Characterization and distribution of esterase activity in activated sludge

The location and activity of esterase enzymes in activated Sludge from three Municipal wastewater treatment plants were characterized using model Substrate, and denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE) Of particulate, freeze thaw (primarily periplasmic enzymes and those associated with outer cell surfaces) and extracellular fractions of activated sludge bacteria. Particulate and freeze thaw fractions had a similar spectrum of substrate specificity and contained significant levels of protein and esterase activity against model substrates, C-2 C-18 monoesters or p-nitrophenol and C-2 C-8 diesters of fluorescein. Esterase activity was highest with substrates that had short alkyl chains (C-4) and decreased as the chain lengths increased beyond C-8. Extracellular fractions contained very low levels of protein (<0.1 mg/l) and showed no esterase activity against any of the model substrates tested. Multiple hands were observed upon analysis of particulate and freeze-thaw fractions by non-denaturing PAGE in combination with activity staining using various alpha -naphthol ester substrates (C-2 C-8). Our results indicate that esterase cnzyrnes in activated sludge are fairly diverse from a structural standpoint but exhibit a high level of functional redundancy, with different enzymes catalyzing the same reactions in different sludges. Extracellular esterase activity was totally absent tor the substrates we tested and the esterase activity that we observed was closely linked to a particulate floc or cellular Material. (C) 2001 Elsevier Science Ltd. All rights reserved.