Myosin is the most important protein in surimi. Reasonably, we employed ATPase activity as an indicator for evaluation of quality of frozen surimi. We established anew ATPase assay system using pH stat. ATP hydrolysis generates H+ together with inorganic phosphate (Pi). The principle of the method is to measure the H+ by titrating with NaOH solution. Consequently, ATP hydrolysis was recorded as a continuous NaOH consumption. Surimi homogenate prepared on Polytron was directly used for the protein content measurement and ATPase assay. Surimi samples with various grades were analyzed. There was a tendency that high grade surimi had a higher ATPase activity. There was also a positive relationship between ATPase activity and the increase in the breaking force and strain by setting for the gel made from the analyzed surimi.

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According to the three-dimensional contour maps showing the gel-forming properties of surimi, 8 freshwater fish species surimis were classified into two types. The V-valley type surimi (silver carp, big-head carp, Chinese snake head and blunt snout bream) shows easy setting, low resistance to gel collapse, high enhancement ability with two-step heating, and narrow optimum heating temperature and time area, which are the same characteristics as walleye pollack surimi. In contrast, the Plateau type surimi (tilapia, grass carp, mud carp and common carp) illustrates difficult set, high resistance to gel collapse, no enhancement ability with two-step heating, and wide optimum heating temperature and time area.
There are seasonal changes of gelling properties of silver carp surimi, and the setting ability of surimi gel is higher in winter and lower in summer. In addition, thermal stability of myofibrillar protein from silver carp surimi is higher in summer and lower in winter. Therefore, the setting ability of surimi gel is evidently affected by thermal stability of myofibrillar protein of surimi, that is, the larger the inactivation rate of myofibrillar protein, the higher the gelation rate of setting.

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The quality of kamaboko gel prepared from walleye pollack surimi with sodium gluconate (Na-gluconate) was investigated in connection with the changes in myofibrillar protein accompanied with gelation. The gel stiffness of the kamaboko gel formed with Na-gluconate was higher than that with sorbitol at the same breaking strength, suggesting that the kamaboko gels with Na-gluconate were harder but not elastic. This characteristic of the kamaboko gel was mainly developed by the subsequent heating at 90°C, which caused no cross-linking reaction of myosin heavy chain. Furthermore, Na-gluconate strongly suppressed the progress of the myosin denaturation in the salt-ground meat. It was, therefore, suggested that Na-gluconate affected the formation of noncovalent intermolecular interaction between proteins through suppressing the myosin denaturation during preheating and promoted the production of the kamaboko gels with higher gel stiffness.

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Milkfish flesh is light pink due to the presence of myoglobin (Mb). The amino acid composition of milkfish Mb shows higher content of thr, glu, and arg, but lower content of ala and lys than those of mackerel, sardine, skipjack, and tuna Mb. The tristimulus color value (L, a, and b value) of Mb solutions changed significantly depending upon the form of Mb. When incubation time increased, the L value of Mb solution increased gradually with the increase in the percentage of metmyoglobin (metMb), while a value decreased and Mb aggregation gradually increased. Washing with acidic media could improve the lightness of milkfish meat paste than those in neutral or alkali pH. The reduction in pH value of flesh might enhance the autoxidation reaction of Mb that consequently improved the lightness of milkfish meat products.

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Species-specificity of fish fast skeletal myosin in physicochemical changes during thermal treatment was investigated for white croaker and walleye pollack. Temperature sweep analysis showed that white croaker myosin exhibited a sol-gel transition in three steps at 30-36 C, at 40-52 C, and over 60 C. On the other hand, walleye pollack myosin formed a thermal gel in two steps in a wide temperature range of 29-46 C and over 58 C. Since these temperature ranges were roughly consistent with those shown for endothermic peaks in DSC measurment, it is most likely that the thermal unfolding of myosin and subsequent hydrophobic binding are involved in gel formation. Frequency sweep analysis at 80 C showed that the shear force of rabbit heat-induced myosin gel as a control was the strongest, followed by those of white croaker and walleye pollack. These results suggest that different myosin gel profiles are derived from species-specific intrinsic properties of myosin.

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Inspections of the skeletal muscle of walleye pollock Theragra chalcogramma caught in Alaska revealed the presence of cysts of an unidentified microsporian and multi-nucleate bodies of lchthyophonus hoferi. This is the first study of the effects of lchthyophonus infestation on the quality of walleye pollock. The infested muscle contained protease, which has the ability to degrade walleye pollock myofibriliar proteins, mainly the myosin heavy chain at temperatures around 50 to 60°C, in vitro. Cysteine protease inhibitors were able to reduce the enzyme activity whereas specific inhibitors of serine proteases and aspartic proteases were ineffective.

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Differences in gel-forming ability of two species of bigeye snapper, P. tayenus and P. macracantus are associated with the differences in some intrinsic properties of muscle. Superior gel-forming ability of P. tayenus to P. macracanthus is attributable to higher thermal stability, higher rate of aggregation via the formation of both hydrophobic interaction and disulfide bonding. In addition, the differences in both extent and molecular properties of endogenous muscle transglutaminase and proteinases between two fish species may be responsible for the differences in gel-forming ability of surimi due to gel setting and gel weakening. Corresponding to the optimum temperatures of crude muscle transglutaminases, P. tayenus and P. macracanthus surimi exhibited optimal setting temperature at 40 and 25°C, respectively. Higher activity of both sarcoplasmic and myofibril-associated proteinases active at elevated temperature of P. macracanthus muscle can lead to higher extent of protein degradation during storage and gel softening during thermal gelation. Deterioration rate of P. tayenus muscle during iced-storage is much slower than P. macracanthus muscle. As a consequence, breaking force of surimi produced from the P. tayenus decrease at a slower rate, compared to that obtained from the P. macracanthus Pretreatment of fish before storage by deheading and evisceration can improve storage stability of fish and slow down the loss of surimi gel quality.

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Enzymes are biological catalysts accelerating chemical reactions and are important tools for improving product quality or processing operations. Among the enzymes used in food processing, hydrolases consist of the largest portion, and proteases mark the second largest group following carbohydrases. Most proteases are used in the variety of food products such as agricultural, dairy, meat, and fish products. Among the potential applications, the proteases from marine animals have been most successfully used as seafood processing aids such as accelerating fermentation of fish sauce and fish protein hydrolysates, improving quality of seafood flavoring agents, improving yields of caviar products, and simplifying deskinning and descaling process. The advantages of using proteases from marine animals in specific applications are due to their unique properties.

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Because of its numerous industrial and research application trypsin is by far the most thoroughly characterized enzyme. Interestingly, trypsins from cold-adapted marine fish show substantially higher catalytic efficiencies than their mammalian equivalents. Two isoforms of trypsin purified from anchovy appeared to be catalytically more active than bovine trypsin with one isoform being as much as 35 times more efficient. Primary structural alignment revealed some noteworthy features, which were further substantiated by comparative three-dimensional structural modeling. Two unique deletions in autolysis loop, lack of Tyr-151 pointing toward the S1 pocket and electrostatic surface potentiality around S1 pocket of anchovy trypsin have been discussed with regard to their structural significances compared to bovine trypsin. The molecular mechanisms underlying this uniqueness are still under speculation, but such knowledge would be invaluable in understanding the structural plasticity of enzyme function as well as for the development of an enzyme with novel specificity.

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In order to clarify the function of fish collagenase in tissues, We investigated substrate specificities of its recombinant enzymes. The enzymes were expressed in E. coil with cDNAs of collagenases such as matrix metalloproteinases 2 and 13 (MMPs-2 and -13), which have been isolated from a rainbow trout fibroblast cDNA library. MMPs-2 and -13 were found to have limited activities against rainbow trout type V collagen and to cleave rainbow trout type I collagen and its gelatin together with severe fragmentation, respectively, at a low temperature. Moreover, expression analysis has revealed the distribution of these MMPs in several tissues of rainbow trout. Therefore, they appear to play a crucial role in the collagen catabolism of rainbow trout.

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Tissue-type transglutaminase (TGase: EC 2. 3. 2. 13) is a calcium-dependent enzyme that catalyzes the cross-linking and amine incorporation of proteins by an acyl transfer reaction. It is widely distributed in various tissues of fish and aquatic invertebrates, as well as mammals. The TGases from marine invertebrates such as bivalves and gastropods are strongly activated with NaCl and Ca²⁺ concentrations found in normal seawater. For example, the activity of scallop adductor muscle TGase increases 11-fold in 0.5M NaCl and 10mM CaCl₂, while the activity of carp muscle enzyme is independent of salt concentration. Freshwater shellfish TGases are not activated with increased NaCl. These findings suggest that the physiological functions of marine invertebrate TGases are closely related to their surroundings. As marine bivalves and gastropods are osmoconformers and have an open blood-vascular system, their muscle cells are exposed to hemolymph that is isosmotic to seawater. When the muscle membrane is damaged by mechanical wounding or biochemical destruction, tissue TGase may be activated by contact with hemolymph and/or seawater, and thereby participate in the healing process.

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Arylsulfatase from Sphingomonas sp. nov. was purified through ionic exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 64 kDa by SDS-PAGE. The enzyme showed an optimum reaction condition at pH 7.0 and 45°C. With the reaction of agar with arylsulfatase in the ratio of 1:10^-6 (w/w) for 2 hr at 50°C, gel strength of agar was increased to 2.3 fold and 97% (p<0.01) of sulfate in agar was removed. The result suggests that arylsulfatase could be applicable to the production of value-added products such as electrophoretic grade agarose.

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We show the distribution, characterization, and application of chitinolytic enzymes in squid, especially chitinase. Measurement of chitinase and β-N-acetylhexosaminidase activities was investigated in the various organs of four species of squid. Both enzyme activities were detected in the digestive organs and liver of all species examined. Chitinases were purified from the liver of Japanese common squid and arrow squid by ammonium sulfate fractionation and column chromatography. The purified enzymes were basic chitinases with molecular masses of 38-42 kDa. The chitinases hydrolyzed glycol chitin and colloidal chitin, but not pNp-GlcNAc and Micrococcus lysodeikticus. Squid liver chitinases hydrolyzed GlcNAc₄ to two molecules of GlcNAc₂, GlcNAc₅ to GlcNAc₂ plus GlcNAc₃, and GlcNAc₆ to two molecules of GlcNAc₃, and GlcNAc₂ plus GlcNAc₄. Preparation of GlcNAc, which is used in food industry, was investigated using crude enzyme solution prepared from the liver of Japanese common squid. The enzyme decomposed insoluble chitin and produced reducing sugar. GlcNAc was detected in the enzymatically hydrolyzed colloidal chitin solution.

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For more than 20 years we have been trying to isolate metabolites with medicinally important activities from Japanese marine invertebrates, including sponges, soft corals, nudibranchs, and tunicates, which resulted in the discovery of more than 300 new compounds; some of them are structurally unique and show potent biological activities. Although none of our compounds entered clinical trials, several compounds whose modes of action were elucidated to some extent are used as research tools. Problems in drug development from marine organisms and their solutions are discussed.

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Inhibitors obtained from macroalgae against α-glucosidase and urease were investigated. Alpha-glucosidase inhibitors obtained from the red algae of Rhodomelaceae family were identified as bromophenols. Symmetric bisbenzyl ethers strongly inhibited Saccharomyces cerevisiae α-glucosidase, compared with the others. Inhibitors against α-glucosidase and urease were obtained from the brown algae Pelvetia babingtonii (Fucaceae) and Alaria crassifolia (Alariaceae). The inhibitors were identified as phlorotannins. P. babingtonii phlorotannin decreased blood glucose levels in sucrose-loaded rats.

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Thirty-five clones belonging to twelve dinoflagellate species have been isolated from coastal waters of Taiwan and cultured since 1996. Crude extracts of them were screened for bioactive compounds by mouse and brine shrimp toxicity assays. Fifteen of the 35 tested clones (42%) showed potent mouse toxicity and 19 of the 34 tested clones (56%) showed brine shrimp toxicity. Clones belonging to Amphidinium carterae, A. klebsii, Gambierdiscus toxicus, Gyrodinium instriatum, Ostreopsis lenticularis, Prorocentrum lima, and an unknown Prorocentrum species were found toxic in one or both animal assays, but not all the clones of the same species were toxic. From P. lima PL01 and P. sp1. PM08, several derivatives of okadaic acid and prorocentrolide were isolated to be active components. Among them, spiro-prorocentrimine was purified in crystal and its relative configuration resolved as the first one in this group of dinoflagellate toxins. 14-O-acetyl-4-hydroxyprorocentrolide, 4-hydroxyprorocentrolide and prorocentrolide were also found scattered in different species of Prorocentrum, and not limited to P. lima only. From P. lima, OA-D9d, DTX-4b and DTX-6 were isolated as new okadaic acid diol esters, together with several known OA analogues and derivatives in this research.

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Four new triterpenoid saponins, erylosides G-J (1-4), were isolated from the sponge Erylus nobilis collected from Jaeju Island, Korea. Based upon the results of combined chemical and spectral analyses, the structures of the aglycones were determined to be lanostane-based, modified penasterols. The oligosaccharide portions were composed of one unit each of L-arabinose, D-galactose, and 2-N-acetyl-D-glucosamine (1 and 3) or two units of L-arabinose and one unit of 2-N-acetyl-D-glucosamine (2 and 4). These compounds exhibited moderate cytotoxicity against a human leukemia cell-line K 562.

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A monoclonal antibody-based immunoassay for localization of tetrodotoxin (TTX) in the skin of a brackishwater puffer Tetraodon steindachneri (“hachinojifugu”) was investigated. Deparaffinized skin sections were incubated with 25% goat serum for 30min to block non-specific binding sites. Blocked sections were incubated with monoclonal TTX antibody diluted 1: 100 in D-PBS for 1h, and incubated again with goat anti-masse lgG biotin conjugate diluted 1:200 goat serum for 30min. Specific binding sites ware detected by incubating the section in a mixture of 20mg DAB and 0.1ml of 5% H₂O₂ in 100ml of 0.05M Tris-HCl buffer (pH 7.6) for 10-12 sec. TTX was recognized in the undifferentiated basal cells and succiform cells in the skin under light microscope. Malphigian cells of the skin did not exhibit any TTX antigen. Neither gland nor enclosed gland-like apparatus possessing TTX was observed in the skin. On the otherhand, anatomical distribution of TTX in T. steindachneri was also reported in this paper, showing the highest toxicity (90-220 MU/g) in the skin, followed by muscle (5.4-15 MU/g).

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cDNA encoding the sea hare-derived antitumour-antibacterial protein Aplysianin A (APA) was expressed on Escherichia coll. APA displays sequence similarity to L-amino acid oxidase (LAAO) found in the snake (Crotalus atrox) venom. Spectrophotometric analysis along with thin layer chromatography of APA indicated that one flavin adenine dinucleotide, a cofactor of LAAO, bound to each subunit of the homotetramer. APA also displayed LAAO activity. Specifically, treatment of L-phenylalanine with APA resulted in the generation of hydrogen peroxide. This finding indicated that the antibacterial activity of APA is mainly caused by hydrogen peroxide production.

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