Partial Purification and Characterization of Transglutaminase From Threadfin Bream (Nemipterus Sp.) Liver

Abstract

Transglutaminase (TGase) from the threadfin bream ([TB]Nemipterus sp.) liver was partially purified using ion exchange, size exclusion and affinity chromatography. Three protein bands with molecular weight (Mw) of 95, 63 and 43 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were observed. Only one distinct fluorescent band appeared on the TGase activity staining of the native-PAGE. When the protein band was eluted and analyzed on the SDS-PAGE, it showed a single band with an Mw of 95 kDa. The enzyme required Ca 2+ up to 1 mM for full activation. TGase was also activated by 10 mM Sr 2+ . Dithiothreitol had no effect on activity. TGase activity was unaffected by NaCl up to 0.6 M and reduced to 75% at 1.2 M NaCl. Optimum pH and temperature was 8.5-9.0 and 50C, respectively. TGase activity was markedly inhibited by the sulfhydryl reagents. The enzyme catalyzed the cross-linking of the TB myosin heavy chains. PRACTICAL APPLICATIONSThreadfin bream (TB) liver transglutaminase (TGase) exhibited Ca 2+dependent characteristic similar to muscle TGase. The enzyme catalyzed the cross-linking of myosin, and therefore, could be applied to improve the textural properties of muscle proteins. The enzyme remained its high activity up to 1.2 M NaCl (ª7% w/v), indicating its potential application in foods containing a relatively high NaCl. The biochemical characteristics of the TB liver TGase are critical information leading to more understanding in the nature of the enzyme as well as determining the optimum conditions for food applica-1 Corresponding

Protein cross-linking ability of sarcoplasmic proteins extracted from threadfin bream

“…Purified tilapia TGase did not require additional DTT for its catalytic incorporation of MDC into DMC (Worratao & Yongsawatdigul, 2005). DTT also had no effect on activity of partially purified TGase from TB liver (Hemung & Yongsawatdigul, 2008). TGase activity in TBSP increased with NaCl concentration, reached the maximum at 0.2 mol/L NaCl and gradually decreased at higher concentrations (Fig.…”

“…An approximately 40% decrease in activity was found at 1.2 mol/L NaCl. Partially purified TGase from TB liver retained about 75% activity at 1.2 mol/L NaCl (Hemung & Yongsawatdigul, 2008). In general, TGases extracted from marine creatures exhibited higher NaCl stability than those from freshwater animals (Nozawa et al, 1997).…”

“…It could, therefore, contain more soluble protein components, which were easily precipitated when heated >40 C. It is important to note that TBSP prepared in this study would more likely resemble sarcoplasmic proteins recovered from a surimi processing plant by low centrifugal force than those prepared using ultracentrifugation. Optimum temperature of TGase partially purified from TB liver was 50 C, pH 8.5-9.0 (Hemung & Yongsawatdigul, 2008). Purified TGase from Japanese oyster showed optimum activity at 25 and 40 C (Kumazawa et al, 1997).…”

“…Molecular weight of TGases from various fish species and oyster ranged from 80 to 90 kDa (Kishi et al, 1991;Kumazawa et al, 1997;Worratao & Yongsawatdigul, 2005). Using a similar approach, Hemung and Yongsawatdigul (2008) reported that the MW of TB liver TGase was 95 kDa. TGases extracted from scallop hemocytes and adductor muscle also showed different mobility on native-PAGE, suggesting possible existence of isozymes (Nozawa, Mori, Kimura, & Seki, 2005).…”

Trypsin Inhibitory Activity and Gel‐Enhancing Effect of Sarcoplasmic Proteins from Common Carp

“…However, addition of 0.1% Ca 2+ did not further improve textural properties of threadfin bream surimi gels treated under other heating regimes. Fish endogenous TGase is a Ca 2+dependent enzyme and shows the highest catalytic activity at 50 • C for threadfin bream liver (Hemung and Yongsawatdigul 2008) and 37 to 50 • C for tilapia (Worratao and Yongsawatdigul 2005). Addition of 0.1% Ca 2+ probably activated endogenous TGase activity at 40 • C. Protein cross-linking mediated by TGase and degradation of myofibrillar proteins induced by Ca 2+ -activated proteinases were likely to occur simultaneously during pre-incubation at 40 • C, but overall textural properties were governed by the Vol.…”