In Situ Monitoring of Cellulase Activity by Microgravimetry with a Quartz Crystal Microbalance

Abstract

Quartz crystal microgravimetry (QCM) was used to investigate the interactions between cellulase enzymes and model cellulose substrates. The substrates consisted of thin films of cellulose that were spin-coated onto polyvinylamine (PVAm) precoated quartz crystal sensors carrying conductive gold surfaces. In QCM the quartz crystals are piezoelectrically driven and the frequency and dissipation shifts allow monitoring of substrate hydrolysis at various temperatures and enzyme concentrations in situ and in real time. The changes in frequency of cellulose-coated quartz resonators during their incubation in cellulase solutions were related to contributions from the liquid phase properties, the adsorptions of cellulase enzymes, and the hydrolysis of the substrate. Cellulase adsorption was found to be nonspecific and irreversible on gold-, PVAm-, and cellulose-coated quartz crystal sensors. The contribution to frequency shifts due to the bulk fluid properties of the cellulase solutions (at concentrations lower than 0.5 mg/mL) was minimal compared to the frequency shifts produced by cellulase binding. The maximum frequency decreases were fitted to a Langmuir model. The adsorption constant and the maximum adsorption were estimated by the fitting parameters of this model. The hydrolysis process was modeled by using a dose-response model that was then used to estimate the maximum hydrolysis rate, to compare the relative effects of temperature on adsorption and hydrolysis rate, and to obtain the apparent activation energy of cellulose hydrolysis. The hydrolysis rate increased with incubation temperature while apparent adsorption decreased. The apparent activation energy for the hydrolysis of the cellulose films employed was calculated to be 37 kJ/mol.