This study evaluates the activity of a recombinant chitinase from the leaf-cutting ant Atta sexdens (AsChtII-C4B1) against colloidal and solid α- and β-chitin substrates. ¹H NMR analyses of the reaction media showed the formation of N-acetylglucosamine (GlcNAc) as the hydrolysis product. Viscometry analyses revealed a reduction in the viscosity of chitin solutions, indicating that the enzyme decreases their molecular masses. Both solid state ¹³C NMR and XRD analyses showed minor differences in chitin crystallinity pre- and post-reaction, indicative of partial hydrolysis under the studied conditions, resulting in the formation of GlcNAc and a reduction in molecular mass. However, the enzyme was unable to completely degrade the chitin samples, as they retained most of their solid-state structure. It was also observed that the enzyme acts progressively and with a greater activity on α-chitin than on β-chitin. AsChtII-C4B1 significantly changed the hyphae of the phytopathogenic fungus Lasiodiplodia theobromae, hindering its growth in both solid and liquid media and reducing its dry biomass by approximately 61%. The results demonstrate that AsChtII-C4B1 could be applied as an agent for the bioproduction of chitin derivatives and as a potential antifungal agent.

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 Atta sexdens were cloned, expressed in Pichia pastoris, 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 Candida albicans and Aspergillus fumigatus, as well as the phytopathogen Lasiodiplodia theobromae. Distinct inhibition profiles were observed: AsChtII-C5B1 (150 µg/mL) showed the highest activity against C. albicans (87.6% inhibition), while AsChtII-C3C4 (25 µg/mL) was most effective against A. fumigatus (60% inhibition). Notably, only AsChtII-C2B3 inhibited L. theobromae growth, inducing severe hyphal deformations observed by scanning electron microscopy (SEM). These findings demonstrate that recombinant A. sexdens chitinases exhibit species-specific antifungal properties, underscoring their potential as biotechnological tools for medical and agricultural applications.