The kinetically controlled synthesis of cephalexin in aqueous two-phase systems was studied, using immobilized penicillin acylase, 7-amino 3-desacetoxycephalosporanic acid as nucleophile and phenylglycine methyl ester as acyl donor. The organic phases used were 80% (v/v) polyethyleneglycol 400 and 600 and the aqueous phase was 2.5 M (NH(4))(2)SO(4). 7-amino 3-desacetoxycephalosporanic acid and cephalexin partition coefficients were determined at pH 7.4 and 7.8, at 14 C and 20 C. Highest partition coefficient for cephalexin was obtained for polyethyleneglycol 400-(NH(4))(2)SO(4) at pH 7.4 and 20 degrees C, while the lowest partition coefficient for 7-amino desacetoxycephalosporanic acid was obtained in the same system at pH 7.8 and 14 degrees C. No significant effect of pH was observed on conversion yield and productivity of cephalexin synthesis: however, higher values were obtained with polyethyleneglycol 400 as organic phase. Higher conversion yields with both biphasic systems were obtained at the lowest temperature, where product hydrolysis was lower; volumetric productivity was higher for the fully aqueous medium (control), being higher at 20 degrees C. All parameters of synthesis were improved at higher substrates concentrations, obtaining conversion yields of 78.2% and 65.4%, with 60 mM 7-amino desacetoxycephalosporanic acid for the polyethyleneglycol 400-(NH(4))(2)SO(4) system and the control, respectively. (C) 2010 Published by Elsevier Ltd.