Enhancement of Cellulolytic Enzyme Activity by Clustering Cellulose Binding Domains on Nanoscaffolds

Cellulose, one of the most abundant carbon resources, is degraded by cellulolytic enzymes called cellulases. Cellulases are generally modular proteins with independent catalytic and cellulose‐binding domain (CBD) modules and, in some bacteria, catalytic modules are noncovalently assembled on a scaffold protein with CBD to form a giant protein complex called a cellulosome, which efficiently degrades water‐insoluble hard materials. In this study, a catalytic module and CBD are independently prepared by recombinant means, and are heterogeneously clustered on streptavidin and on inorganic nanoparticles for the construction of artificial cellulosomes. Heteroclustering of the catalytic module with CBD results in significant improvements in the enzyme’s degradation activity for water‐insoluble substrates. In particular, the increase of CBD valency in the cluster structure critically enhances the catalytic activity by improving the affinity for substrates, and clustering with multiple CBDs on CdSe nanoparticles generates a 7.2‐fold increase in the production of reducing sugars relative to that of the native free enzyme. The multivalent design of substrate‐binding domain on clustered cellulases is important for the construction of the artificial cellulosome, and the nanoparticles are an effective scaffold for increasing the valence of CBD in clustered cellulases. A new design is proposed for artificial cellulosomes with multiple CBDs on noncellulosome‐derived scaffold structures. Heteroclustering of recombinant cellu‐lase modules on streptavidin and nanoparticles results in significant improvements in the enzyme’s degradation activity for water‐insoluble substrates. The clustering of multiple cellulose‐binding domains (CBDs) on CdSe nanoparticles generates a significant increase in activity relative to that of the native free enzyme. A new design for artificial cellulosomes with multiple CBDs on noncellulosome‐derived scaffold structures is proposed.