Vol.1 No.1 2008

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Research paper : Mass preparation and technological development of an antifreeze protein (Y. Nishimiya, et al.)−9−Synthesiology - English edition Vol.1 No.1 (2008) further, we conducted an extensive survey of cold-adapted organisms in Japanese coastal waters with assistance of the Marine Bio-Institute of Sapporo Medical University and the Federation of Fisheries Cooperative Association of Notsuke. Various samples were also purchased from food stores and insect shops in Sapporo City. AFP activity was assayed using a photomicroscope system that enables detection of the ice-shaping ability using only 1 µL of sample [11]. Over 160 species of Japanese fish were examined, of which at least 50 species contained AFP. In addition, AFP was found in tissues of Japanese plants (wheat), insects (stag beetles), and fungi (basidiomycete).Interestingly, AFP activity was detected in fish fillets, minced fish, and dried fish purchased from food markets. On detailed examination, it was found that AFP could be purified from fish muscle as well as from serum (Figure 2A). The figure shows that larger amount of AFP was purified from samples obtained from heart tissue. Since presence of high amounts of contaminants such as lipids, enzymes, and sticky substances necessitated additional purification steps, which lowered the preparation efficiency of AFP, fish muscles may represent the best choice for mass preparation of AFP.AFP isolated from the Japanese fish was compared with the established version recovered from cold-adapted polar species for DNA and amino acid sequences, 3D structure, and ice binding ability. Although these analyses are ongoing, so far it was found that the amino acid sequences were similar, allowing their grouping as Types I~III. Similar result was also obtained for AFP from Japanese insects. Every Japanese organism expressed AFP as isoform mixture, as was the case for AFP from polar organisms. Especially, sample of eelpout (a species that lives off the east coast of Japan) was found to produce at least 13 isoforms of type III AFP. Initially, the presence of such large number of isoforms was perplexing. However, isoform mixtures of AFP possess higher antifreeze activity (thermal hysteresis) than single isoform [12]. Even an isoform with poor individual activity displayed enhanced activity in the presence of small amount of higher activity isoform (Figure 2B). Such cooperative enhancement was also identified for cell preservation activity of AFP in hypothermic conditions [13]. One should note that it is difficult to produce isoform mixture of AFP by genetic engineering and chemical synthesis.The above findings prompted the scenario summarized in Figure 3 for practical use of AFP. The characterization and functional analysis of Japanese AFPs define the direction of the development of AFP technology (Figure 3A). Next, preparation of relatively large amounts of AFP isoform mixture can be achieved using fish muscles as raw material (Figure 3B). The isoform mixture can be used to perform various tests (Figure 3C), and holds potential for scale-up to industrial quantities. 4 Factors that determine technology Highly purified sample is indispensable for research. In contrast, even poorly purified sample is generally sufficient for industrial and commercial use. In fact, enzyme used in detergents and food processing is partially purified product of cultivation [9], offering an attractive saving in preparation costs. Partially purified AFP can also be utilized in food industry and in ice slurries, since AFP functions in a solution containing large amount of impurities. In the rules and regulations of the Japanese food industry, partially purified AFP is categorized as natural extract while highly purified AFP is considered food additive. The former can be applied directly to various foods after careful safety check Fig. 3 Example of “Full Research” for AFP. A: Biochemistry and molecular biology of AFP (Type 1 Basic Research). The ribbon representation shows a structure of type III AFP (PDB code = 1MSI) where polar atoms colored in red and blue constitute an ice-binding surface. B: Development of mass preparation technique of AFP (Type 2 Basic Research). C: High yields of AFP allow research collaborations with researchers from different backgrounds, leading to practical use and industrialization of AFP. Fig. 4 Evaluations of factors such as activity and resource availability for AFP species (isoform mixture) from fish A, B, C, and so on, needed for technological developments. We divided the evaluation results into four grades: ++ (excellent), + (good), − (poor), and −− (very poor) (see text). The evaluation for element indicated by thick-framed box is an ordinary biochemical study. Activity and resource availability are found to be especially important for advancing the technological developments of AFP.Collborations with many different fieldsCryo preservation ofwater-containing materialACNCBFigure 3Ice slurry techniqueAFP-assembled materialpromoting ice-nucleationHypothermic preservationof cells and tissuesPractical use, IndustrializationA・・・・・・DCBFigure 4+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +++++ActivityResource availabilityMolecular sizeStructural rigidityInfrastructureAcid/alkaline toleranceHeat Stability

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