Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-D-glucosamine (GlcNAc) units. Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates.
Reference: “Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity”. P.E. Kidibule, P. Santos-Moriano, E. Jiménez-Ortega, M. Ramírez-Escudero, M.C. Limón, M. Remacha, F.J. Plou, J. Sanz-Aparicio, M. Fernández-Lobato. Microbial Cell Factories 17:47 (2018). doi:10.1186/s12934-018-0895-x