Abstract
The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.
Highlights
Disruptive functions of carbohydrate-binding modules (CBMs) toward crystalline cellulose and their synergism with hydrolases are important
The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose
The r.m.s.d. values for the entire molecules of the chimeric cellulases fluctuated wildly and never converged perhaps because the two modules linked by the flexible linker could move with relatively little restriction in the same molecule. These results suggest that the moieties of FnCel5A and CBMs could retain their respective independent structures, and their relative spatial positions could be shifted relatively freely in the same chimeric cellulase molecule
Summary
Disruptive functions of carbohydrate-binding modules (CBMs) toward crystalline cellulose and their synergism with hydrolases are important. Several proteins and modules have disruptive functions toward crystalline cellulose via non-hydrolytic mechanisms [12, 13], including expansins [14], expansin-like proteins [15], swollenins [16], and carbohydrate-binding modules (CBMs) [17,18,19] They non-hydrolytically loosen, peel, split, or disrupt the packaging of crystalline cellulose and convert it into its amorphous form, which can be readily hydrolyzed by cellulase. They do not directly hydrolyze cellulose, they can significantly increase the efficiency of enzymatic hydrolysis as a moiety within a cellulase molecule, as a subunit within a cellulosome [19, 20], or as an independent component [21, 22]. By investigating the activities of these chimeric cellulases, we could investigate the characteristics of CBMs
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