Cross-linked enzyme aggregates (CLEAs) are novel type biocatalysts well suited to catalyze reactions of organic synthesis. Penicillin acylase is a versatile enzyme that can both hydrolyze and synthesize beta-lactam antibiotics. CLEAs and CLEAs covered with polyionic polymers (polyethyleneimine and dextran sulfate at two different enzyme to polymer ratios) were prepared at varying cross-linking agent to enzyme ratio: 0.15 and 0.25. Results are presented on the effect of such variables on immobilization yield, specific activity, stability and performance of penicillin acylase CLEAs in the kinetically controlled synthesis of cephalexin. The cross-linking agent to enzyme ratio had no significant effect on the specific activity of the CLEAs, but affected immobilization yield, stability in ethylene glycol medium and conversion yield and productivity in the synthesis of cephalexin, being always higher at the lower crosslinking agent to enzyme ratio. Best results were obtained with CLEAs at 0.15 glutaraldehyde to enzyme protein ratio: specific activity of hydrolysis and synthesis was 708 and 325 UI/g(CLEA), respectively, conversion yield was 87%, specific productivity was 5.4 mmol cephalexin/(g(CLEA).h) and 90% of the enzyme remained active after 170 h at operating conditions. (C) 2008 Elsevier Ltd. All rights reserved.