Purification and sensor applications of an oxygen insensitive, thermophilic diaphorase

An intracellular diaphorase was isolated from a strain of Bacillus stearothermophilus (LLD-R, NCIMB 12403). The enzyme was extracted and purified to homogeneity by a combination of ion exchange, gel filtration and affinity chromatography in a 37% overall yield with a purification factor of 150. The molecular weight of the enzyme was found to be 30000 by gel filtration chromatography and was shown to be monomeric by gel electrophoresis. Using dichlorophenol as an electron acceptor the purified enzyme had a specific activity of 280 U mg−1; other electron acceptors included ferrocene carboxylic acid and hexacyanoferrate(III), but not dioxygen. In order to use the enzyme in an amperometric biosensor, optimal immobilisation conditions were investigated. Immobilon affinity membrane was found to give the best performance in terms of current sensitivity, analytical range (1 × 10−7 to 1 × 10−3 mol l−1 for NADH) and 3σ limit of detection (LoD) (5 × 10−8 mol l−1 NADH). Co-immobilisation of the diaphorase with different dehydrogenases resulted in amperometric sensors for glucose (LoD 5 × 10−7 mol l−1), ethanol (LoD 1 × 10−6 mol l−1), lactate (LoD 1 × 10−6 mol l−1) and β-hydroxybutyrate (LoD 5 × 10−7 mol l−1).