The production of extracellular and mycelia-associated penicillin G acylase (maPGA) with Mucor griseocyanus H/55.1.1 by surface-adhesion fermentation using Opuntia imbricata, a cactus, as a natural immobilization support was studied. Enzyme activity to form 6-aminopencillanic acid (6-APA) from penicillin G was assayed spectrophotometrically. The penicillin G hydrolysis to 6-APA was evaluated at six different times using PGA samples recovered from the skim milk medium at five different incubation times. Additionally, the effect of varying the penicillin G substrate concentration level on the PGA enzyme activity was also studied. The maximum reaction rate, V (max), and the Michaelis constant, K (M), were determined using the Michaelis-Menten model. The maximum levels for maPGA and extracellular activity were found to be 2,126.50 international unit per liter (IU/l; equal to 997.83 IU/g of support) at 48 h and 755.33 IU/l at 60 h, respectively. Kinetics of biomass production for total biomass showed a maximum growth at 60 h of 3.36 and 2.55 g/l (equal to 0.012 g of biomass per gram of support) for the immobilized M. griseocyanus biomass. The maPGA was employed for the hydrolysis of penicillin G to obtain 6-APA in a batch reactor. The highest quantity of 6-APA obtained was 226.16 mg/l after 40-min reaction. The effect of substrate concentration on maPGA activity was evaluated at different concentrations of penicillin G (0-10 mM). K(M) and V(max) were determined to be 3.0 x 10(-3) M and 4.4 x 10(-3) mM/min, respectively.