β- Lactam antibiotics, in particular penicillins and cephalosporins have the annual sales of 65% of the total antibiotics market. Therefore the annual expenditure of penicillin G acylase (PGA) is estimated to be in the range of 10-30 million tons. PGA immobilization preparations (both enzyme and cells) to successfully produce 10,000 tons of total 6-aminopenicillanic acid (6-APA) provide a novel direction to the antibiotics industries promoting a safer and cleaner environment. In the present study, different bacterial samples were isolated from soil of different places of Dibrugarh and screened for biotransformation ability to produce 6-APA. Among ten of them, three gram positive bacterial samples designated as AKDD-2, AKDD-4 and AKDD-6 showed the production of 6-APA from penicillin G. Assessment of 6-APA production after incubation in penicillin G (2mg/ml) by three different samples separately in free and agar immobilization state was done by HPLC analysis. Reusability of immobilized cell was found successful up to 14 days. The work focuses comparison of catalytic properties to produce 6-APA of some industrially valuable soil microorganisms entrapped in agar with the free form.