Purification and characterization of transglutaminase from Tropical tilapia (Oreochromis niloticus)

Transglutaminase (TGase) from Tropical tilapia (Oreochromis niloticus) was purified to electrophoretic homogeneity using successive chromatographies of DEAE-Sephacel, Sephacryl S-4 HR and HiTrap Heparin with a yield and purification-fold of 12.9% and 69.8, respectively. The molecular weight (MW) of the purified tilapia TGase was estimated to be 85 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point (pI) of tilapia TGase was 6.53. Optimal temperature and optimal pH of tilapia TGase were 37–50 °C and 7.5, respectively. Optimal concentrations of CaCl2 and dithiothreitol (DTT) were at 1.25 and 5 mM, respectively. The activity of TGase towards monodansylcadaverine (MDC) decreased as the NaCl concentration increased. Chelating agents, ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA), inhibited TGase activity. Tilapia TGase was strongly inactivated by ρ-chloromercuribenzoic acid (PCMB), N-ethylmaleimide (NEM), iodoacetamide (IAA), Cu2+, and Zn2+, suggesting a thiol group at the active site.