Bi-Enzyme Alcohol Biosensors Based on Genetically Engineered Alcohol Oxidase and Different Peroxidases

摘要

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 Imax app of 572–940 nA, an apparent Michaelis constant KM 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 × 10−4min−1.