Vol.1 No.1 2008

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Research paper−7−Synthesiology - English edition Vol.1 No.1 (2008) the internal structure and allow preservation of quality and/or activity of these materials in the deep freezer or LN2. Special refrigerators that mechanically inhibit ice crystal growth have also been developed. Although these techniques are useful in sample preservation, they generate carbon dioxide (CO2). Global reduction of CO2 is a priority in addressing global warming. In seeking an alternative cryopreservation strategy, we began considering 1 Objective Antifreeze protein (AFP) can inhibit ice-growth by accumulating on the surface of ice nuclei formed in near-freezing water, and can preserve cell function under hypothermic conditions (-0 °C). The objective of this study was to develop new technologies so these properties of AFP can be applied in industry and medicine. Figure 1 compares the principles of current cryotechnology without AFP and the expected cryotechnology utilizing AFP. Common perception is that water freezes at 0 °C; however, spontaneous freezing at 0 °C hardly occurs when liquid water is placed in cold environment such as in the refrigerator (-18 °C). Unfrozen water below 0 °C is generally called supercooled water [1]. Freezing is triggered by natural formation of numerous ice nuclei in supercooled water (Figure 1A, top). The generated ice nuclei successively undergo crystal growth by adsorption of surrounding water molecules, and finally occupy the whole space (Figure 1A, bottom). Thus, a common block of ice is inevitably polycrystalline and not a single crystal of water molecule. The temperature range between -7 °C~0 °C is generally called the “zone of maximum ice crystal formation”. This means that within this temperature range, water-containing materials such as foods, cells, and tissues rapidly develop internal ice crystals [2]. Since rapid growth effectively destroys the inner structure of the materials, this temperature zone is not preferable for freezing storage. Conventionally, this problem was overcome using deep freezers that operate at - 60 °C ~ -80 °C or liquid nitrogen (LN2, -196 °C), as they reduce the exposure time of the materials to temperatures that promote ice crystal formation thereby minimizing crystal formation (Figure 1B). The minute ice crystals that form at these lower temperatures do not effectively destroy - Toward the practical use of biomolecules -Yoshiyuki Nishimiya, Yasuhiro Mie, Yu Hirano, Hidemasa Kondo, Ai Miura and Sakae Tsuda*Research Institute of Genome-based Biofactory, AIST Tsukisamu-Higashi 2-17-2-1, Toyohira-ku, Sapporo 062-8517, Japan *E-mail : s.tsuda@aist.go.jp.Antifreeze protein isolated from the blood serum of Arctic and Antarctic fish is an extraordinary biomolecule that binds to ice and preserves cell structure. We recently discovered that Japanese edible fish species also contain an antifreeze protein, and established a method of isolating the protein from fish muscles. We determined that the isolated antifreeze protein consists of a mixture of many isoforms that together are more active than any single isoform. Mass preparation of antifreeze protein is currently under investigation to provide material for a variety of studies and industries. Fig. 1 Comparison between current and expected cryotechnologies. Large circles in panels A~D represent supercooled water, and small hexagons indicate ice nuclei. The small circles shown in panels C~E represent AFP. Two large squares illustrated in C represent ice-nucleation plate by assembly of AFP. Large circle in E represents cell-preservation fluid containing AFP. A: Infinite numbers of ice nucleus are naturally created in supercooled water between -18 ºC and 0 ºC in a general freezing device. Each ice nucleus undergoes crystal growth, and the resultant polycrystalline occupies the whole space of water. B:Use of extremely low temperature (-196 ºC to -60 ºC) can effectively inhibit ice crystal growth, although it needs high-energy cost. C: AFP-assembled plate effectively freezes the attached water near 0 ºC. D: AFP can inhibit the ice crystal growth strongly even in general freezing device. E: AFP increases the viability of various cells near 0 ºC.Mass preparation and technological development of an antifreeze protein0 ℃-18 ℃〜-60 ℃-196 ℃〜-196℃〜0 ℃-3 ℃〜0 ℃-18 ℃〜4 ℃0 ℃〜AEDCBCurrent CryotechnologyFigure 1Expectative CryotechnologyKeywords : Antifreeze protein, 3D structure, ice-binding, mass preparation, cell preservation, ice nucleus plate[Translation from Synthesiology, Vol.1, No.1, p.7-14 (2008)]

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