17-01: Application of electrochemical sensors in kinetic studies of cellulolytic enzymes

One of the challenges in fundamental understanding of enzymatic hydrolysis of cellulosic biomass lies in the limited selection of simple, fast and quantitative activity assays for cellulolytic enzymes. This is partly because of the experimental challenges associated with the insoluble and heterogeneous nature of cellulose but also because there is a shortage of assay technologies, particular continuous methods. We have shown that enzyme-modified electrochemical sensors can be utilized for continuous monitoring cellulase action on both model cellulose substrates and lignocellulosic biomass. In comparison with conventional assay methods the electrochemical measurements have the advantage of being free from the influence of optical properties of the reaction mixture and provide monitoring in real-time. In this presentation we compare sensors modified by enzymes with different specificities. One application of these enzyme sensors is that they can capture the transient kinetics for cellobiohydrolases acting on its insoluble substrate. The analysis of the transient regime of cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) by explicit modeling has provided insight into the rates of different steps in the enzymatic reaction. We also show how a novel continuous electrochemical assay for the activity of cellulases against soluble substrates may be used to distinguish the populations of productively- and non-productively bound cellulase on cellulose. We believe that these methods has potential in fundamental cellulase research to probe the molecular mechanism behind enzymatic cellulose degradation and open the door for rational protein engineering of more efficient cellulase variants to meet conditions in industrial applications.