Purification and properties of an ice-nucleating protein from an ice-nucleating bacterium, Pantoea ananatis KUIN-3.
An ice-nucleating protein (INP) from the extracellular ice-nucleating matter (EIM) of Pantoea ananatis (Erwinia uredovora) KUIN-3 was purified and characterized. The EIM produced by the strain KUIN-3 was purified by ultrafiltration, sucrose density-gradient ultracentrifugation and gel filtration. The INP was purified using of column chromatography on hydroxyapatite and Superdex 200 in the nondenaturing detergent of 0.1% (w/v) Triton X-100. The purified INP was composed of one subunit of 117 kDa according to SDS-PAGE. It has become apparent that the INP was the ice-nucleating lipoglycoprotein based on the reaction of carbohydrate stain and lipid stain with the INP. It was inhibited by p-mercuribenzoate and N-bromosuccinimide. The activity of the INP gradually decreased from 65 degrees C. The pH stability was held between pH 7.0 and pH 11.0. The INP had a lower ice-nucleating temperature below pH 6.0. It has become apparent that the INP consisted of the class C structure in the EIM based on its freezing difference spectrum in D2O versus H2O.
Muryoi
Naomi
N
Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka 564-8680, Japan. gm0d801@ipcku.kansai-u.ac.jp
Matsukawa
Kenji
K
Yamade
Kazuhiro
K
Kawahara
Hidehisa
H
Obata
Hitoshi
H