Despite attention devoted to prolyl-hydroxyproline (Pro-Hyp) in nutraceuticals and pharmaceuticals, its enzymatic synthesis has not been achieved. This study investigated aminolysis reaction from proline esters or amide and hydroxyproline using prolyl aminopeptidases from Streptomyces aureofaciens TH-3 (PAP TH-3). The effects of pH, buffer concentration, reaction temperature, and the type and concentration of the acyl donor were examined for the aminolysis activity of PAP TH-3. The Pro-Hyp synthesis was conducted in alkaline conditions at low temperature. The type of acyl donor also affected the yield. Finally, optimum conditions were established as 5 mu l of 1 M proline amide, 50 mu l of 2 M hydroxyl-proline, 40 mu l of 1200 mM boric buffer (pH 11), 5 mu l of water containing 10 mu g PAP TH-3, 4 degrees C and 3 h. Based on the acyl donor. 30% of the maximum yield was obtained. (C) 2011 Elsevier Ltd. All rights reserved.