Purification and Characterization of a New Anti-Nucleating Protein Isolated from Acinetobacter calcoaceticus KINI-1

抄録

Strain KINI-1 capable of producing an anti-nucleating agent was isolated from a camphor leaf. Strain KINI-1 was identified as Acinetobacter calcoaceticus from its characteristics and taxonomics; the optimum temperature and pH for the production of the anti-nucleating agent were 30°C and 7.0, respectively. As the activity of this agent disappeared with the application of protease treatment, this agent, which was an anti-nucleating protein (ANP) was partially purified from the culture to an electrophoretically main band state by ultrafiltration, acetone precipitate, QA-52 column chromatography and Toyopearl HW 55S and Superose 12 gel filtration chromatography. The molecular weight of the ANP estimated by sodium dodecylsulfatepolyacrylamide gel electrophoresis was 55 kDa. The ANP had various substrate specificities for ice nuclei from various ice-nucleating bacteria and AgI. It was demonstrated that the antinucleating activity of ANP increased in proportion to the protein concentration and the antinucleating activity of ANP against ice nucleus from the cells of Erwinia uredovora KUIN-3 was 2.2°C at a concentration of 12.5μg/ml. Furthermore, we found that this ANP was bound to the surface of the ice nuclei, thereby causing anti-nucleating activity. The surface hydrophobicities of the ice nuclei were increased after binding with ANP, whereas the surface charge was changed from 6.6 to 6.1. Also, we confirmed that this action by ANP could give rise to a supercooling point at -5.0°C in the cell suspension of Erwinia uredovora KUIN-3.