Adsorption and Hydrolytic Activity of the Polycatalytic Cellulase Nanocomplex on Cellulose

Abstract

The formation of polycatalytic enzyme complexes may enhance the effectiveness of enzymes due to improved substrate interaction and synergistic actions of multiple enzymes in proximity. Much effort has been made to develop highly efficient polycatalytic cellulase complexes by immobilizing cellulases on low-cost polymer or nanoparticle scaffolds, aiming at their potential applications in biomass conversion to fuels. However, some key cellulases carry out the hydrolytic reaction on crystalline cellulose in a directional, processive manner. A large, artificial polycatalytic complex is unlikely to undergo a highly coordinated motion to slide on the cellulose surface as a whole unit. The mechanism underlying the activity enhancements observed in some artificial cellulase complexes and the limit of this approach remain elusive. Herein, we report the synthesis of polycatalytic cellulase complexes bound to colloidal polymer nanoparticles with a magnetic core and describe their unique adsorption, hydrolytic activities, and motions on cellulose. The polycatalytic clusters of cellulases on colloidal polymers show an increased rate of hydrolytic reactions on cellulose, but this was observed mainly at relatively low cellulase-to-cellulose ratios. Enhanced efficiency is mainly attributed to increased local concentrations of cellulases on the scaffolds and their polyvalent interactions with cellulose. However, once bound, the polycatalytic complexes can only carry out reactions locally and are not capable of relocating to new sites rapidly due to their lack of long-range surface mobility and their extremely tight binding. The development of highly optimized polycatalytic complexes may arise by developing novel nanoscaffolds that induce concerted motion of the complex as a whole.

Kinetic Study on Enzymatic Hydrolysis of Cellulose in an Open, Inhibition-Free System

“…reesei on the surface of cellulose. The preparation and characterization of the cellulases (Cel7A and Cel5A) and cellulose films can be found in our previous publication and are briefly described in the Methods Section. , The cellulose film has a typical thickness of 80–100 nm and a crystallinity of ∼60%, as measured by AFM and wide-angle X-ray diffraction (Figure S1). The cellulose films were pre-equilibrated with the running buffer (50 mM sodium acetate, pH 5) before the SPR measurement.…”

“…All other chemicals were of analytical grade and purchased from Fischer Scientific. Cel7A and Cel5A were purified from the Celluclast mixture using GE FPLC equipped with ion exchange columns as described previously (see Supporting Information for Figure S1A). , The purity of Cel7A and Cel5A was verified using SDS-PAGE based on their molecular weights (Figure S1B) and by their specific activities against fluorogenic substrate 4-methylumbelliferyl β- d -cellobioside at 50 °C . The extinction coefficients of 78,800 and 78,000 M –1 cm –1 were used to determine the concentrations of Cel7A and Cel5A in solution, respectively.…”

“…20,46 The purity of Cel7A and Cel5A was verified using SDS-PAGE based on their molecular weights (Figure S1B) and by their specific activities against fluorogenic substrate 4-methylumbelliferyl β-D-cellobioside at 50 °C. 49 The extinction coefficients of 78,800 and 78,000 M −1 cm −1 were used to determine the concentrations of Cel7A and Cel5A in solution, respectively.…”

Enzymatic Activities of Polycatalytic Complexes with Nonprocessive Cellulases Immobilized on the Surface of Magnetic Nanoparticles

“…The MNPs were then modified with methacryloxy­propyl­trimethoxy­silane (MPS) to obtain MNP-MPS nanoparticles, and then poly­(methacrylic acid) (PMAA) layer on the MNP-MPS was synthesized by distillation–precipitation polymerization of methacrylic acid (MAA) in acetonitrile, with N , N ′-methylene­bis­(acrylamide) (MBA) as the cross-linker and 2,2′-azobis­(iso­butyronitrile) (AIBN) as the initiator. The detailed experimental procedures and analysis can be found in our previous studies , and will not be repeated here.…”

“…We are interested in understanding whether the formation of artificial polycatalytic complexes from endoglucanase will further improve their capability in producing soluble sugars, as a consequence of both improved substrate binding and the proximity of multiple enzymes that may act on cellulose substrate in a concerted manner. − Polycatalytic cellulase complexes made by immobilization of industrial cellulases on polymer or nanoparticle scaffolds are also technologically attractive due to the low cost and recyclability of these synthetic nanoscaffolds and the scale-up potential for biorefinery applications. − However, the relatively large, polycatalytic complexes could possess quite a different adsorption behavior and hydrolytic activity, in comparison to the corresponding free cellulases . First, the surface area of cellulose available for binding of the polycatalytic complexes may decrease considerably, as most of the internal surface area of pores may not be accessible to large complexes when the particle size is comparable to the average pore size. − Second, once bound, the polycatalytic complexes may tend to stay on the surface of cellulose as a consequence of multivalent contacts between the polycatalytic complex and the cellulose substrate. , Third, distinct reaction activities or product patterns may be observed in the polycatalytic complexes due to the proximity of enzymes present in these complexes and their collective actions .…”