Penicillin G acylase (PGA; E.C. 3.5.1.11) is an important enzyme which has broad applications in industries of beta-lactim antibiotics production. In this study, a promising PGA gene from Alcaligenes faecalis (afpga) and another pcm gene encoding protein isoaspartate methyltransferase (PIMT) were constructed into pET43.1a((+)) and pET28a((+)), respectively. The recombinant plasmids pETAFPGA and pETPCM were transformed into the same host cell Escherichia coli BL21 (DE3). Results suggested that the two plasmids could peacefully exist in the host cell and the two genes could be efficiently expressed after induction. The product of pcm gene could function as a helper molecule for enzyme AFPGA. PIMT increased the enzymatic activities in supernatant of ferment broth (1.6 folds) and cell lysate (1.8 folds), while it did not significantly affect the expression level of penicillin G acylase.