MALATE DETERMINATION BY FLOW INJECTION ANALYSIS ( FIA ) USING A COUPLED DEHYDROGENASE/TRANSFERASE SYSTEM

Abstract

The determination of L-malate was based on its oxidation by NADt catalyzed by immobilized malate dehydrogenase (MDH, EC 1.1.1.37) in a flow injection system. The NADH produced in an enzyme reactor was detected fluorimetrically. Malate dehydrogenases from different sources were immobilized on aminoderivatized controlled pore glass by glutardialdehyde. Hence the equilibrium of the MDH catalyzed reaction is far in favour of L-malate only little response could be obtained by malate injection. To achieve higher substrate conversion, aspartate aminotransferase (AST, EC 2.6.1.1) was coimmobilized to transform the reaction product oxaloacetate. Enzyme reactors with and without AST were compared and optimized. With the bienzyme reactor, malate concentrations between 5 and 500 uM could be monitored.