PURIFICATION AND CHARACTERIZATION OF EXTRACELLULAR ICE-NUCLEATING MATTER FROM ERWINIA-UREDOVORA KUIN-3

The extracellular ice-nucleating matter (EIM) produced by the ice-nucleating bacterium, Erwinia uredovora KUIN-3, was purified to apparent homogeneity by ultrafiltration, sucrose density-gradient ultracentrifugation, and gel filtration. The purified EIM was sphercial matter (0.2-0.4 mum) by examination using a transmission electron microscope. The ice-nucleating activity from the EIM was equal to that of the cells in this strain. It had become apparent that the EIM had class A (approximately -5-degrees-C), B (-5 approximately -8-degrees-C), and C (-8-degrees-C approximately) structures as judged by its freezing difference spectrum in D2O versus H2O. The enzyme treatments by protease K, alpha-mannosidase, beta-galactosidase, and phospholipase C decreased the activity of the EIM as well as of the cells. Further. the activities of class A and B from the EIM were decreased by treatment with diamine oxidase. Also, the activity of the purified EIM was stable from pH 5.0 to 10.0 and at temperatures below 25-degrees-C. It was demonstrated that the components of the EIM were lipid (10%), protein (43%), saccharide (35%), and polyamine (12%).