A procedure is described for the immobilization of glutaryl-7-ACA acylase from recombinant E. coli on Amberlite XAD7 modified by transamidation with I,2-diaminoethane and activated with glutaraldehyde. The determination of the optimal operative conditions such as enzyme loading, effect of pH, temperature, and product inhibition is described for the hydrolysis of 7-beta-(4-carboxybutanamido) cephalosporanic acid (glutaryl-7-ACA) to give 7-aminocephalosporanic acid (7-ACA). The inactivation kinetics of the immobilized preparations determined in a continuous fixed-bed reactor are also reported.