Abstract: Two novel amperometric biosensors for the determination of L-malic acid in food samples have been compared. Both sensors make use of a Clark-type O-2-electrode but differ in the enzymes used. The first sensor is composed of malate dehydrogenase (decarboxylating), also known as 'malic enzyme' (MDH(dec.), EC 1.1.1.40) and pyruvate oxidase (POP, EC 1.2.3.3). It covers a linear detection range from 1 µmol L-1 to 0.9 mmol L-1 L-malate, with a response time of 1.5 min (t(90)) and a relative standard deviation of 3.5%. Measurements with real samples offered a good correlation with the standard enzymatic assay (difference±7%) Stored at room temperature, the response of the sensor is constant for 8 days. The second biosensor is based on the three enzyme sequence malate dehydrogenase (MDH, EC 1.1.1.37), oxaloacetate decarboxylase (OAC, EC 4.1.1.3) and pyruvate oxidase (POP, EC 1.2.3.3). It has a non-linear calibration curve. Concentrations from 5 µmol L-1 to 1 mmol L-1 L-malate can be detected, within a response time of 1.5 min and with a relative standard deviation of 20%. The lower detection limit for L-malate is 2 µmol L-1. The response is constant for 10 days when the sensor is stored at room temperature. 11 References

Abstract: The coimmobilization of a NAD(P)(+)-dependent dehydrogenase with salicylate hydroxylase (SHL, EC 1.14.13.1) in front of a Clark-electrode yields a flexible new design for dehydrogenase based biosensors. The feasibility of the approach has been tested with malic enzyme (MDH; EC 1.1.1.40) as the dehydrogenase, resulting in a novel L-malate sensor. It had substantial advantages over the biosensor approaches reported earlier: effective re-oxidation of NADPH by SHL yielded an extended linear range from 0.01 to 1.2 mmol L-1 L-malate and strongly reduced NADP(+)-requirement (< 0.025 mmol l-1), while the working stability was increased to more than 30 days. The results obtained from six real samples showed a close correlation with the standard enzymatic method. The presented scheme with SHL and the Clark-electrode can be employed together with any NAD(P)(+)-dependent dehydrogenase.

Abstract: 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.