Penicillin acylase (PA) is an industrial enzyme used for hydrolysis of penicillin to ?-lactam nucleus 6-aminopenicillanic acid (6-APA) using for manufacturing semi-synthetic penicillins. Microbial enzyme transformation processes are generally preferred over the conventional chemical conversion process. Bacterial strains such as Escherichia coli are widely used for industrial production of 6-APA. The study provides a comprehensive resource for i) screening/characterization of PA producing E. coli strains from different habitats, ii) optimization of conditions for PA production and iii) improvement of PA producing E. coli strains by genetic manipulations like chemical/radiation mutagenesis and transformation. The cost-effective and environment friendly enzymatic methodology developed through the concrete approach of fermentation technology and molecular biology and the high-yielding PA producing local E. coli strains/mutants achieved during this study will be of many fold significance for industrial synthesis of 6-APA at economical rates. Contribution to establishment of culture collection will be an added facility available to researchers/students.