Application of fluorescence spectroscopic techniques and probes to the detection of biopolymer degradation in natural environments

The activities and substrate specificities of extracellular enzymes in natu ral systems are not well understood, despite their critical role in microbi al remineralization of organic carbon. These enzymes initiate organic carbo n degradation by selectively hydrolyzing high molecular weight substrates t o lower molecular weight products which can be transported into cells. A se t of single- and dual-labeled fluorescent polysaccharides was synthesized a nd characterized to explore a variety of approaches for measuring enzymatic hydrolysis of biopolymers via photophysical techniques, focusing particula rly on rapid and robust optical techniques which are amenable to field meas urements in remote locales. A shotgun-labeling approach yielded dual-labele d probes that exhibited substantial donor fluorophore quenching. The photop hysical response of these probes to hydrolysis via purified enzymes was inv estigated in the lab, and fluorescence polarization proved to be a rapid an d reliable technique for monitoring probe hydrolysis. Initial field results were also obtained from hydrolysis experiments in sediment porewaters. Bec ause polarization measurements are rapid and simple, this approach could be used to follow the extracellular enzymatic hydrolysis of a wide range of b iopolymers which fuel microbial metabolism. (C) 2000 Elsevier Science B.V. All rights reserved.