Papain was modified with the anhydrides of various monocarboxylic (acetic or propionic) and dicarboxylic (citraconic, maleic or succinic) acids. 7-10 of the 11 primary amino groups of the enzyme were modified. The organic solvent tolerances of the modified enzyme forms were increased (especially in the concentration range of 10-60%) in comparison with the unmodified enzyme. Acylation enhanced the catalytic activity and stability of papain both in buffer and in aqueous organic solvents (ethanol and acetonitrile). Decrease of the positive charges on the surface of papain resulted in a higher enzyme stability than when they were replaced by negative charges. The kinetic parameters revealed that in aqueous ethanol the maximum rates (V(max)) and Michaelis constants (K(M)) of the modified papain forms were increased, and higher catalytic efficiencies (k(cat)/K(M)) were detected as compared with the native enzyme. The results of near-UV circular dichroism and tryptophan fluorescence spectroscopic studies suggested that the modifications caused only local changes around the aromatic residues. The modified enzyme forms led to higher N-acetyl-L-tyrosine ethyl ester synthesis conversions in aqueous ethanol; acetyl and propionyl papain furnishing the highest productivity. (C) 2008 Elsevier Ltd. All rights reserved.