Characterization and mathematical modeling of a bienzyme electrode for L-malate with cofactor recycling

The coimmobilization of a NADP(+)-dependent dehydrogenase with p-hydroxyben zoate hydroxylase (PHBH, EC 1.14. 13.2) in front of a Clark electrode yield s a flexible design for highly selective, dehydrogenase-based biosensors, T he use of L-malate dehydrogenase (decarboxylating, EC 1.1.1.40) as a model enzyme resulted in a novel L-malate sensor. It had improved characteristics compared with those of earlier sensor approaches: a strongly reduced NADP( +) requirement (0.01 mmol L-1), an extended linear range from 0.005 to 1.1 mmol L-1 L-malate, and a working stability of more than 30 days. Only inexp ensive chemicals (p-hydroxybenzoate, MgCl2) were needed in millimolar amoun ts. A linear mathematical model for the steady state helped to elucidate th e sensor operation. Both experimental and simulation results indicated that the bienzyme sensor behaved like a quasi monoenzyme electrode with a hypot hetical "L-malate hydroxylase": The response was determined by the substrat e concentration and diffusivity only, indicating the perfect coupling of bo th enzyme reactions by the intermediate NADPH. The presented scheme based o n PHBH and the Clark electrode is a promising and reliable approach for oth er NADP(+)-dependent dehydrogenases.