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

Abstract

Polycatalytic enzyme complexes made by immobilization of industrial enzymes on polymer- or nanoparticle-based scaffolds are technologically attractive due to their recyclability and their improved substrate binding and catalytic activities. Herein, we report the synthesis of polycatalytic complexes by the immobilization of nonprocessive cellulases on the surface of colloidal polymers with a magnetic nanoparticle core and the study of their binding and catalytic activities. These polycatalytic cellulase complexes have increased binding affinity for the substrate. But due to their larger size, these complexes were unable to access to the internal surfaces of cellulose and have significantly lower binding capacity when compared to those of the corresponding free enzymes. Analysis of released soluble sugars indicated that the formation of complexes may promote the prospect of having consistent, multiple attacks on cellulose substrate. Once bound to the substrate, polycatalytic complexes tend to remain on the surface with very limited mobility due to their strong, multivalent binding to cellulose. Hence, the overall performance of polycatalytic complexes is limited by its substrate accessibility as well as mobility on the substrate surface.

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.…”

“…Both EG and CBH consist of a catalytic domain (CD) and a cellulose binding domain (CBD), the latter improving their adsorption onto insoluble cellulose polymers for efficient hydrolytic reactions. − Their action mechanisms, however, are considerably different. Tr Cel7A acts on the cellulose chains like a molecular motor, having a continuous, processive motion for many cycles of reactions. ,,,− In contrast, Tr Cel5A may dissociate from the substrate before proceeding to the next cycle of reaction, although a certain extent of “pseudo-processivity” may exist. , …”

“…2,8,10,17−19 In contrast, TrCel5A may dissociate from the substrate before proceeding to the next cycle of reaction, although a certain extent of "pseudoprocessivity" may exist. 20,21 The complex reactions involving multiple, synergistic cellulases at liquid−solid interfaces are not completely understood. Therefore, much effort has been made to quantify the action mechanisms and kinetic behaviors of individual cellulases and their mixtures, for example, by using a variety of bulk assays, single-molecule imaging analyses, and surfacebased sensing techniques.…”

Tuning Enzyme/α-Zr(IV) Phosphate Nanoplate Interactions via Chemical Modification of Glucose Oxidase

“…60–63 Several previous studies have been used. The various immobilization strategies on the conventional Au sensor include the self-assembled (SAM) molecular monolayers, 64 various functional polymers, 65,66 targeted immobilization of bioreceptors, 61–63 gold nanoparticles, quantum dots, 67 and other highly refractive graphene based plasmonic materials, 68,69 which basically improves the sensitivity by means of both surface area and plasmonic properties material. In this study, we spin-coated the α -ZrP onto the Au sensor to get more uniform thin films, which facilitated the more reproducible SPR experiments.…”

“…Surface plasmon resonance (SPR) is a powerful label free and ultrasensitive technique to probe the live monitoring of biomolecular interactions occurring in extremely close vicinities of sensors where the environment is very sensitive to refractive index change of the sensing medium. − Several previous studies have been used. The various immobilization strategies on the conventional Au sensor include the self-assembled (SAM) molecular monolayers, various functional polymers, , targeted immobilization of bioreceptors, − gold nanoparticles, quantum dots, and other highly refractive graphene based plasmonic materials, , which basically improves the sensitivity by means of both surface area and plasmonic properties material. In this study, we spin-coated the α-ZrP onto the Au sensor to get more uniform thin films, which facilitated the more reproducible SPR experiments.…”