The impact of mass transfer limitations on penicillin G acylase immobilized in glyoxyl-agarose particles of different sizes and enzyme loads was evaluated for the reactions of hydrolysis of penicillin G and synthesis of cephalexin under the hypothesis that the impact of internal diffusional restrictions on the catalytic potential of the enzyme will be of a greater magnitude for a fast reaction of hydrolysis than a slower reaction of synthesis. Experimental evidences were obtained from batch reactor operation where increase in enzyme load and particle size has a much stronger impact on the former reaction. Additional evidences were obtained by the impact of the above biocatalyst properties on the apparent Michaelis constants for substrates. Michaelis constants of penicillin G acylase catalysts of different enzyme load and particle sizes varied between 0.73 and 4.55 mM and between 3.0 and 11.1 mM for the hydrolysis of penicillin G and the synthesis of cephalexin respectively. Michaelis constants for penicillin G acylase biocatalysts subjected to progressive size reduction were reduced from 5.0 to 0.46 mM and from 7.2 to 2.1 mM for hydrolysis of penicillin G and synthesis of cephalexin respectively. Higher fluctuation between these values in hydrolysis of penicillin G reflects higher impact of diffusional restrictions on this reaction. Thiele modulus for the substrate was much higher for penicillin G hydrolysis than for cephalexin synthesis in a wide range of substrates concentrations. Ratio between moduli for hydrolysis of penicillin G and synthesis of cephalexin was 15 at saturating concentration of phenylglycine methyl ester and increased at lower concentrations of such substrate. Results highlight the importance of designing the biocatalyst according to the reaction in which it will be used, being particularly important in the case of penicillin G acylase that is currently being used both in reactions of hydrolysis and synthesis. (C) 2010 Elsevier Inc. All rights reserved.