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

Abstract

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 CaChi18B from the novel chitinase-producing facultative anaerobic bacterium Chitinilyticum aquatile CSC-1, designated as CaChi18B_ΔChBD_s. 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 K_m value was 1.159 mg mL^-1 and the V_max was 10.37 μM min^-1 mg^-1. Compared to CaChi18B_ΔChBD₁, which has only the first ChBD truncated at the N-terminus, CaChi18B_ΔChBD_s exhibited minor changes in the optimal temperature and pH, while the K_m and V_max values increased significantly. CaChi18B_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 CaChi18B_ΔChBD₁. These findings indicated that the ChBD influences the enzyme's K_m, V_max, and product distribution, enhancing our understanding of ChBD's roles and advancing chitin utilization.