BIOPHYSICAL CHARACTERIZATION OF CHI18-5, A CHITINASE RESPONSIVE TO pH AND SOLVENT CONDITIONS

Chitinases from Trichoderma spp. have attracted considerable attention due to their relevance in diverse biotechnological applications, including agriculture, medicine, and waste bioprocessing. In this work, we report the cloning, recombinant expression, and purification of Chi18-5, a glycoside hydrolase family 18 (GH18) chitinase from Trichoderma atroviride, and its structural and functional characterization. The enzyme hydrolyzes chitin and chitosan to produce chitooligosaccharides (COS) and N-acetylglucosamine (NAG), compounds with notable industrial potential.

We performed a comprehensive analysis of its stability and conformational dynamics using circular dichroism (CD), fluorescence spectroscopy, and ATR-FTIR. The enzyme exhibits a clear pH-dependent conformational switch, shifting from a compact, inactive structure at neutral pH to a more flexible and catalytically active state under acidic conditions (pH 4–5). This behavior was further supported by Langmuir monolayer studies, where Chi18-5 showed differential interfacial properties and insertion capacity depending on pH, consistent with a structural activation mechanism at acidic pH.

Additionally, preliminary results from ongoing studies show that Chi18-5 retains its secondary structure and enzymatic activity in the presence of ethylene glycol, in contrast to other water-miscible solvents such as isopropanol or acetonitrile, which promote unfolding or aggregation.

These findings provide insights into the structural adaptability of GH18 chitinases and support their formulation and stabilization under non-physiological conditions. Current efforts are focused on demonstrating the enzyme’s biocontrol potential in different biological models, aiming to expand its application scope.

This study was supported by CEPROCOR and CONICET. IDB and SRS are Researchers at CONICET. FER and RR are doctoral and professional training Fellows of CEPROCOR, respectively.