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.
Cellulosomes, which are assemblies
of cellulases with various catalytic
functions on a giant scaffoldin protein with a carbohydrate-binding
module (CBM), efficiently degrade solid cellulosic biomass by means
of synergistically coupled hydrolysis reactions. In this study, we
constructed hybrid nanocellulosomes from the biotinylated catalytic
domains (CDs) of two catalytically divergent cellulases (an endoglucanase
and a processive endoglucanase) and biotinylated CBMs by clustering
the domains and modules on streptavidin-conjugated nanoparticles.
Nanocellulosomes constructed by separately clustering each type of
CD with multiple CBMs on nanoparticles showed 5-fold enhancement in
cellulase degradation activity relative to that of the corresponding
free CDs, and mixtures of the two types of nanocellulosomes gradually
and synergistically enhanced cellulase degradation activity as the
CBM valency increased (finally, 2.5 times). Clustering the two types
of CD together on the same nanoparticle resulted in a greater synergistic
effect that was independent of CBM valency; consequently, nanocellulosomes
composed of equal amounts of the endo and endoprocessive CDs clustered
on a nanoparticle along with multiple CBMs (CD/CBM = 7:23) showed
the best cellulose degradation activity, producing 6.5 and 2.4 times
the amount of reducing sugars produced from amorphous and crystalline
cellulose, respectively, by the native free CDs and CBMs in the same
proportions. Our results demonstrate that hybrid nanocellulosomes
constructed from the building blocks of cellulases and cellulosomes
modules have the potential to serve as high-performance artificial
cellulosomes.
Molecular evolution was used to generate capping molecules that selectively bound to the noncellulose components in cellulosic biomass and facilitated access of cellulolytic enzymes to the substrate components. The peptides, which were selected by means of a phage-display method, strongly promoted the enzymatic degradation of cellulose components in the biomass.Scheme 1 Phage-displayed peptide library method used in this study. † Electronic supplementary information (ESI) available: Experimental procedures, adsorption isotherms against cellulose (Fig. S1), and peptide properties for identified biomass-binding peptides (Table S1). See
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