Functional roles of dual chitin-binding domains in Chitiniphilus eburneus chitinase properties

Chitinase catalyzes the β-(1,4) glycosidic bonds in chitin, demonstrating antifungal and insecticidal properties. Some chitinases contain multiple chitin-binding domains (ChBDs), which enhance catalysis on α-chitin. However, the specific roles of each ChBD in chitinase function remain poorly understood. Herein, Chitiniphilus eburneus Ce0303 and ChBD-truncated chitinases were expressed to investigate the contribution of its ChBDs to binding, stability, and catalytic properties. Conserved aromatic residues including Trp-5, Tyr-19, Trp-119 and Trp-132 within ChBDs are crucial for substrate binding. The interaction between substrate and ChBD1 is primarily mediated by hydrogen bonds, while ChBD2 relies predominantly on hydrophobic interactions. Truncated chitinase Chi-2 containing ChBD1 exhibited enhanced catalytic efficiency toward α-chitin, with specific activity of 2.62 U mg^-1 and k_cat/K_m value of 1.16 mL mg^-1 s^-1, whereas Ce0303 and truncated Chi-1 containing ChBD2 demonstrated superior thermal stability, with half-lives of 3.36 and 3.22 h at 55 °C, respectively. ChBDs reduced the crystallinity of α-chitin to as low as 70.55 %, thus enhancing catalytic domain's accessibility, while the endo- and exo-catalytic activities of Ce0303 remained unchanged. This study advances the understanding of ChBDs function, providing a theoretical foundation for improving α-chitin catalysis and expanding the applications of chitinase in agriculture and aquaculture.