A recombinant plasmid, pPAKSP 1, was constructed to produce Penicillin G acylase using phage P_L promoter. Unexpectedly, thermal induction led to accumulation of the 94KDa polypeptide rather than active Penicillin G acylase in large amount. Using a minicell system we analyzed the 94KDa polypeptide and have found that Penicillin G acylase is made as a full length precursor which may be subsequently processed. The recombinant plasmids, pPAKS63 and pPAKSP_L1, coding for Penicillin G acylase, were expressed in minicell and the plasmid pPAKS63 directed the synthesis of approx. 94KDa protein in minicell. The protein was also identified as the precursor of Penicillin G acylase by western immunoblot analysis.