Cellobiohydrolases are exoacting, processive enzymes that effectively hydrolyze crystalline cellulose. They have attracted considerable interest because of their role in both natural carbon cycling and industrial enzyme cocktails used for the deconstruction of cellulosic biomass, but many mechanistic and regulatory aspects of their heterogeneous catalysis remain poorly understood. Here, we address this by applying a deterministic model to real-time kinetic data with high temporal resolution. We used two variants of the cellobiohydrolase Cel7A from Hypocrea jecorina , and three types of cellulose as substrate. Analysis of the pre-steady-state regime allowed delineation rate constants for both fast and slow steps in the enzymatic cycle and assessment of how these constants influenced the rate of hydrolysis at quasi-steady state. Processive movement on the cellulose strand advanced with characteristic times of 0.15-0.7 s per step at 25 °C, and the rate was highest on amorphous substrate. The cellulose binding module was found to raise this rate on crystalline, but not on amorphous, substrate. The rapid processive movement signified high intrinsic reactivity, but this parameter had marginal influence on the steady-state rate. This was because dissociation and association were slower and, hence, rate limiting. Specifically, the dissociation from the strand was found to occur with characteristic times of 45-100 s. This meant that dissociation was the bottleneck, except at very low substrate loads (0.5-1 g/L), where association became slower.

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM(-1) cm(-2)), low detection limit (25 nM), and fast response time (t(95%) ≈ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β-anomer of cello-oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose.