Artículo de publicación ISIOrganic cosolvents, and among them, polyols, are suitable media to perform the enzymatic synthesis of -lactam antibiotics with immobilized penicillin acylase, because they effectively reduce water activity, depressing hydrolytic reactions in favor of synthesis. Among polyols, ethylene glycol has proven to be particularly suited as reaction medium for their synthesis. Previous studies have shown that pH, temperature, and cosolvent concentration are the most relevant variables in the kinetically controlled synthesis of cephalexin from 7-amino-3-deacetoxy cephalosporanic acid and phenylglycine methyl ester, conversion yield increasing at low temperatures and high cosolvent concentrations. The objective of this work is the optimization of temperature, pH, and ethylene glycol concentration in the kinetically controlled synthesis of cephalexin with immobilized penicillin acylase at lower than ambient temperature in terms of substrate molar conversion yield. Phenylglycine was used as acyl donor and 7-amino-3-deacetoxy cephalosporanic acid was the limiting substrate at 30 mM. Optimization was performed using surface of response methodology, optimum conditions being 12 ◦C, pH 6.8, and 60% (v/v) ethylene glycol, at which cephalexin yield was close to stoichiometric with respect to the limiting nucleophile, which is unattainable in aqueous medium. Stability of the biocatalyst at optimum conditions for cephalexin synthesis was very high, with a projected half-life of 1500 h, making it a suitable catalyst for the large-scale production of cephalexin.