Functional and Structural Insights into Phenoloxidase from Hermetia illucens: A Combined Biochemical and Computational Approach

Abstract

The enzyme phenoloxidase (PO) is an essential component in the immune system of insects, which is responsible for the rapid activation and encapsulation of microbial pathogens. This study focuses on the in vitro and in silico investigation of PO using 4th instar larvae of black soldier fly (BSF), Hermetia illucens. The preliminary assays confirmed the occurrence of PO in the H. illucens larval haemolymph with the highest affinity towards DL-dopa as the substrate within 5 min at a λmax of 480 nm. The relative quantification of the H. illucens PO (HiPO) gene in the larvae showed higher expression in the fat body (FB) compared to haemocyte lysate supernatant (HLS). The PO studied under control and stress-induced conditions showed decreased activity during starvation and increased activity during injury. In silico analysis using molecular docking of various substrates with the reference structure of PO showed the highest affinity towards DL-dopa and L-dopa, followed by tyrosine, dopamine, and catechol, respectively. Hemocyanin and tyrosinase as functional domains were observed in the amino acid sequence of HiPO with an N-glycosylation site. Molecular docking of HiPO revealed a strong affinity for DL-dopa, and molecular dynamics simulation studies for the HiPO-DL-dopa complex provided their functional insights. The findings offer biomedical applications by providing an insight into the innate immune mechanisms that can guide the development of novel antimicrobial, antioxidant, and immunomodulatory strategies. This would help to comprehend the relation between immunity and metabolism, positioning H. illucens as a resilient feed source that supports livestock health, maintaining food safety and quality.