Bioelectrocatalysis of Pyruvate with PQQ-dependent Pyruvate Dehydrogenase Becky L. Treu, Daria Sokic-Lazic, and Shelley D. Minteer * Department of Chemistry, Saint Louis University, St. Louis, Missouri 63103, USA Although pyruvate has not been considered as a fuel for an enzymatic biofuel cell, there are dehydrogenase enzyme capable of oxidizing pyruvate. This paper details the discovery of a pyrroloquinoline quinone-dependent pyruvate dehydrogenase (PQQ-PDH) in Gluconobacter species. A method was developed to isolate and purify PQQ-PDH from Gluconobacter, along with the characterization of the purified enzyme. It was found that the purified enzyme can undergo direct electron transfer at carbon electrodes surfaces, which allowed for its incorporation into a pyruvate biofuel cell. It was also found that PQQ-PDH lacks substrate specificity, which minimizes its usefulness in many sensor applications, but is advantageous for deep oxidation in biofuel cell anodes. Introduction Bioelectrocatalysis is the use of a biological entity to catalyze a redox reaction at an electrode. The biological entity employed as a catalyst could be an enzyme (protein), an organelle, or a complete living cell. Enzymes are efficient catalysts and due to their size, higher catalyst loading is possible, which typically results in higher volumetric catalytic activity. In addition, enzymes are not living entities and do not require media to maintain activity(1). Enzymatic bioelectrocatalysis can be broken down into two main types: mediated bioelectrocatalysis and direct bioelectrocatalysis(2). Direct bioelectrocatalysis involves the ability of the enzyme to directly transfer electrons with the electrode, whereas mediated bioelectrocatalysis involves the use of additional redox species that mediated the electron transfer between the enzyme and the electrode. For simplicity sake, researchers have been interested in direct bioelectrocatalysis, but this has been difficult due to the limitations of enzymes capable of this type of electron transfer, because most enzyme active site is buried in the protein. Several pyrroloquinoline quinone (PQQ)- dependent dehydrogenase enzymes have been shown to undergo direct electron transfer (DET); therefore, this class of enzymes has been of interest to bioelectrochemists. Literature has reported DET of PQQ-dependent glucose dehydrogenase(3, 4), glycerol dehydrogenase(5), alcohol dehydrogenase(6), aldehyde dehydrogenase(7), lactate dehydrogenase(8), and lipoxygenase(9). However, there are far fewer known PQQ- dependent enzymes compared to NAD(P)-dependent enzymes. Many of the PQQ- dependent enzymes that have been studied are isolated from the acetic acid family of bacteria, namely Gluconobacter and Acetobacter species. The Gluconobacter genome contains many currently unclassified PQQ-dependent dehydrogenases(10). Therefore, this paper was focused on the discovery of a pyruvate oxidizing PQQ-dependent dehydrogenase from Gluconobacter followed by the purification of this PQQ-dependent pyruvate dehydrogenase (PQQ-PDH). There are a variety of Gluconobacter species in the literature, so this paper details the differences in enzymatic activity of PQQ-PDH ECS Transactions, 25 (28) 1-11 (2010) 10.1149/1.3309672 ©The Electrochemical Society 1 Downloaded 05 Mar 2010 to 165.134.19.219. Redistribution subject to ECS license or copyright; see http://www.ecsdl.org/terms_use.jsp