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 per-oxidases and alcohol oxidase from Hansenula poly-morpha C-105 as biological recognition elements.The sensor architecture comprises a first layer containing either horseradish peroxidase or royal palm tree per-oxidase crosslinked with an Osmium complex-modified redox hydrogel.On top,a second layer was formed by electrochemically induced precipitation of a cathod-ic 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 1.5-2.0x10~(-4)min~(-1).