DEVELOPMENT OF A BIOSENSOR FOR MONITORING OF GLYCEROL DURING ALCOHOLIC FERMENTATION

A biosensor for the measurement of glycerol in FIA was constructed usi ng covalently immobilized glycerokinase and glycerol-3-phosphate oxida se in conjunction with a Pt based hydrogen peroxide probe. Different i mmobilization strategies have been studied including random and asymme tric immobilization onto a polymeric support and immobilization onto t wo different membranes. The latter resulted in the best configuration for batch measurement. The most effective configuration for measuremen t in FIA was the immobilization of glycerokinase in a glass beads reac tor coupled with glycerol-3-phosphate oxidase on a preactivated Immobi lon AV membrane kept at the electrode surface. Using a 250-mu l inject ion loop, 3 mmol ATP(Mg (+) (2)) in 0.1 M berate buffer pH 8.5 and a f low rate of 0.5 ml/min, a linear response in the 2 x 10(-6)/10(-3) mol /l range and a detection limit of 5 x 10(-7) mol/l were obtained for g lycerol. Lifetime of the glycerol-3-phosphate membrane was extended up to 1 month by storage in the working buffer containing 1% DEAE-dextra n and 5% lactitol. More than 350 samples can be assayed with this syst em. The biosensor was used to monitor off-line glycerol production dur ing alcoholic fermentations carried out at different pHs and temperatu res. (C) 1998 Elsevier Science S.A. All rights reserved.