Hisanori Nozawa

Partial Purification and Characterization of Six Transglutaminases from Ordinary Muscles of Various Fishes and Marine Invertebrates

Six transglutaminases were prepared from ordinary muscles of scallop (Patinopecten yessoensis), botan shrimp (Pandalus nipponensis), squid (Todarodes pacificus), carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), and atka mackerel (Pleurogrammus azonus), and their physicochemical and enzymatic properties were compared with each other. The Km value of carp transglutaminase for monodansyl cadaverine, a kind of primary amines, was 0.33 mM, while the other enzymes had Km values of 0.01-0.03 mM. The Km, values for succinylated casein of scallop, botan shrimp, squid, carp, rainbow trout, and atka mackerel enzymes were 1.2, 0.3, 1.8, 0.3, 0.2, and 0.1 mg/ml, respectively. In the presence of 0.5 M NaCl, the activities of scallop, botan shrimp, and squid transglutaminases were further enhanced about 11-, 2-, and 6-fold, respectively. There was no effect of NaCl on the activities of fish enzymes. These increment in the activities were dependent of NaCl concentrations and could be exhibited by using KCl instead of NaCl. To date there are no reports on types of transglutaminases whose activities are stimulated by salts. These findings suggest there exist a novel type of TGase in marine invertebrate muscles in which the osmotic pressure is isotonic to sea water.

Effect of neutral salts on activity and stability of transglutaminase from scallop adductor muscle

Abstract A sodium chloride-stimulated transglutaminase (TGase) is widely distributed in the muscle of marine invertebrates. The TGase from the adductor muscle of scallop ( Patinopecten yessoensis ) was used to investigate the interaction between the enzyme and neutral salts. At various sodium chloride concentrations, the Michaelis constant values for the substrates, monodansyl cadaverine and succinylated casein, were constant. Enhancement in activity by the addition of sodium chloride occurred instantly and reversibly, suggesting small conformational changes of the TGase. This increased activity was also exhibited by other sodium salts with the following order of effectiveness: Cl − =Br − >I − =NO 3 − . TGase was inactivated when pre-incubated with sodium chloride in the absence of both substrates. Inactivation rates increased with increase in sodium chloride concentration and temperature. It was suggested that enhanced muscle TGase activity is induced by the contact with seawater or body fluids, and may be involved in wound healing.

Effects of microbial transglutaminase and starch on the thermal gelation of salted squid muscle paste

The addition of microbial transglutaminase (MTGase) to salted squid muscle paste greatly strengthened the elasticity of the thermal gel, which was produced by the preferential crosslinking of myosin heavy chains through a two-step heating process that consisted of setting at 40°C and subsequent heating to 80°C or 90°C. Starch increased the breaking strength of thermal squid gels, but decreased the deformation. Thus, the starch-added gels became harder and less elastic. Although, when both MTGase and starch were added to squid muscle paste, the changes in viscoelasticity and myosin cross-linking reaction were similar to those with MTGase alone during setting, the storage and loss moduli of the paste sharply increased with an increase in starch content above 70°C. Subsequently, the thermal gel texture became more brittle or breakable than gels prepared by setting with MTGase only.

Thermally induced gelation of paramyosin from scallop adductor muscle

Thermally induced gelation of paramyosin from scallop smooth adductor muscle was investigated by dynamic rheological measurements under various conditions. The paramyosin thermal gel was produced at pH 6.5 and 7.2 at temperatures above 30°C through a two-step increase in storage (G′) and loss (G″) moduli; these values were higher than in gels produced from actomyosin at a high temperature. The thermal gel properties were very firm and brittle. In contrast, one main peak of G′ was observed during gelation at pH 8.0. The gel produced at pH 8.0 was more transparent and less soluble in a 6 M urea-0.5 M NaCl solution than those formed either at pH 6.5 or 7.2. These differences in the thermal gel properties are presumed to derive from the pH dependence of the gel matrix-forming process, such as oxidative cross-linking between cysteine residues, rather than from the thermal unfolding of the paramyosin molecules. The thermal gelation profile of chymotrypsin-digested paramyosin showed marked depression of G′ at high temperature.

Effects of microbial growth on the preservation of scallop muscle in a vital state

Adductor muscles dissected from live scallop Patinopecten yessoensis were stored in oxygenated artificial sea water. The initial muscle adenosine triphosphate (ATP) level, approximately 7.5 μmol/g, remained longer at 5°C than at either 0 or 10°C. The pH of sea water, decreased continuously and the consumption of dissolved oxygen increased even after muscle ATP was almost exhausted. The number of viable microbes, measured as colony-forming units (c.f.u.) in the muscle, increased to reach a plateau at approximately 107–108 c.f.u./g, while muscle ATP remained at high levels. After this time, muscle ATP sharply decreased. Antibiotics or sorbate added into the oxygenated sea water effectively inhibited both the growth of microbes and the decrease in the pH of sea water. Under these conditions, the retention period of muscle ATP was greatly extended. Thus, it seems most likely that scallop adductor muscle cells are suffocated by the limitation of oxygen supply caused by aerobic microbes grown on the surface of muscle tissue.