A QCM-D-based continuous assay for the study of the enzymatic degradation of insoluble calcium-polygalacturonate

Pectin is a major component of plant cell walls and constitutes a significant fraction of dicot plant materials. Pectin primarily consists of linear stretches of α-1,4-linked galacturonic acid residues, and is present in plant cell walls as insoluble calcium-crosslinked hydrogels. Most of the enzyme assays developed to date to follow pectin degradation by enzymes are based on using soluble pectic substrates, notably polygalacturonic acid in solution, and therefore lack the specificities of the solid-liquid interface. In this work, we developed a continuous assay to assess the enzymatic degradation of insoluble calcium-polygalacturonate. The substrate was immobilized on the surface of quartz sensors for a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and the assay involves using these functionalized sensors for enzymatic rate measurements. We show that the enzymatic rates respond in a dose-dependent manner to the frequency increase during QCM-D measurements and demonstrate that such an assay can detect enzyme attack characteristics that a colorimetric assay using soluble polygalacturonic acid cannot. This QCM-D based assay can be useful for better selection, characterization, and engineering of enzymes for action on insoluble plant matrices.