Boosting Biocatalytic Efficiency: Engineering of Chitinase Chit33 with Chitin and Cellulose Binding Domains for Sustainable Chitin Conversion

Endochitinase Chit33 has shown great potential in converting chitin, a recalcitrant waste, into bioactive chitooligosaccharides (COS). This study evaluates how cellulose-binding domain (CBD) and chitin-binding domain (ChBD) affect the hydrolytic activity and product specificity of Chit33. Recombinant proteins were produced and isolated with a simple yeast extracellular medium concentration. The domain functionality was proved using chitin and cellulose supports. ChBD provided more stable immobilization than CBD but reduced the Chit33 activity. CBD enhanced the enzyme activity on both colloidal (α-/β-allomorphs) and crystalline chitin, doubling it on α-chitin, although not on their deacetylated forms. Besides, CBD increased the COS production from the colloidal forms of α-/β-chitin (by 30% and 85%, respectively) and expanded the product diversity from 1 to 9 <i>N</i>-acetylglucosamine units. In contrast, Chit33-ChBD predominantly yielded chitin tetrasaccharides. These findings highlight the importance of selecting appropriate binding domains to tailor product specificity, as polymerization and acetylation degrees directly impact the COS biological properties.