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

## TL;DR

Researchers characterized antifungal chitinases from leaf-cutter ants, showing they can inhibit human and plant pathogens, suggesting potential medical and agricultural uses.

## Contribution

The study provides the first biochemical and antifungal characterization of chitinases from the leaf-cutter ant Atta sexdens.

## Key findings

- Recombinant chitinases from Atta sexdens showed optimal activity at pH 4–5 and 50 °C.
- AsChtII-C5B1 inhibited Candida albicans by 87.6%, while AsChtII-C2B3 inhibited Lasiodiplodia theobromae with hyphal deformations.
- AsChtII-C3C4 was most effective against Aspergillus fumigatus at low concentrations.

## Abstract

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.