Study of extracellular ice-nucleating matter from ice-nucleating bacteria

采用超滤、蔗糖密度梯度离心和凝胶过滤等方法对噬夏孢欧文氏菌（Erwinia uredovora KUIN-3）胞外冰核物质（EM）进行纯化。用透射电镜观察，纯化的EM为球形物质（0.2- 0.4 μ m）。EM的冰核活性与该菌株的细胞相当。根据其在D_2O和H_2O中的冷冻差谱，可以明显地看出EM具有A（-5 ℃）、B（-5--8 ℃）和C（-8 ℃）类结构。用蛋白酶K、α-甘露糖苷酶、β-半乳糖苷酶和磷脂酶C处理EM和细胞都降低了活性。此外，从EM的A类和B类的活性降低处理与二胺氧化酶。纯化后的EM在pH5.0 ~ 10和25 ℃以下的温度下活性稳定，其主要成分为脂类（10%）、蛋白质（43%）、糖类（35%）和多胺（12%）。加入抑制S-腺苷甲硫氨酸脱羧酶活性的丙酮醛双胍基腙（MGBG），抑制了该菌的冰核化活性。MGBG处理后，细胞和外膜中的多胺含量下降。结果表明，多胺是该菌冰核物质的主要成分。

The extracellular ice-nucleating matter (EM) produced by the ice-nucleating bacterium, Erwinia uredovora KUIN-3, was purified to apparent homogeneity by ultrafiltration, sucrose density-gradientultracentrifugation, and gel filtration. The purified EM was sphercial matter (0.2-0.4mum) by examination using a transmission electron microscope. The ice nucleating activity from the EM was equal to that of the cells in this strain. It had become apparent that the EM had class A (--5ﾟC) , B ( -5--8ﾟC) , and C (-8ﾟC) structures as judged by its freezin differencespectrum in D_2O versus H_2O.The enzyme treatments by protease K,alpha-mannosidase, beta-galactosidase, and phospholipase C decreased the acyivity of the EM as well as of the cells. Further, the activities of class A and B from the EM were decreased by treatment with diamine oxidase. Also the activity of the purified EM was stable grom pH 5.0 to 10 and at temperatures below 25ﾟC.It was demonstrated that the components of the EM were lipid (10%) , protein (43%) , saccharide (35%) , and polyamine (12%) .The effects of polyamines on the ice-nuceating activity of an ice-nuceating bacterium, Erwinia uredovora KUIN-3, were investigated. The addition of methylglyoxal bis (guanylhydrazone) {MGBG} which inhibited the activity of S-adenosylmethionine decaboxylase, inhibited the ice-nucleating activity of the cell of this bacterium. The amounts of polyamines in the cells and outer membranes after MGBG treatment decreased. The results suggested that polyamine was the most important component of the ice-nucleating material in this bacterium.