Purification and characterization of transglutaminase from squid gill

Abstract

The present study used squid gill as a source of transglutaminase (TGase) because it has extremely high TGase activity compared with other tissues. The enzyme was purified using successive chromatographies of Sephacryl S-300 and hydroxyapatite columns. The yield and purification-fold of the enzymatic activity was 12.6% and 14.1-fold, respectively. The molecular mass of the purified enzyme was estimated to be 94 kDa by using sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Enzyme activity was enhanced 15-fold with an increase in NaCI concentration. Although the activity was dependent on Ca2+ concentration, it was not sufficiently activated even by 50mM CaCl2 in the absence of NaCl, but could be fully activated with 10mM CaCl2 in 0.7 M NaCl. However, in the absence of substrates, the enzyme was rapidly inactivated. The pH and temperature optima of the enzyme were approximately pH 8.0 and 20°C, respectively. It was stable in the absence of Ca2+ at pH 7.5-9.0 and had a rate constant (KD) of 1.6 × 10-5s-1 for thermal inactivation at 50°C. These results in which squid gill TGase could be activated at higher concentrations of Ca2+ and NaCl than at a physiological concentration, suggest that contact with seawater or body fluid seems to activate the enzyme if the tissue is disrupted.