Changes in the enzymatic hydrolysis rate of Avicel cellulose with conversion

Abstract

The slow down in enzymatic hydrolysis of cellulose with conversion has often been attributed to declining reactivity of the substrate as the more easily reacted material is thought to be consumed preferentially. To better understand the cause of this phenomenon, the enzymatic reaction of the nearly pure cellulose in Avicel was interrupted over the course of nearly complete hydrolysis. Then, the solids were treated with proteinase to degrade the cellulase enzymes remaining on the solid surface, followed by proteinase inhibitors to inactive the proteinase and successive washing with water, 1.0 M NaCl solution, and water. Next, fresh cellulase and buffer were added to the solids to restart hydrolysis. The rate of cellulose hydrolysis, expressed as a percent of substrate remaining at that time, was approximately constant over a wide range of conversions for restart experiments but declined continually with conversion for uninterrupted hydrolysis. Furthermore, the cellulose hydrolysis rate per adsorbed enzyme was approximately constant for the restart procedure but declined with conversion when enzymes were left to react. Thus, the drop off in reaction rate for uninterrupted cellulose digestion by enzymes could not be attributed to changes in substrate reactivity, suggesting that other effects such as enzymes getting "stuck" or otherwise slowing down may be responsible.

Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

“…Obstacle-based kinetic interpretations have been repeatedly suggested for cellobiohydrolases (12,20,25,(37)(38)(39) and recently also for processive endoglucanases (40), and the present results support the general validity of this approach. We emphasize, however, that obstacles have not yet been directly identified and that their structural origin remains obscure.…”

A steady‐state theory for processive cellulases

“…The conventional criteria for a constant reaction rate cannot be applied for cellulolytic enzymes because they exhibit a continuous slow-down (so-called non-linear kinetics) even if the substrate is plentiful and product inhibition is negligible. This behavior relies on factors that are not accounted for in Scheme 1, but the loss of activity generally develops much more slowly than the processes defined in Scheme 1 [4,[21][22][23][24]. It follows that the impact of these putative inactivation processes is low in experiments with short reaction times.…”

Evolution and impact of cellulose architecture during enzymatic hydrolysis by fungal cellulases

“…Restarting hydrolysis with fresh enzyme is an adequate experiment to measure the losses of substrate reactivity [35,[40][41][42]. It is still difficult to explain the decrease of the reaction rate vis a vis the evolution of morphological features of the substrate.…”