Bi-enzyme alcohol biosensors based on genetically engineered alcohol oxidase and different peroxidases

We report on the development of a bi-layer bi-enzyme biosensor architecture using different peroxidases and alcohol oxidase from Hansenula polymorpha C-105 as biological recognition elements. The sensor architecture comprises a first layer containing either horseradish peroxidase or royal palm tree peroxidase crosslinked with an Osmium complex-modified redox hydrogel. On top, a second layer was formed by electrochemically induced precipitation of a cathodic electrodeposition paint simultaneously entrapping alcohol oxidase isolated from a genetically modified strain of Hansenula polymorpha C-105. The sensor architecture was optimized with respect to effective electron transfer and stability of the enzyme. The main characteristics of the biosensors are an apparent maximal current I-max(app) of 572-940 nA, an apparent Michaelis constant K-M(app) of 9.5 mM, a sensitivity of 60-98 nA mM(-1) and an improved operational stability represented by a deactivation constant of 1.5-2.0 x 10(-4) min(-1).