A bienzyme electrode probe for malate

A new amperometric malate enzyme electrode probe has been constructed using a hydrogen peroxide-based sensor coupled with malic and pyruvate oxidase enzymes. The first enzyme catalyzes the oxidation of malic acid, which in the presence-of NADP(+) yields pyruvate as product. The oxidation of pyruvate is catalyzed by pyruvate oxidase, which yields H2O2 as product in the presence of O-2 and phosphate as cosubstrates and thiamine pyrophosphate and Mg2+ as cofactors. The H2O2 is then detected by the electrochemical transducer, and the output current changes are correlated to the concentration of malic acid in solution. Analytical parameters such as pH, temperature, buffer, substrate and cofactor concentrations, and response time have been optimized, Probe stability and reproducibility have been evaluated. The malic enzyme was used first in solution and then coimmobilized with pyruvate oxidase, Coimmobilization of the oxidase and dehydrogenase enzymes has been performed both randomly and asymmetrically on different supports, Calibration curves for malate have been constructed with all the analytical parameters optimized. The detection limit for this newly designed probe was 5 x 10(-7) mol/L, with a broad linear range between 10(-6) and 5 x 10(-4) mol/L. Recovery studies of malate in a wine matrix have been carried out. Malic acid has been determined in grape musts during grape maturation, Results correlated well when compared with those from a spectrophotometric procedure.[...]