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 natural systems are not well understood, despite their critical role in microbial remineralization of organic carbon. These enzymes initiate organic carbon degradation by selectively hydrolyzing high molecular weight substrates to lower molecular weight products which can be transported into cells. A set of single- and dual-labeled fluorescent polysaccharides was synthesized and characterized to explore a variety of approaches for measuring enzymatic hydrolysis of biopolymers via photophysical techniques, focusing particularly on rapid and robust optical techniques which are amenable to field measurements in remote locales. A shotgun-labeling approach yielded dual-labeled probes that exhibited substantial donor fluorophore quenching. The photophysical response of these probes to hydrolysis via purified enzymes was investigated in the lab, and fluorescence polarization proved to be a rapid and reliable technique for monitoring probe hydrolysis. Initial field results were also obtained from hydrolysis experiments in sediment porewaters. Because polarization measurements are rapid and simple, this approach could be used to follow the extracellular enzymatic hydrolysis of a wide range of biopolymers which fuel microbial metabolism.