Characterization and distribution of esterase activity in activated sludge

The location and activity of esterase enzymes in activated Sludge from thre e Municipal wastewater treatment plants were characterized using model Subs trate, 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 activa ted sludge bacteria. Particulate and freeze thaw fractions had a similar sp ectrum of substrate specificity and contained significant levels of protein and esterase activity against model substrates, C-2 C-18 monoesters or p-n itrophenol and C-2 C-8 diesters of fluorescein. Esterase activity was highe st with substrates that had short alkyl chains (C-4) and decreased as the c hain lengths increased beyond C-8. Extracellular fractions contained very l ow levels of protein (<0.1 mg/l) and showed no esterase activity against an y of the model substrates tested. Multiple hands were observed upon analysi s of particulate and freeze-thaw fractions by non-denaturing PAGE in combin ation 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 o f functional redundancy, with different enzymes catalyzing the same reactio ns in different sludges. Extracellular esterase activity was totally absent tor the substrates we tested and the esterase activity that we observed wa s closely linked to a particulate floc or cellular Material. (C) 2001 Elsev ier Science Ltd. All rights reserved.