H202-forming NADHoxidase from Thermus thermophilus HB8 for cofactor recycling in biosensor applications: molecular cloning ofthe gene and its expression in E.coli

Abstract

Oxidoreductases represent a great potential for the construction of amperometric biosensors for the measurement of clinically and biotechnologically important substrates. In many enzymatic redox processes, NAD(P)t serves as cofactor and is consumed in stoichiometric amounts. The consumption of the cofactor makes the application economically unfeasible. Efficient recycling of the cofactor is therefore of great importance for the application of a lot of oxidoreductases in biosensors. Some dehydrogenases have been usedfor cofactor recycling in coupled enzyme reactions [1, 2]. However, additional substrates of these enzymes are again required for this type of cofactor regeneration. An attractive alternative was suggested by using the NAD(P)H oxidase (EC 1.6.99.3) which catalyzes the oxidation of NAD(P)H.This enzymeuses dioxygen from air as a substrate and reducesit with the formation of hydrogen peroxide [3]. It can be applied for the measurement ofsubstrates in amperometric enzyme electrodes which are enzymatically coupled to NAD(P)*- reducing dehydrogenases. The NAD(P)H oxidase from thermophilic bacteria is particularly interesting for the development of amperometric biosensors, since the high stability of proteins promises enzyme electrodes with a longer lifetime. We have recently reported the purification and some properties of an NADH oxidase from Thermus thermophilus HB8 [4]. Since only minute amounts of the NADH oxidase are present in T. thermophilus HB8cells, we have cloned the NADHoxidase gene from T. thermophilus HB8 and efficiently expressed in E. coli and purified the enzyme forits application in biosensors[5].