In vitro assembly and cellulolytic activity of a β-glucosidase-integrated cellulosome complex.

The cellulosome is a supramolecular multi-enzyme complex formed by protein interactions between the cohesin modules of scaffoldin proteins and the dockerin module of various polysaccharide-degrading enzymes. In general, the cellulosome exhibits no detectable β-glucosidase activity to catalyze the conversion of cellobiose to glucose. Because β-glucosidase prevents product inhibition of cellobiohydrolase by cellobiose, addition of β-glucosidase to the cellulosome greatly enhances the saccharification of crystalline cellulose and plant biomass. Here, we report the in vitro assembly and cellulolytic activity of a β-glucosidase-coupled cellulosome complex comprising the three major cellulosomal cellulases and full-length scaffoldin protein of Clostridium (Ruminiclostridium) thermocellum, and Thermoanaerobacter brockii β-glucosidase fused to the type-I dockerin module of C. thermocellum. We show that the cellulosome complex composed of nearly equal numbers of cellulase and β-glucosidase molecules exhibits maximum activity toward crystalline cellulose, and saccharification activity decreases as the enzymatic ratio of β-glucosidase increases. Moreover, β-glucosidase-coupled and β-glucosidase-supplemented cellulosome complexes similarly exhibit maximum activity toward crystalline cellulose (i.e. 1.7-fold higher than that of the β-glucosidase-free cellulosome complex). These results suggest that the enzymatic ratio of cellulase and β-glucosidase in the assembled complex is crucial for the efficient saccharification of crystalline cellulose by the β-glucosidase-integrated cellulosome complex.

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