Glycerol has drawn increasing attention as a possible fuel, because it has many desirable qualities and is abundant due to the fact that it is a byproduct of biodiesel production. Previous research has shown that non-natural enzyme cascades can be used to create a bioanode that can stepwise oxidize glycerol to carbon dioxide. Two of these enzymes are pyrroloquinoline quinone (PQQ) dependant alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) derived from Gluconobacter. The third enzyme, which is responsible for carbon bond cleavage, is oxalate oxidase (OxOx) derived from barley. Previous research has shown that all three enzymes have demonstrated the ability to undergo direct electron transfer to a carbon electrode which allows for a simple and efficient bioanode that completely oxidizes glycerol. In this study, each enzyme was individually immobilized within modified Nafion(R) on a glassy carbon rotating disc electrode (GC-RDE) and voltammetric analysis was performed employing different rotation rates in a solution containing each enzyme's respective substrate. This substrate was glycerol for alcohol dehydrogenase, glyceraldehyde for aldehyde dehydrogenase, and mesoxalic acid for oxalate oxidase. From the voltammograms, Levich plots were produced and the solution diffusion coefficient (D-soln). the membrane diffusion coefficient (D-film), k(CAT), K-M, and V-MAX were determined. (C) 2009 Elsevier Ltd. All rights reserved.