Effects of Linker Length and Flexibility on the Activity of GH18 Chitinases in Bacteria

Chitinases that play pivotal roles in chitin biodegradation for high-value bioconversion often adopt multimodular architectures with catalytic and binding domains connected by linkers. The linkers have been proven to possess functional roles, yet their mechanistic contributions remain poorly defined. In this study, a systematic analysis of 1242 bacterial glycoside hydrolase family 18 chitinases revealed a preferred linker length of 30–45 amino acids and an enrichment in threonine–proline (TP) motifs. Engineered Serratia marcescensSmChiC variants with controlled lengths (15–60 residues) demonstrated that a 45-residue linker (GS9) optimally enhances hydrolytic efficiency (22.4–39.8% activity increase) across α-/β-/colloidal chitins, with QCM-D confirming enhanced substrate binding/hydrolysis dynamics. Strikingly, engineered SmChiC variants with different rigidities demonstrated that the TP-linker variant outperformed the wild-type, flexible (GS9), and rigid (EK) variants, exhibiting rapid substrate association as revealed by HS-AFM. These findings highlight the critical role of linker design in optimizing chitinase function and provide a foundation for engineering enhanced glycoside hydrolases.