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

The coimmobilization of a NADP(+)-dependent dehydrogenase with p-hydroxybenzoate hydroxylase (PHBH, EC 1.14. 13.2) in front of a Clark electrode yields a flexible design for highly selective, dehydrogenase-based biosensors, The 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 inexpensive chemicals (p-hydroxybenzoate, MgCl2) were needed in millimolar amounts. A linear mathematical model for the steady state helped to elucidate the sensor operation. Both experimental and simulation results indicated that the bienzyme sensor behaved like a quasi monoenzyme electrode with a hypothetical "L-malate hydroxylase": The response was determined by the substrate concentration and diffusivity only, indicating the perfect coupling of both enzyme reactions by the intermediate NADPH. The presented scheme based on PHBH and the Clark electrode is a promising and reliable approach for other NADP(+)-dependent dehydrogenases.