The deactivation behavior observed in enzymatic hydrolysis of cellulose substrate could be due to several different substrate and enzyme characteristics. A full understanding of the physical and structural changes in cellulose microfibrils during the hydrolysis is essential in figuring out the substrate characteristics which largely contribute to the rate limitation at longer hydrolysis durations. In this study, several different analytical techniques like Fourier transform Infrared (FTIR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and sum frequency generation (SFG) spectroscopy are used to investigate the physical changes which are apparent during cellulase action on Avicel, bleached softwood and bacterial cellulose. XPS showed that accessibility of cellulose microfibril surface by the enzymes could play bigger role in determining the hydrolysis rate compared to typical substrate characteristics like crystallinity and degree of polymerization (DP). Surface coverage of cellulose microfibrils by inactivated protein and/or the hydrolysis product generated during the longer hydrolysis period could have limited the exposure of cellulose chains to the binding sites of the enzymes. The cellulose source chosen for enzymatic hydrolysis could manifest difference in hydrolysis progression. SFG showed changes in meso-scale arrangement of cellulose microfibrils in bleached softwood and bacterial cellulose with the increasing hydrolysis duration, but this did not change the final reaction rate between different cellulose substrate. FTIR, SFG and XRD also confirmed that cellulose Iα is preferentially hydrolyzed by the enzymes in bacterial cellulose.

Physical and structural changes in cellulose microfibrils responsible for enzymatic hydrolysis rate deactivation studied by FTIR, XRD, XPS and SFG

249th National Meeting (2015)

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