Purification, characterization, and in silico modelling of peroxidase from Lagenaria siceraria (bottle gourd) for biotechnological applications

Peroxidases are widely used oxidoreductase biocatalysts in oxidative transformation and environmental remediation. This study presents an integrated characterization of a previously uncharacterized peroxidase from Lagenaria siceraria (bottle gourd), including purification, biochemical and kinetic analysis, electrochemical behavior, azo dye decolorization, and in silico structural validation. The enzyme was purified by ammonium sulfate fractionation, dialysis, DEAE-cellulose chromatography, and sucrose density-gradient centrifugation, achieving 17.07-fold purification with 26.79% recovery. SDS-PAGE and MALDI-TOF analysis indicated a molecular mass of approximately 63 kDa. Maximum activity was observed at pH 5.0 and 60°C. The apparent Km values were 1.66 mM for guaiacol and 0.45 mM for H2O2. Electrochemical analysis confirmed redox activity during substrate oxidation. The enzyme achieved 53.3% decolorization of methyl red and 70.0% decolorization of methyl orange within 12 h. In silico modeling indicated a structurally organized, α-helix-rich architecture with favorable stereochemical properties. Overall, L. siceraria represents a promising plant-derived peroxidase source for wastewater treatment and green biocatalysis.