Different carrier-free and carrier-bound penicillin acylases were evaluated in the thermodynamically controlled enzymatic synthesis in organic medium of deacetoxycephalosporin G (using phenylacetic acid and 7-amino-deacetoxycefalosporanic acid) used as a model reaction system. Stability of all biocatalysts was determined in a strong (dioxane) and a moderately weak cosolvent (diglyme) to select the best biocatalysts and reaction medium for performing synthesis. Diglyme was selected as cosolvent, while cross-linked enzyme aggregates with polymeric microenvironment (CLEAs-DP) and glyoxyl agarose immobilized penicillin acylase (GAPA) were selected as biocatalysts to perform the thermodynamically controlled synthesis of deacetoxycephalosporin G at different concentrations of cosolvent. Half-lives of CLEA-DP and GAPA were 666 h and 71 h, respectively, being their activities similar (242 and 254 IU/g, respectively). At the best conditions (70% diglyme), conversion yields were closer to 90%. Productivities of CLEA-DP and GAPA were 2.80 mM/h and 2.54 mM/h, respectively. Stability of both biocatalysts during synthesis was tested in sequential batch reactor operation being significantly higher for CLEA-DP where no reduction in equilibrium conversion was observed after four sequential batches. Higher stability also reflected in higher productivity. (C) 2008 Elsevier Inc. All rights reserved.