Cellulose Thin Film Hydrolysis by Cellulase Enzymes in the Presence of Inhibitors – a QCM Study

Cellulose thin film hydrolysis by cellulase enzymes in the presence of inhibitors – a QCM study

Utshab Chakravorty, Barbara Knutson, Stephen E. Rankin, Sue Nokes

Abstract

Conversion of biomass to chemicals and biofuels using the products of cellulose hydrolysis is an important area of research for the production of the next generation of biofuels.  However, the saccharification and biochemical conversion of cellulose from lignocellulosic biomass is potentially limited by the release of inhibitors during the processing steps which are used to make the cellulose accessible.   Understanding the role and sensitivity of these inhibitors in altering the activity of cellulase enzymes is necessary to the design of pretreatment and separation approaches that maximize cellulose conversion.  Bulk studies of cellulose degradation do not provide sufficiently detailed time-dependent and concentration-dependent information to delineate the effect of inhibitors on the adsorption of cellulases to cellulose surface and the subsequent hydrolytic activity of the cellulases.  This study uses the frequency response of model cellulose thin films cast onto Quartz Crystal Microbalance (QCM) sensors to model the binding and  hydrolysis of cellulose by cellulases (from Trichoderma reesei)  in the presence of furfural and acetic acid.   The sensitivity of the frequency response during cellulose hydrolysis to the specific inhibitor and inhibitor concentration is demonstrated.  The concentration-dependent inhibition frequency response data are modeled using a reaction network that accounts for enzyme adsorption, hydrolysis, and several modes of enzyme inhibition and deactivation.  The model results can be used to interpret the mode of inhibition.  The rate constants of the binding and hydrolysis reactions determined by regression to sets of QCM data with varying inhibitor concentration indicate a strong binding affinity of the enzyme to the substrate and consistent rate coefficient for hydrolysis.  The QCM data are used to understand the possible roles of various modes of active site inhibition and inhibition of binding to cellulose on changes in overall conversion in the presence of the inhibitors.