Experimental methodology for evaluating enzyme kinetics and biocatalytic activity of antioxidant systems in pigmented Ficus leaves exposed to green-synthesized silver nanoparticles

Abstract

Plant-derived biomaterials provide a sustainable platform for the development of functional nanostructures and biocatalytic systems. In this study, the kinetic properties and biocatalytic efficiency of catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO) were evaluated in green and red leaves of Ficus benghalensis and Ficus amplissima using Michaelis–Menten kinetics. Red leaves of both species consistently exhibited lower Km values and comparable or higher Vmax values than green leaves, indicating enhanced substrate affinity and catalytic efficiency. For catalase, the Km value decreased from 34.88 to 13.23 mM in F. benghalensis and from 21.47 to 10.44 mM in F. amplissima in red leaves. Similarly, peroxidase activity in red F. amplissima leaves showed a higher Vmax (µmol min⁻¹ mg⁻¹ protein) than green leaves (17.27 µmol min⁻¹ mg⁻¹ protein) demonstrating improved catalytic turnover. Phytochemical analysis revealed elevated levels of phenolics, flavonoids, tannins, alkaloids, and saponins in red leaves, while elemental profiling confirmed the presence of Fe, Cu, Mn, and Zn, which support enzymatic activity. FTIR analysis confirmed the presence of hydroxyl, carbonyl, amide, and aromatic functional groups associated with phenolics, flavonoids, proteins, and polysaccharides, indicating their role in biocatalytic activity and nanoparticle synthesis . Leaf extracts were further utilized as reducing and stabilizing agents for the green synthesis of AgNPs. The synthesized AgNPs exhibited characteristic surface plasmon resonance (SPR) peaks between 413 and 443 nm, confirming nanoparticle formation. AgNPs showed enhanced biological activity compared with crude extracts, with DPPH radical scavenging activity reaching 81.16% in red F. benghalensis leaves and antibacterial activity producing inhibition zones up to 14.9 mm against Enterococcus . Summarized F. benghalensis exhibited stronger antioxidant enzyme activity, whereas F. amplissima demonstrated higher biomass yield and peroxidase catalytic efficiency. These findings identify pigmented Ficus leaves as effective biomaterials for sustainable AgNP synthesis and enzyme-based biocatalytic applications in biotechnology, agriculture, and environmental systems.