Two protease inhibitors of 67 and 18 kDa, respectively, were purified from glassfish, Liparis tanakai, eggs by affinity chromatography. The smaller protein was purified with a yield and purity of 0.25% and 49.69-fold, respectively, and was characterized for further study. The glassfish egg protease inhibitor exhibited stability between 50 and 65 degrees C in an alkaline environment (pH 8). It was shown to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 4.44 nM. Potent glassfish protease inhibitor with N-Val-Gly Ser-Met-Thr-Gly-Gly-Phe-Thr-Asp-C amino acid residues was synthesized and its inhibitory activity was compared. Moreover, the 18-kDa protein inhibited cathepsin, a cysteine protease, more effectively than did egg white protease inhibitor, whereas the reverse was true for papain. Glassfish egg protease inhibitor is classified as a member of the family I cystatins.

α-Glucosidase inhibitory activities of the various solvent fractions (n-hexane, CHCl3 , EtOAc, BuOH, and water) of sea cucumber internal organ were investigated. 1,3-Dipalmitolein (1) and cis-9-octadecenoic acid (2) with potent α-glucosidase inhibitory activity were purified from the n-hexane fraction of sea cucumber internal organ. IC50 values of compounds 1 and 2 were 4.45 and 14.87 μM against Saccharomyces cerevisiae α-glucosidase. These compounds mildly inhibited rat-intestinal α-glucosidase. In addition, both compounds showed a mixed competitive inhibition against S. cerevisiae α-glucosidase and were very stable at pH 2 up to 60 min. The KI values of compounds 1 and 2 were 0.48 and 1.24 μM, respectively. Therefore, the internal organ of sea cucumber might be a potential new source of α-glucosidase inhibitors suitably used for prevention of obesity and diabetes mellitus.

Microalga Chlorella vulgaris has a wide application as a source of biomass fuel, natural food col oring agent and dietary supplement. Its cell wall consists of multiple layers including cellulose and in order to utilize microalgae, the pre treatment is necessary to breakdown rigid cell walls. β glucosidase (EC 3.2.1.21) is a crucial enzyme to hydrolyze cellulose efficiently. In this study, β glucosidase produced by Trichoderma citrinoviride cultivated on microalgae C. vulgaris was purified to homogeneity with a recovery of 8.5% and spe cific activity of 168.7 U/mg. The purified enzyme was obtained as a single band with the molecular mass of 110 kDa on SDS-PAGE. The optimum pH and temperature for enzyme activity and stability were 4.0 and 50°C, and 8.0 and 30°C, respectively. Metal ions (Mg 2+ , and Zn 2+ ) activated the enzyme activity, whereas SDS inhibited moderately. K m , V max , K cat , and K i values of β glucosidase were 0.96 mM, 300.42 μmol min -1 mg -1 , 2.73 min -1 and 2.83 mM using p nitrophenyl β D glucoside as a substrate, whereas they were 4.30 mM, 24.34 μmol min -1 mg -1 , 0.022 min -1 and 0.53 mM using cellobiose, respectively.

Reactive oxygen species (ROS) are a toxic product of aerobic metabolism which is generated abundantly in inflammatory tissues. Oxidative stress induces the production and the release of mast cell mediators. H 2 O 2 upregulates IL-4 and IL-6 genes expression and cytokine secretion through an apurinic/apyrimidinic endonulease (APE/Ref-1)-dependent pathway in mast cell (1) . Tumor necrosis factor-a (TNF-a) can increase the level of ROS and activate NF-kB which is responsible for up-regulation of matrix metalloproteinases (MMPs) (2) . MMPs belong to the family of neutral endopeptidases which participate in many physiological processes including tissue remodeling and inflammation. In this study, the biofunctional activities of squid milt hydrolysates on antioxidant and MMPs inhibitory activity were investigated.Squid milt was hydrolyzed with trypsin at 50 C for 5 h in 0.05 M phosphate buffer, pH 8. The squid milt hydrolysate was purified using 10, 5 and 3 kDa cut-off ultrafiltration membranes. Ultrafiltration fractions were freeze-dried and further used for antioxidant and MMPs inhibitory activity assessment (3) . Fig. 1. Superoxide radical scavenging activity of squid milt hydrolysate (100 mg/mL). Fig. 2.Hydroxyl radical scavenging activity of squid milt hydrolysate (100 mg/mL). Fig. 3. MMPs inhibitory activity of squid milt hydrolysate (100 mg/mL).There was no significant difference between < 3 kDa and 5-10 kDa molecular weight of squid milt hydrolysates (p > 0.05) on superoxide radical scavenging activity (Fig. 1), but significantly different on hydroxyl radical scavenging activity (Fig. 2). The high molecular weight of fraction (5-10 kDa) had highest activity on MMPs inhibition (p < 0.05). In conclusion, the squid milt hydrolysate with high molecular weight (5-10 kDa) had strong antioxidant and MMPs inhibitory activity. These results may useful to promote squid milt which is fishery industry by-product as material for biofunctional food resources with the benefits as antioxidant and antiinflammation.