Processive cellulases such as Trichoderma reesei cellobiohydrolase I (TrCel7A) is a two-domain enzyme consisting of a small cellulose-binding domain (CBD) and a catalytic domain (CD). The two domains of the cellulase work synergistically at liquid-solid interfaces and carry out the hydrolytic reactions in a processive manner - i.e., the enzyme remains bound and “slide” along the polysaccharide chain that has been previously separated from the crystalline cellulose lattice. Due to the complexity of the molecular mechanism, quantitative analysis of the binding and hydrolysis of processive cellulases on cellulose remains to be a challenge. Herein, we use surface plasmon resonance (SPR) spectroscopy to in-situ monitor the binding and hydrolysis processes of regenerated, model cellulose films by TrCel7A, under different enzyme concentrations and different experimental conditions, such as with continuous flow of enzyme solution, stopped flow of enzyme solution and replenishment of the flow cell with buffer after adsorption. Kinetic parameters were then obtained by achieving a “global” fit of the experimental data with a kinetic model. We also investigate the dependence of these adsorption, desorption and hydrolysis rates on the temperatures and discuss the implication of these results. The study may be useful for obtaining more quantitative understanding of enzymatic degradation of cellulose.

Kinetic study on enzymatic hydrolysis of model cellulose films using surface plasmon resonance

251st National Meeting (2016)

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