An inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), is a reversible inhibitor of esterases. The reversible inhibition of the enzyme activity is thought to be examined after separation and immobilization of the enzyme under non-denaturing conditions. Hydrolytic changes of phosphatidylcholine by carboxylesterase were obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the esterase was separated by non-denaturing two-dimensional electrophoresis, was immobilized to membranes and was stained by Ponceau S. The changes were inhibited after the enzyme on the membrane was treated by tacrine. Furthermore, the hydrolytic activity of the esterase was recovered after the inhibitor was washed with aspartic acid solution. These results indicate that the phosphatidylcholine hydrolysis activity of the isolated and immobilized enzyme is reversibly inhibited under non-denaturing conditions. Furthermore, this method can be developed to the production of an enzyme reactor able to regulate amounts of lipids.