The term ice nucleation describes the initiation of the phase transition of water from a liquid to a solid state. When a water sample of moderate size is cooled, it will normally not freeze at 0°C. If the water is pure, it can be cooled to temperatures near to -40°C before it freezes. Liquid water at temperatures lower than 0°C is termed supercooled water, and this supercooled state is metastable. To enable ice formation to take place, water molecules must cluster in an ice-like pattern and this cluster must reach a critical size. If the initial aggregation of water molecules takes place on a foreign structure, the process is termed heterogeneous ice nucleation. If the water molecules aggregate without the help of another structure, the nucleation is termed homogenous.
A structure that organizes water into an ice-like pattern so that nucleation takes place is called an ice nucleator and responsible for heterogeneous ice nucleation. However, the term is usually not used for substances inducing ice nucleation at temperatures lower than -10°C. Many bacteria have ability to minimize freezing injury due to extracellular ice formation and impose an ice like arrangement on the water molecule in contact with their surface and lower the energy necessary for the initiation of ice formation. Ice nucleation activity of bacteria provides cold protection from the release heat of fusion or establish protective extracellular freezing instead of lethal intracellular freezing. The "ice plus" bacteria posses INA protein (Ice nucleation-active protein) found on the outer bacterial wall acts as the nucleating center for ice crystals. This protein located on the outer membrane of these bacteria is responsible for ice nucleation. Genes conferring ice nucleation activity have sequenced from many bacterial strains showed INPs of 120~180 kDa, with similar primary structure. This protein was lipoglycoproteins complexes that form large membrane-bound aggregates. INPs comprises of continuous repeat of a consensus octapeptide (Ala-Gly-Thr-Gly-Ser-Thr-Leu-Thr) and function as templates for the formation of small ice crystal seeds termed "ice nuclei". This facilitates ice formation at high subzero temperature, while "ice minus" bacteria do not posses Ina proteins and lower the ice nucleation temperature. Turner et at. (1990) have classified ice nucleating protein into three chemically distinct class depending on A, B, C structure. The class C structure was composed of aggregates of ice- nucleating protein (INP), the class B structure was a glycoprotein with sugar residue including glucose, mannose etc., attached to the protein core, and the class A structure was a lipoglycoprotein that was covalently anchored to the cell surface via a mannose-PI (phosphotidylinositol) that is similar to the anchoring of many proteins to cell membranes I eukaryotic cells.
The ice nuclei activity has been classified by the range of temperature in which they initiate freezing: type 1 ice nuclei are active between -2°C to -5°C, type 2 are active between -5°C to -7°C and type 3 are active between -7°C to -10°C. Very potent ice nucleators, active at high subfreezing temperature, are produced by bacteria such as Erwinia herbicola. Other bacterial genera viz., Pseudomonas, Pantoea (Erwinia) and Xanthomonas can nucleate the crystallization of ice from super-cooled water.
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