Direct extraction of catalytic proteins from natural microbial communities

A method was developed for rapid investigation of enzymes in natural microbial communities without isolation or cultivation of individual species. Conservative extraction of total proteins present in cells concentrated from up to 1 l of freshwater or wastewater sludge samples was performed by sonication and centrifugation. The protein extracts were immobilized on 0.2 μm pore size nitrocellulose membranes using a vacuum-driven compartmentalized filtration apparatus. Reaction-dependent colorimetric staining of the membranes allowed the detection of enzymatic activities attributable to acid phosphatase, catechol oxidase, cellulase, nitrate reductase, peroxidase, and xanthine dehydrogenase in protein extracts from the wastewater sludge samples. Only catechol oxidase, nitrate reductase, peroxidase, and xanthine dehydrogenase activities were detected in protein extracts from the freshwater samples. Control experiments with Pseudomonas putida indicated a detection limit of enzyme activity of 10 3 cells or 3.3 ng of total protein. The method described precludes the detection of allozyme or isozyme multiplicity, but it facilitates rapid screening of several different enzymes and could be adapted for quantitative assessment of spatial and temporal distribution of enzymes in many heterogenous aquatic microbial communities.