The partially purified chlorophyllase, obtained from the alga Phaeodactylum tricornutum, was further purified by preparative native gel electrophoresis. The purification procedure provided the recovery of large amounts of a single purified chlorophyllase fraction. However, the electrophoretic analyses of the purified enzymatic fraction under denaturing conditions demonstrated the presence of two bands with mol wt of 43 +/- 3 and 46 +/- 3 kDa. The purification procedure resulted in 2- and 195-fold increases in chlorophyllase activity compared to that of the partially purified and crude enzymatic extracts, respectively. The optimum pH for chlorophyllase hydrolytic activity was found to be 8.0. The,optimum incubation time and temperature for the hydrolytic activity of the purified chlorophyllase were found to be 2 h and 31 degrees C, respectively. The optimum concentrations of magnesium chloride and dithiothreitol, used as activators, were 4 and 5 mM, respectively. The addition of individual plant membrane lipids, including phosphatidylcholine, phosphatidylglycerol, and beta-carotene, to the reaction media increased the enzyme activity markedly. The purified enzyme fraction displayed preferential specificity toward selective substrates with an order of activity as follows : purified chlorophyll b > purified chlorophyll a > partially purified chlorophyll > crude chlorophyll. Diisopropyl fluorophosphate and phytol, respectively, showed noncompetitive and competitive inhibitory effects on chlorophyllase activity with K-i values of 0.78 mM and 3.75 mM, respectively.