L-Malate Determination in Wines and Fruit Juices by Flow Injection Analysis Adaptation of a Coupled Dehydrogenase/Transferase System

Abstract

A flow injection system for the enzymatic determination of L-malate is described. It is adapted for the analysis of wines and fruit juices by in-line sample dilution. Malate dehydrogenase (MDH) and aspartate aminotransferase (AST) were coimmobilized on controlled pore glass. The NADH generated in the packed bed bienzyme reactor was detected fluorimetrically. Sufficient L-malate conversion could be achieved using the coupled transferase reaction to transform the reaction product oxaloacetate. The linear range of the unmodified flow injection system, 5 – 100 μ M of L-malate, could be increased up to 50 mM by means of zone sampling so that untreated samples could be analyzed directly. From 12 – 20 samples per hour could be analyzed with a standard deviation of less than 2 % depending on variation in the degree of dilution. The activity of the bienzyme reactor decreased less than 4% over an analysis period of 3 weeks with more than 1200 injections.

Key Words:

• FIA
• malate dehydrogenase
• aspartate aminotransferase
• immobilized enzymes
• coupled enzyme reaction
• zone sampling (gradient dilution)