How to quantify enzyme activity and kinetics in "non-bulk" systems? An example through the enzymatic hydrolysis of hemicellulose thin films
Résumé
In the carbohydrate-based bioindustry, enzymes are often used in conditions where they have to work on solid surfaces and/or penetrate within structures that are locally highly concentrated. The effect of such physical constraints on the enzyme activity and kinetics is however poorly understood; mostly because following and quantifying the hydrolysis in such conditions is still a challenge. With this work, our intention is to provide a detailed characterization of an enzyme's activity when its substrate is both concentrated and immobilized at a solid interface. This is essentially done by monitoring the in-situ degradation of a thin film of a model hemicellulose using a Quartz Crystal Microbalance with Dissipation (QCM-D). The thin film is composed of a unique arabinoxylan, extracted from wheat bran, and that is chemically modified for covalently binding onto gold.1 The film is partly swollen by water, and its water content (hence its local dry concentration) can be tuned by partially removing the Larabinofuranosyl units that decorate the xylan chain.2 The film is then put into contact with a solution containing an endo-1,4-β-xylanase (NpXyn11A3 ), into the QCM-D cell, and the loss of mass in the film is followed with time as degradation occurs. Using mathematical models that are under development in our laboratory, we aim at converting the raw QCM-D data into kinetics curves that give the reaction rate as a function of the polymer concentration in the film. Such a procedure would allow us to accurately compare the behavior of the enzyme in a film with its "bulk" behavior; the latter having been characterized classically with a dilute solution of the same substrate. Our results should reveal precious indications about the effect of substrate immobilization and conformation/concentration on the action of an enzyme.