Bacterial enzymes such as penicillin acylases are used for biosynthesis of betalactam antibiotics. The present study highlights the importance of manipulation of microbiological media and methods for isolation and characterization of bacterial strains having potential for penicillin acylases. Simple approach has been adopted for developing mutants with enhanced production of penicillin acylase. Bacterial strains collected from different ecological niches have unique properties and manifests diverse levels of enzyme activity. The high diversity in the natural population demonstrates that broad sampling of the bacterial population and strain improvement are necessary to select promising penicillin acylase producing bacterial strains that might be used in the industry for the synthesis of antibiotics.