A model system for a fluorometric biosensor using permeabilized Zymomonas mobilis or enzymes with protein confined dinucleotides

Abstract

Using permeabilized Zymomonas mobilis or glucose-fructose oxidoreductase isolated from this microorganism a model system for biosensors with a protein confined NADP(H) cofactor for the determination of glucose, fructose, gluconolactone, and sorbitol was developed. Either permeabilized microorganisms containing the oxidoreductase or the pure enzyme were confined via membrane separation in a small measuring chamber, that was integrated into a flow injection analysis system (FIA). The measuring principle was the monitoring of the NAD(P)H fluorescence, excited at 360 nm and measured at 450 nm. NADP(H), which is confined in the protein complex, was oxidized or reduced during the enzymatic reactions and the changes in the fluorescence intensity were related to the substrate concentration. The sensitivity of the system covered a range from 0.001 to 100 g/L of the analyte depending on substrate and operating conditions. The applicability of this model system for bioprocess monitoring was proved using samples from a Pseudomonas pseudoflava cultivation.

Intrinsic fluorescence of enzymes and fluorescence of chemically modified enzymes for analytical purposes: a review

“…Substrates determined in this way include ethanol, lactate, pyruvate, phenylalanine, malate, glutamate, glycerol or aminobutyric acid, lactate dehydrogenase being an example of an enzyme. This methodology is of great use, has been the object of considerable research and new applications are still being found (10,11). However, NADH does not have suitable fluorescent properties for the application of EFMs to real samples; in the first place, its relatively low molar absortivity and quantum yield result in determinations with medium sensitivity; second, the spectral region where its fluorescent properties are displayed frequently gives rise to interference problems, especially when minimal sample treatment is desired.…”