A systematic approach for the selection of potential biocatalysts from a natural source was developed and then a practical application was addressed. The approach that involves systematically combined conventional screening methods and current tools comprises the following consecutive steps: strain enrichment for activity screening, identification of positive strains, choosing whole genome-sequenced strains as candidates, gathering information about responsible enzymes, bioinformatic analyses and gene mining, probing genetic molecules and then functional expression. The target compound (R,S)-ketoprofen ethyl ester was to be resolved into an enantiomer, and a potential esterase from Pseudomonas fluorescens KCTC 1767 was prepared by the proposed procedure. The enzyme had a high activity and also strict selectivity for the enantiomer (S)-ketoprofen and was suitable therefore as a biocatalyst for practical use. The result achieved by the combined approach could not easily be obtained using other approaches with typical procedures. Hence the approach proposed here should be of considerable use for the screening of potential enzymes, particularly for enzymes with desired activity to unnatural substrates, from conditionally expressed and/or repressed proteins that are distributed widely in natural pools under normal conditions.