The production of penicillin G acylase (PGA, an important industrial enzyme) from a wild strain of Bacillus megaterium using a pool of free amino acids as substrate was studied in a bench-scale bioreactor. Experiments carried out in shakers showed that the substitution of casein for free amino acids in the presence of cheese whey was the culture medium that provided the highest productivity. Several cultivations were carried out in a bioreactor operated in either batch or fed-batch mode. Batch runs showed that enzyme production is associated with microorganism growth. The following set of amino acids was preferentially consumed: Ala, Arg, Asp, Gly, Lys, Ser, Thr and Trp. On the other hand, the rates of consumption of His, Ile, Leu, Met, Phe, Pro, Tyr and Val were lower.