BACKGROUND: Activation and stabilization of enzymes have received considerable attention. In recent years, chemical modification has re-emerged as a powerful complementary approach to site-directed mutagenesis and directed evolution because it is simple and effective. Chemical modification of enzymes by dicarboxylic acid anhydride enhanced their activities and stabilities. RESULTS: Lipase, alpha-chymotrypsin and subtilisin modified by citraconic anhydride were examined. Lipase and alpha-chymotrypsin were successfully modified by citraconic anhydride, while subtilisin showed poor reactivity with it. Optimum pH range for lipase and alpha-chymotrypsin, and the secondary structure of alpha-chymotrypsin was found to be unchanged by the modification process. Effect of the modification on the activity and thermostability of lipase was small. On the other hand, modification of lysine residues on the enzyme surface enhanced the catalytic activity and thermostability of alpha-chymotrypsin. Further modification of lysine residues caused the reduction of thermostability. Modification of more hydrophobic dicarboxylic acid anhydride resulted in an increase in thermostability. CONCLUSION: The modification of the lysine residues on the enzyme surface with citraconic anhydride shows a small effect on the catalytic reactivity and enhances the thermostability of the enzymes. (c) 2014 Society of Chemical Industry