IDENTIFICATION OF D. MELANOGASTER ENZYMES POTENTIALLY INVOLVED IN PENTACHLOROPHENOL DEGRADATION

M. CASTALDO^1,2, D.M. MONTI², A. TESTA³, J. VONTAS^4,5, D. TSAKIRELLI^4,5, M.M. MONTI¹, P.A. PEDATA¹ | ¹Institute for Sustainable Plant Protection (CNR-IPSP), Portici (NA), Italy; ²Department of Chemical Sciences, University of Naples “Federico II”, Naples, Italy; ³Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, Caserta, Italy; ⁴Pesticide Science Lab, Agricultural University of Athens, Athens, Greece; ⁵Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece

To date, microplastics and other environmental pollutants continue to cause increasing contamination of natural matrices. In this context, there is a growing need to identify new eco-friendly and cost-effective methods for their degradation. Enzyme-based degradation of environmental pollutants represents a promising strategy to mitigate toxic substances and promote their breakdown. Insects may serve as a valuable source of enzymes for biodegradation due to their well-developed detoxification systems. The aim of this study is to identify novel enzymes capable of degrading pentachlorophenol (PCP), using a Drosophila melanogaster strain resistant to this pollutant at 2000 ppm. RNA-seq analysis was performed on the resistant strain reared with and without PCP exposure. The analysis revealed several enzymes known to be involved in detoxification pathways. Among these, two cytochrome P450s (CYP6W1 and CYP6D5) and one glutathione S-transferase (GSTD2), showing fold changes of 8.95, 8.19, and 9.80, respectively, were selected for heterologous expression. All sequences were analyzed for single nucleotide polymorphisms (SNPs): only CYP6W1 harbored a missense SNP, which was included in the final sequence. Escherichia coli BL21 Star™ cells were co-transformed with plasmids encoding either Cyp6d5 or Cyp6w1, along with a plasmid encoding Musca domestica cytochrome P450 reductase (MdCPR). For GSTD2 expression, E. coli Lemo21 (DE3) cells were used. Following successful expression, bacterial membranes co-expressing DmCYP6D5–MdCPR and DmCYP6W1–MdCPR were isolated. Protein integrity and concentration were assessed using CO difference spectroscopy and CPR activity assays. All membrane preparations exhibited the characteristic CO-reduced spectrum, and CPR activity against cytochrome c was confirmed, indicating the presence of functional enzymes. For DmGSTD2, protein expression resulted in a high yield, with most of the protein present in the soluble fraction. The enzymatic activity of purified DmGSTD2 was tested against cumene hydroperoxide (CHP), yielding an activity of 0.46 μmol/min/mg protein and indicating a functional enzyme. To assess their potential for bioremediation, the enzymes will be tested for PCP degradation in both in vitro assays and in vivo through bacterial expression systems.