores underwent evaluation through docking studies and pharmacophore analysis. Among them, four phytochemicals—donepezil, protopine, 3',4',5,7-tetramethoxyflavone, and piperine—demonstrated favorable binding affinity. The Ha006a-ligand complexes were subsequently validated through molecular dynamics simulations. Furthermore, biochemical analysis encompassing the determination of IC50 values was conducted, complemented by analysis of thermostability through Differential Scanning Calorimetry and interaction kinetics through Isothermal Titration Calorimetry. Thus, bioinformatics, biochemical, and biophysical methods were employed to characterize Ha006a-ligand complexes. This investigation highlights 3',4',5,7-tetramethoxyflavone as the most stable inhibitor, suggesting its potential for synergistic testing with OPs. Consequently, these inhibitors offer a promising solution to OP resistance and addresses environmental concerns associated with excessive insecticide usage, enabling a significant reduction in their overuse. CHAPTER 5 reports the characterization of one of the haloacid dehalogenase (HAD) superfamily proteins (SLHAD1) from Staphylococcus lugdunensis. The mechanisms underpinning enzyme activity involve assembling conserved motifs in the active site, forming the well-conserved hydrolase core domain, which resembles the α/β Rossmann fold. Hydrolases are well-known for their diverse function including bioremediation of several environmental pollutants and the execution of cellular metabolic processes. The functional analysis in this chapter revealed that SLHAD1 is a metal-dependent acid phosphatase, which catalyzes the dephosphorylation of phosphorylated metabolites of cellular pathways, including glycolysis, gluconeogenesis, nucleotides, and thiamine metabolism. Based on the substrate specificity and genomic analysis, the physiological function of SLHAD1 in thiamine metabolism has been tentatively assigned. The crystal structure of SLHAD1 has been determined at 1.7 Å resolution with a homodimer in the asymmetric unit. It was observed that SLHAD1 exhibited time-dependent cleavage at a specific point, occurring through a self-initiated process. A combination of bioinformatics, biochemical, biophysical, and structural studies explored the unique features of SLHAD1. Overall, the study revealed a detailed characterization of a critical enzyme of Staphylococcus lugdunensis, which is associated with several life-threatening infections and is significantly involved in several cellular pathways.