A chemiluminometric FIA procedure for the enzymatic determination of l-aspartate

Abstract

An enzymatic FIA procedure was developed for the chemiluminometric determination of l-aspartate in a medium for mammalian cell cultivation. Packed bed flow reactors containing aspartate:aminotransferase and l-glutamate oxidase, respectively, immobilized on sieved porous glass beads were combined with a peroxidase modified fibre optic sensor to detect the produced hydrogen peroxide. Peroxidase from Arthromyces ramosus was immobilized on a preactivated microporous nylone membrane and catalyzes the light generating oxidation of luminol by hydrogen peroxide. To improve the selectivity of the l-aspartate determination a l-glutamate eliminating reactor was prepared by coimmobilization of l-glutamate oxidase and catalase in a packed bed enzyme reactor. Under FIA conditions at least 0.5 mM l-glutamate can be quantitatively eliminated from the preconditioned sample solution. l-Aspartate can be determined in the range between 5 and 1000 μM under FIA conditions. The determination of l-aspartate was optimized with respect to pH, cosubstrate concentration and residence time in the packed bed enzyme reactors. The proposed procedures show high recoveries between 96 and 100% for the l-aspartate determination in a medium for mammalian cell cultivations after the elimination of l-glutamate.