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Applications and Production of an Extracelllular Ice-Nucleating Substance by the Ice-Nucleating Bacteria

冰核细菌胞外冰核物质的应用和生产

基本信息

批准号：
01550766
负责人：
OBATA Hitoshi
金额：
$ 0.9万
依托单位：
Kansai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

When Erwinia uredovora (KUIN-3) was cultured aerobically in a medium consisting of Simmons-citric acid broth (pH 6.1) for 24 hr at 18 ^ﾟC, the ice-nucleating activity of strain KUIN-3 cells was obtained. The ice-nucleating temperature, T_<50>(^ﾟC) was detected at -2.8 ^ﾟC in cell suspensions (1.1 x 10^8 cells/ml) of KUIN-3.The production of an extracellular ice-nucleating substance by KUIN-3 was examined. In an ice-nucleating medium containing yeast extract, 30 g ; sucrose, 10 g ; DL-serine, 2 g ; DL-alanine, 2 g ; K_2SO_4, 8.6 g and MgSO_4・7H_2O, 1.4 g, in one liter of distilled water, maximum growth was obtained with 1.1 mg/ml of the extracellular ice-nucleating substance at 18 ^ﾟC after 48 hr of shaking.Cell-free ice nuclei were shed from Pseudomonas viridiflave (KUIN-2) cells with 2 % Triton X-100 in the presence of 10 mM EDTA-2Na. KUIN-2 shed cell-free ice nuclei (0.22 mg protein/ml) active at -4.9 to -5.8 ^ﾟC. The ice-nucleating activity was greatly decreased by treating the cell-free ice nuclei for 30 min at 40 ^ﾟC, and was completely lost by heating at 90 ^ﾟC. The ice-nucleating activity of the cell-free ice nuclei was inhibited by the addition of proteases, protein-modifying reagents and denaturants. It is suggested that the ice-nucleating activity sites of cell-free ice nuclei contain proteins. The ice-nucleating activity of cell-free ice nuclei containing Mg^<2+> occurred within the temperature range -3.3 to -3.6 ^ﾟC.

将噬夏孢欧文氏菌（Erwinia uredovora，KUIN-3）在pH 6.1的Simmons-柠檬酸肉汤培养基中于18 ℃有氧培养24小时，获得了KUIN-3细胞的冰核活性。在<50>KUIN-3细胞悬浮液（1.1 × 10^8个细胞/ml）中测得冰核温度为-2.8 ℃。在含有酵母提取物30 g，蔗糖10 g，DL-丝氨酸2 g，DL-丙氨酸2 g，K_2SO_4 8.6 g和MgSO_4·7H_2O 1.4 g溶于1 L蒸馏水中，1.1 mg/ml的细胞外冰成核物质在18 ℃下振荡48小时后获得最大生长。在10 mMEDTA-2Na存在下，用2%Triton X-100从绿黄假单胞菌（KUIN-2）细胞中脱落游离冰核。KUIN-2脱落无细胞冰核（0.22 mg蛋白质/ml）在-4.9至-5.8 ℃下有活性。无细胞冰核在40 ° C处理30 min后，冰核活性大大降低，在90 ° C加热后，冰核活性完全丧失。无细胞冰核的冰成核活性被蛋白酶、蛋白质修饰剂和变性剂抑制。这表明，无细胞冰核的冰成核活性位点含有蛋白质。含Mg^<2+>的无细胞冰核的冰核成核活性发生在-3.3至-3.6 ° C的温度范围内。