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

Abstract

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.

Origins of Activity Enhancement in Enzyme Cascades on Scaffolds

“…26À28 While the concept of increasing throughput by placing enzymes close together is intuitively appealing, 29,30 we show that, for freely diffusing reactants, cascade throughput is only enhanced in very large containers, such as mammalian cells 31 or the extracellular environment of the cellulosome, 32,33 and only for a limited time. Of course, cellular scaffolds often support enzymatic cascades in which reactants are not permitted to diffuse freely, 10,34 but recent engineered scaffolds aim to support reaction sequences with freely diffusing substrates.…”

Nanoscale Engineering of Designer Cellulosomes

“…[234,239] Parallel to these activities in the production of minicellulsomes, Nanosized platforms, based on a variety of different types of materials, are currently being assessed as potential cellulosomal scaffolds. Kim and colleagues [240] used the high affinity streptavidin-biotin binding pair to form clusters of organic nanoparticles with expressed CBM modules and cellulosome enzymatic domains. In some cases, streptavidin conjugated cadmium selenide nanospheres were used as a cellulosomal scaffold.…”

Size‐modulated synergy of cellulase clustering for enhanced cellulose hydrolysis

“…Unfortunately, the use of native cellulosomes for large‐scale biomass processing has not been economically viable because full‐length cellulosomes cannot be efficiently recovered [7]. Several approaches have emerged in recent years to immobilize cellulases onto non‐cellulosomal scaffolds to potentially improve the stabilities, storage properties, and enzyme synergies [8–11]. Among them, nanoparticles, which are known for their minimum diffusional limitation, maximum specific surface area, and effective enzyme loading, are ideal supports for cellulase immobilization.…”