High-level expression of recombinant penicillin acylase (PAC) using the strong trc promoter system in Escherichia coli is frequently limited by the processing and folding of PAC precursors (proPAC) in the periplasm, resulting in physiological stress and inclusion body formation in this compartment. Periplasmic heat-shock proteins with protease or chaperone activity potentially offer a promise for overcoming this technical hurdle. In this study, the effect of the two genes encoding periplasmic heat-shock proteins, that is degP and fkpA, on pac overexpression was investigated and manipulation of the two genes to enhance the production of recombinant PAC was demonstrated. Both DeltadegP and DeltafkpA mutants showed defective culture performance primarily due to growth arrest. However, pac expression level was not seriously affected by the mutations, indicating that the two proteins were not directly involved in the pathway for periplasmic processing of proPAC. The growth defect caused by the two mutations (i.e., DeltadegP and DeltafkpA) was complemented by either one of the wild-type proteins, implying that the function of the two proteins could partially overlap in cells overexpressing pac. The possible role that the two heat-shock proteins played for suppression of physiological stress caused by pac overexpression is discussed.