Mackerel Trypsin Purified from Defatted Viscera by Supercritical Carbon Dioxide

Viscera of mackerel (Scomber sp.) were defatted by supercritical carbon dioxide (SCO2) treatment. Trypsin (SC-T) was then extracted from the defatted powder and purified by a series of chromatographies including Sephacryl S-200 and Sephadex G-50. The purified SC-T was nearly homogeneous on SDS-PAGE, and its molecular weight was estimated as approximately 24,000？Da. N-terminal twenty amino acids sequence of SC-T was IVGGYECTAHSQPHQVSLNS. The specific trypsin inhibitors, soybean trypsin inhibitor and TLCK, strongly inhibited the activities of SC-T. The pH and temperature optimums of SC-T were at around pH 8.0 and , respectively, using Nα-p-tosyl-L-arginine methyl ester as a substrate. The SC-T was unstable below pH 5.0 and above , and it was stabilized by calcium ion. These enzymatic characteristics of SC-T were the same as those of other fish trypsins, especially spotted mackerel (S. borealis) trypsin, purified from viscera defatted by acetone. Therefore, we concluded that the SCO2 defatting process is useful as a substitute for organic solvent defatting process. 1. Introduction Fish viscera are one of the sources of digestive enzymes that may have some unique properties of fascinate with both basic research and industrial applications. Their survival in waters required adaptation of their enzyme activity to low temperatures of their habitats. That is to say, fish proteinases have higher catalytic efficiency at low temperatures than those from warm-blooded animals [1, 2]. In addition, the strong positive correlation between the habitat temperature of marine fish and the thermostability of its trypsin has been demonstrated [3–11]. High activity at low temperatures and instability against heat, low pH, and autolysis of fish proteinases are interesting for some industrial applications [12]. Cod trypsin is already practically used in food production and cosmetics [13, 14]. Furthermore, Pacific cod and Atlantic cod trypsins were utilized as catalyst of enzymatic peptide synthesis [9, 15]. On the other hand, lipids in the tissue prevent from extracting, preparing, and purifying enzymes [16]. Conventional methods for the removal of lipids from materials involve cooking, pressing, and liquid extraction. On liquid extraction for enzyme preparation, it is usually used with organic solvents, such as hexane, ethanol, and acetone, and so forth [16, 17]. However, The removal of lipids with organic solvents causes protein denaturation and/or loss of functional properties [18]. Organic solvents are also highly flammable and are toxic for human health. Consideration of