Artículo de publicación ISIThe presence of organic cosolvents was previously considered necessary to obtain high conversion yields in the synthesis of β-lactam antibiotics with immobilized penicillin acylase, and it is so when working at moderate substrate concentrations. Conversion yields close to stoichiometric and high productivities were recently reported for the synthesis of cephalexin at high substrate concentrations in ethylene glycol medium. Under such conditions, the effect of cosolvent concentration on yield is not significant so we raised the hypothesis that stoichiometric yields and high productivities are attainable at very high substrate concentrations in fully aqueous medium leading to substantial process improvement in terms of costs and environment. To test the hypothesis, the kinetically controlled synthesis of cephalexin with immobilized penicillin acylase was conducted in aqueous medium at substrates concentrations up to and beyond their solubilities at varying temperature, pH, enzyme to substrate and acyl donor to nucleophile ratios. At the best conditions, 99% conversion yield was attained with volumetric productivity of 300 mM/h and specific productivity of 7.8 mmol/h g cat. These values are slightly higher than those previously obtained under optimized conditions in organic medium so that the hypothesis has been confirmed, which opens up the possibility of efficiently produce the antibiotic through an environmentally friendly process.