The Impact of Chitinase Binding Domain Truncation on the Properties of CaChi18B from Chitinilyticum aquatile CSC-1

The chitinase binding domain (ChBD) plays a crucial role in the properties of enzymes. To assess its impact, we cloned a truncated mutant of the chitinase gene <i>CaChi18B</i> from the novel chitinase-producing facultative anaerobic bacterium <i>Chitinilyticum aquatile</i> CSC-1, designated as <i>CaChi18B_ΔChBD_s</i>. The recombinant chitinase was successfully expressed and purified, exhibiting a specific activity of 3.48 U/mg on colloidal chitin, with optimal conditions at 45 °C and pH 6.0, and retaining over 80% activity at temperatures up to 40 °C. Kinetic analysis revealed that the <i>K_m</i> value was 1.159 mg mL^-1 and the <i>V_max</i> was 10.37 μM min^-1 mg^-1. Compared to <i>Ca</i>Chi18B_ΔChBD₁, which has only the first ChBD truncated at the N-terminus, <i>Ca</i>Chi18B_ΔChBD_s exhibited minor changes in the optimal temperature and pH, while the <i>K_m</i> and <i>V_max</i> values increased significantly. <i>Ca</i>Chi18B_ΔChBD_s exhibited tolerance to various metal ions, with K⁺ and NH₄⁺ enhancing activity, while Cu²⁺ significantly inhibited it. Most organic reagents had minimal impact, except for formic acid, which severely reduced activity. The primary hydrolysis product in the initial phase was GlcNAc, contrasting with (GlcNAc)₂ for <i>Ca</i>Chi18B_ΔChBD₁. These findings indicated that the ChBD influences the enzyme's <i>K_m</i>, <i>V_max</i>, and product distribution, enhancing our understanding of ChBD's roles and advancing chitin utilization.