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_ΔChBD(1), 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 NH(4)(+) enhancing activity, while Cu(2+) 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_ΔChBD(1). 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.