(260d) 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.