A cytochrome b-glucose dehydrogenase chimeric enzyme capable of direct electron transfer

The development of third generation biosensors depends on the availability of direct electron transfer (DET) capable enzymes. A successful strategy is to fuse a cytochrome domain to an enzyme to fulfil the function of a built-in redox mediator between the catalytic center and the electrode. In this study, we fused the cytochrome domain of Neurospora crassa CDH IIA (NcCYT) N-terminally to glucose dehydrogenase from Glomerella cingulata (GcGDH) to generate the chimeric enzyme NcCYT-GcGDH in a large amount for further studies. Heterologous expression in P. pastoris and chromatographic purification resulted in 1.8 g of homogeneous chimeric enzyme. Biochemical and electrochemical characterization confirmed that the chimeric enzyme is catalytically active, able to perform interdomain electron transfer (IET) and direct electron transfer (DET) via the fused cytochrome domain. The midpoint redox potential of the fused b-type cytochrome is 91 mV vs. SHE at pH 6.5 and the specific current obtained on a porous graphite electrode is 2.3 microA cm^-2. The high current obtained on this simple, unmodified electrode at a rather low redox potential is a promising starting point for further optimization. The high yield of NcCYT-GcGDH and its high specific activity supports the application of the chimeric enzyme in bioelectrocatalytic applications.