Functional Characterization and Antifungal Activity of Insect-Derived Chitinases Expressed in Pichia pastoris

Chitinases catalyze the hydrolysis of β-1,4-glycosidic bonds in chitin, a structural biopolymer synthesized by numerous organisms. Although these enzymes have been widely investigated, studies focusing on insect-derived chitinases remain limited. In this study, three recombinant chitinases from the leaf-cutter ant <i>Atta sexdens</i> were cloned, expressed in <i>Pichia pastoris</i>, and biochemically characterized. The enzymes-AsChtII-C2B3 (one catalytic and three chitin-binding domains), AsChtII-C3C4 (two catalytic domains), and AsChtII-C5B1 (one catalytic and one binding domain), exhibited optimal activity at pH 4-5 and 50 °C using colloidal chitin as substrate. Chitinase activity on colloidal α-chitin was confirmed by ¹H NMR (proton nuclear magnetic resonance) spectroscopy, revealing GlcNAc concentrations of 0.41, 0.48, and 0.56 mmol L^-1 for AsChtII-C3C4, AsChtII-C2B3, and AsChtII-C5B1, respectively. Their antifungal activities were evaluated against the human pathogens <i>Candida albicans</i> and <i>Aspergillus fumigatus</i>, as well as the phytopathogen <i>Lasiodiplodia theobromae</i>. Distinct inhibition profiles were observed: AsChtII-C5B1 (150 µg/mL) showed the highest activity against <i>C. albicans</i> (87.6% inhibition), while AsChtII-C3C4 (25 µg/mL) was most effective against <i>A. fumigatus</i> (60% inhibition). Notably, only AsChtII-C2B3 inhibited <i>L. theobromae</i> growth, inducing severe hyphal deformations observed by scanning electron microscopy (SEM). These findings demonstrate that recombinant <i>A. sexdens</i> chitinases exhibit species-specific antifungal properties, underscoring their potential as biotechnological tools for medical and agricultural applications.