Abstract: The aim of present study was to optimize conditions for conversion of penicillin G into 6-APA using intact crude cells of locally collected PGA producing bacterial strains as biocatalyst. Corn steep liquor medium supplemented with phenylacetic acid was used for PGA production. For enzymatic conversion of penicillin G into 6-APA by PGA impregnated bacterial cells, a maximum reaction time of 4 h was found adequate. The procedure for extraction and crystallization of 6-APA from the enzyme reaction mixture was standardized. Isolation process was carried out under controlled pH conditions and 6-APA crystals were recovered from the reaction mixture via filtration, concentration and drying. The maximum PGA activity was observed in Escherichia coli strain BDCS-N-FMu12 (6.4 mg 6-APA h-1 mg-1 wet cells) whereas Bacillus megaterium (ATCC 14945 used as check) exhibited only 2.4 mg 6-APA h-1 mg-1 wet cells. The overall yield of 6-APA crystals obtained after enzymatic conversion of penicillin G ranged between 37-55 and 47-68% in foreign and local strains, respectively. BDCS-N-FMu12 was identified as the best PGA producer with 68% 6-APA conversion whereas ATCC 14945 showed the lowest conversion (37%). The recovery of 6-APA (68%) obtained by using crude intact cells as cheap biocatalyst appeared promising. The process of enzyme fermentation and 6-APA crystallization optimized during this study seems cost-effective and environment-friendly. However, further studies are required to scale up the 6-APA biosynthesis reaction for achieving 80-90% conversion of penicillin G into 6-APA by PGA hyper-producing locally collected strains of E. coli.