DIVERSITY OF CUCUMBER CHITINASE ISOFORMS AND CHARACTERIZATION OF ONE SEED BASIC CHITINASE WITH LYSOZYME ACTIVITY

Up to six bands with chitinase activity could be detected in seeds or young seedlings after native polyacrylamide gel electrophoretic (PAGE) separation of proteins at pH 8.9 in the Davis system. Two root-specific acidic chitinases were found between 2 and 11 days after germination. Up to eight bands with chitinase activity were observed after native PAGE separation of proteins at pH 4.3 in the Reisfeld system. Since one chitinase activity migrated with the lowest electrophoretic mobility in both polyacrylamide gel systems, a total of 13 chitinases could be found. Seeds (0, 1 and 2 days after germination) were the best source of basic chitinase activities. The strongest activity had a relative mobility (Rm) value of 0.80 when compared to the mobility of purified hen egg white lysozyme set at 1.00. This activity (Rm 0.80) was partially purified by ammonium sulfate fractionation, ion-exchange chromatography and preparative PAGE. The enzyme had an estimated molecular weight of 27 000 after sodium dodecyl sulfate (SDS)-PAGE. It exhibited lysozyme activity as determined by lysis of Micrococcus cells. Its lysozyme activity was optimal at pH 4.5 and at low ionic strength. Its chitinase activity was typical of an endochitinase type as estimated by the relative efficiency of hydrolysis of 4-methylumbelliferyl-chitotriose versus 4-methylumbelliferyl-chitobiose Cucumber seeds thus contain a highly basic endochitinase/lysozyme which disappears almost completely 3 days after germination. The role of this seed hydrolase has yet to be determined. © 1990.