Transglutaminase from Asian seabass liver: purification, characterization and cross-linking activity against natural actomyosin

Abstract

Abstract The study investigated the purification and characterization of transglutaminase (TGase) from Asian seabass liver. In addition, protein cross-linking ability of partially purified TGase was also elucidated against the natural actomyosin extracted from threadfin bream surimi. During ammonium sulfate fractionation, the highest specific activity (11.31 ± 0.17 units/mg protein) was obtained from the 80–100% ammonium sulfate (AS) fraction (partially purified TGase; pTGase). Purity increased 1.46 times compared to crude samples with a 73% yield. Further purification using DEAE-Sepharose enhanced the specific activity (904.71 ± 4.65units/mg protein) by 117.19-fold as compared to the crude sample. The molecular weight (MW) of the purified TGase was 43.4 kDa, which was slightly different than the MW determined by SDS-PAGE (41.1 kDa). Optimal TGase activity was found at 45–50 °C and pH 8, with significant declines beyond these ranges. Furthermore, TGase maintained activity (80%) when heated till 55 °C, whereas it showed wide-range pH stability. NaCl and CaCl2 at 3% and 20 mM, respectively enhanced TGase activity. When natural actomyosin (NAM) was treated with pTGase at various concentrations (1–10 unit/mg protein), decreasing solubility and ɛ-amino groups confirmed its cross-linking ability. The result was also supported by the formation of aggregates of NAM proteins as determined by transmission electron microscopic images. Thus, Asian seabass liver could be a new source of purified and partially purified TGase, which can be employed for various food and pharmaceutical applications.

Keywords

• Enzyme activity
• Protein cross-linking
• DEAE-Sepharose