We describe two general methodologies, based on filter-sandwich assays, for isolating enzymatic activities from a large repertoire of protein variants expressed in the cytoplasm of E. coli cells. The enzymes are released by the freezing and thawing of bacterial colonies grown on a porous master filter and diffuse to a second "reaction" filter that closely contacts the master filter. Reaction substrates can be immobilized either on the filter or on the enzyme itself (which is then, in turn, captured on the reaction filter). The resulting products are detected with suitable affinity reagents. We used biotin ligase as a model enzyme to assess the performance of the two methodologies. Active enzymes were released by the bacteria, locally biotinylated the immobilized target substrate peptide, and allowed the sensitive and specific detection of individual catalytically active colonies.