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P006 Effects of amino acids on the prevention of freeze-dried enzyme inactivation * Paveena Srirangsan1, Kiyoshi Kawai2, Naoko Hamada-Sato3, Manabu Watanabe1and Toru Suzuki1 1 Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan 2 Department of Biofunctional Science and Technology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan 3 Course of Safety Management in Food Supply Chain, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan Enzymes (and other biological materials) are commonly stabilized by freeze-drying with protectant molecules. Disaccharides (e.g., sucrose, trehalose) are the most popular among them because they stabilize enzymes both thermodynamically and kinetically. Similar protective effects of amino acids are attracting increased attention because of the varied physical (e.g., crystallinity, glass transition temperature) and chemical (e.g., forming complexes with other molecules) properties of the freeze-dried amino acid . In addition, some amino acids (e.g., arginine, glycine, glutamic acid, lysine) also prevent aggregation in aqueous solutions prior to the drying process and after reconstitution [2, 3]. However, up until now there have been few studies on the stabilizing effects of amino acids on freeze-dried enzyme. In the present study, therefore, a model enzyme (xanthine oxidase: XOD) was freeze-dried with 10 different kinds of amino acid, sucrose, trehalose, and their mixtures, and consequently, activities of the enzyme remaining after freeze-drying of each formulation were measured using UV-VIS spectrophotometer. Freeze-drying of XOD in the absence of stabilizing additive resulted in significant reduction of the activity to approximately 20%. Higher activity was remained for the enzyme dried with sucrose, trehalose or amino acids. Sucrose maintained XOD activity of 70%. Trehalose was less effective which about 50% of the activity was remained. Among 10 amino acids tested, only arginine showed the greater protective effects on the dried XOD than the disaccharides which 80-90% of the activity was preserved. The other amino acids, including glutamine, glycine, histidine, leucine, lysine, phenylalanine, proline, and valine, stabilized the enzyme in the same or less degree to those of the disaccharides. On the other hand, glutamic acid, the negatively charged amino acid, conversely failed to protect the enzyme. Combined using of the disaccharides and the positively charged amino acids (i.e., arginine, histidine and lysine) exhibited the relatively high stabilizing effect comparing to the use of disaccharide individually, but showed no different protective effect in comparison with those of the arginine alone formulations. On the other hand, the activity of XOD freeze-dried with the disaccharides and the remaining amino acids was maintained in the same or less degree to those of the disaccharide alone formulations, but greater or equally well preserved depending on the type of the disaccharide used, than the amino acid alone formulations.  Izutsu, K., Kadoya, S., Yomota, C., Kawanishi, T., Yonemochi, E., and Terada, K. (2009). Freeze-drying of proteins in glass solids formed by basic amino acids and dicarboxylic acids. Chem. Pharm. Bull., 57, 43-48.  Tsumoto, K., Umetsu, M., Kumagai, I., Ejima, D., Philo, J.S., and Arakawa, T. (2004). Role of arginine in protein refolding, solubilisation, and purification. Biotechnol. Prog., 20, 1301-1308.  Zhang, M.Z., Pikal, K., Nguyen, T., Arakawa, T., and Prestrelski, S.J. (1996). The effect of the reconstitution medium on aggregation of lyophilized recombinant interleukin-2 and ribonuclease A. Pharmcol. Res., 13, 643-646.
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