Molecular characterization of thermostable chitinases from Indian Pleurotus ostreatus and their effectiveness against the plant pathogenic Alternaria solani and Fusarium oxysporum

Chitinase has diverse applications across agriculture, energy production, food processing, pharmaceuticals, and human healthcare. Characterizing chitinase is essential for understanding its potential uses in these fields. This study reported the synthesis and characterization of chitinases from three different cultures of Pleurotus ostreatus, designated as KUFC112, KUFC113, and KUFC114. Evaluated their effectiveness against the plant pathogenic fungi Alternaria solani and Fusarium oxysporum. Enzyme yield and activity were optimized using response surface modelling. Chitinases purified through DEAE-cellulose column chromatography exhibited stability at elevated temperatures (up to 90 °C), pH range 4–8, in the presence of metals (Ca²⁺, Fe²⁺, Mg²⁺, and Mn²⁺), sodium dodecyl sulfate, and β-mercaptoethanol. The full-length Chi1 gene (1188 bp) obtained from all three cultures was cloned and encoded 395 amino acids. The amino acid sequences of CHI1 protein from KUFC112 and KUFC113 were identical, whereas that of KUFC114 had differences at the amino acid positions 321 (D in P. ostreatus KUFC112/V in P. ostreatus KUFC114), 329 (A/T), and 351 (T/S). Calculated molecular weight and the extinction coefficient of all three CHI1 proteins were estimated as 42.90 kDa and 52370 M⁻¹cm^−1, respectively. The phylogenetic tree based on deduced amino acid sequences of CH1 confirmed the relatedness of all three isolates with reported P. ostreatus strains. The predicted 2D structure of CHI1 of KUFC112/KUFC113 contained 11 α-helices and 12 β-sheets, whereas KUFC114 contained 12 α-helices and 11 β-sheets. The 3D structure of the CH1 proteins was developed using the template structure of endochitinase (Uniport: P29029) and validated. Through molecular simulation, the stability or flexibility of proteins was evaluated.