Vol.1 No.1 2008

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Research paper : Mass preparation and technological development of an antifreeze protein (Y. Nishimiya, et al.)−11−Synthesiology - English edition Vol.1 No.1 (2008) technique, and purification processes. The arrows connect our present choices of elements, thereby indicating procedures to obtain highly purified AFP (thick arrow) and partially purified AFP (broken arrow). The open circles (〇) indicate detailed methods to purity AFP such as heat denaturation, centrifugation, filtering, and chromatography, which were carried out using suspension fluid of minced muscle of AFP-containing species of fish. These methods were initially chosen based on Figure 4 and were further optimized by considering cost and time required to improve the preparation efficiency. Removal of certain procedures and rearranging others also contributed to improvement. For example, high-performance liquid chromatography (HPLC) was not selected because of its inefficiency and high operational costs. As described, we chose AFP-containing fish as source material. After considering the quantity of fish resources and their price, we selected three species of edible fish which were captured at a fishery located on the east coast of Hokkaido and for which involvement of AFP types I~III were identified in our laboratory. These fish were not captured as merchandise but were “by-catches” of scallops and shrimps (i.e. they were food for by-catch fish). The by-catch fish are typically discarded as they have no market value. We were able to secure at least three tons of by-catch fish during the winter. Minced muscle was transferred to a cold storage warehouse for storage, and samples were transported to the laboratory as needed. For type III AFP, our efficiency of purification was approximately 3 g (99 % purity)/5 days/1 person. The collaborating company prepared crude AFP samples at 200-times higher efficiency than our laboratory, but this was still not the achievable upper limit. Figure 6A shows the appearance of approximately 10 g of high purity sample of type III AFP. Currently, we have amassed 240 g with market value of approximately USD 3 million. Raw material and preparation costs were extremely low.Acquisition of gram quantities of AFP enabled us to examine the cryopreservation effect of AFP on various water-containing substances such as processed food, soups, ice-sweets, noodles, bread, soft drinks, alcohol beverages, drugs, cosmetics, inks, polymer gels, polymer membranes, vegetables, fruits, seeds, meats, and seafood. Although the examinations are not complete, in principle, they are expected to become freeze tolerant by addition of AFP. For substance such as meat whose inner structure is complex, it is difficult to transfer AFP internally. Once the meat is minced, however, distribution of AFP is facilitated, and freeze tolerance can be expected.Figure 6 shows some examples. Figure 6B1 is a photograph of agarose gel after overnight storage at -18 ºC in a home freezer. As frozen gel began to thaw, water flowed out from the inside of the gel. This occurred because the agarose network of gel was destroyed by growth of ice crystals (Figure 1A). Similar phenomenon was observed in the thawing of frozen meats, fruits, and vegetables, and this water flow is called drip in meats. We found that addition of slight amount of AFP effectively preserved the inner structure of frozen gel and stopped the water flow, as shown in B2. This was attributed to the inhibition of ice crystal growth by strong binding of AFP to crystal surface (Figure 1D). It should be noted that strong preservation effect of AFP was observed in the temperature zone of maximum ice crystal formation, between -7 ºC and 0 ºC, implying that AFP can replace LN2 for high quality preservation of water-containing materials, and this may further save energy and reduce CO2 emissions. In this study, type III AFP was chemically or physically immobilized on a surface plane of metal by spraying a solution of highly purified protein (Figure 3). This type of AFP can form an ice-binding surface (represented by red and blue CPKs in Figure 3A), which binds specifically to a set of oxygen atoms of ice crystals [14]. This ice-binding site is located on the opposite side of the N-terminal end of this molecule. Given this knowledge, we assumed that if we connected the N-terminal end of type III AFP to the metal surface, the ice-binding surface would be directed outside of the metal surface. When performing this experiment, numerous AFP molecules should, in practice, be connected to the metal surface in that manner, creating a fairly large ice-binding surface on metal due to assembly of type III AFP molecules. Our assumption is that such large ice-binding surface will cause the assembly of ice-like structure in water placed on the surface. In other words, the AFP-assembled surface will have ice-nucleation function. Figure 6C is a photograph of an aluminum plate on which approximately Fig. 6 Outcomes of the present study. A: Approximately 11 g of highly purified type III AFP. The preparation efficiency of this sample is 3 g/5 days/person. B: Photographs of agarose gel in absence (B1) and presence of AFP (B2) after cold storage (-18 ºC). Addition of AFP can provide freeze tolerance to many water-containing materials. C: AFP-assembled aluminum plate that exerts ice-nucleation function. D: Cell preservation fluid containing AFP, which dramatically improves cell viability under hypothermic condition (0 ºC). B1B2ACDFigure 6

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