Determination of d- and l-alanine concentrations using a pyruvic acid sensor

The concentrations of d- and l-alanine in bivalves are useful as indicators of environmental pollution. Amino acid oxidase with a low substrate specificity catalyzes the oxidation of various amino acids. Among the various amino acids, pyruvic acid can be generated from alanine only by the catalytic oxidative reaction of this oxidase. Therefore, in this study, the concentrations of d- and l-alanine were determined from the concentration of pyruvic acid, which was determined from the consumption of oxygen based on the oxidative reaction of pyruvate oxidase. From this point of view, there is a very strong possibility that biosensors utilizing enzymes with a low substrate specificity can be developed. The results obtained were as follows. (1) The optimum conditions for the use of pyruvic acid sensor were as follows: temperature of 25 °C, pH of 6.8, flow rate of 0.1 ml/min, thiamin diphosphate concentration of 1.5 mM, and injection volume of 50 μl. (2) d-Alanine and l-alanine optimally reacted with d- and l-amino acid oxidase at 30 °C, pH 8.2, for 30 min and at 37 °C, pH 7.8, for 90 min, respectively. (3) The linear relationships between the concentrations of d- and l-alanine and the output of the sensor were obtained at 3.56–106.8 μg of d-alanine and 5.34–71.3 μg of l-alanine. (4) The concentrations of d- and l-alanine in Meretrix iusoria, Patinopecten yessonsi, and Corbicula leana obtained by the proposed assay were in good agreement with those determined by a conventional method.