Purification and characterization of trypsin from the pyloric caeca of brownstripe red snapper (Lutjanus vitta)

Trypsin was purified from the pyloric caeca of brownstripe red snapper (Lutjanus vitta) by ammonium sulphate (40–60% saturation) precipitation, soybean trypsin inhibitor (SBTI)-Sepharose 4B column and DEAE-Sephacel column chromatography. Purified trypsin showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) and native-PAGE. A yield of 4.9% with the purification-fold of 20 was obtained. Trypsin had an apparent molecular weight of 23 kDa. SBTI and N-ρ-tosyl-l-lysine-chloromethylketone (TLCK) showed a strong inhibitory effect on the purified trypsin, while other protease inhibitors exhibited negligible inhibition. Trypsin had maximal activity at pH 8.5 and 60 °C for the hydrolysis of α-N-benzoyl-dl-arginine-ρ-nitroanilide (BAPNA). It was stable within the temperature range of 25–55 °C and pH range of 7.0–10.0. Purified trypsin had a Michaelis–Menten constant (K_m) and catalytic constant (k_cat) of 0.507 mM and 4.71 s⁻¹, respectively, when BAPNA was used as the substrate. For the hydrolysis of α-N-ρ-tosyl-l-arginine methyl ester (TAME), K_m and k_cat were 0.328 mM and 112 s⁻¹, respectively.     

The effects of pretreatments on antioxidative activities of protein hydrolysate from the muscle of brownstripe red snapper (Lutjanus vitta)

Different pretreatments of mince from brownstripe red snapper (Lutjanus vitta) including 1) washing; 2) membrane separation; 3) washing followed by membrane separation and 4) membrane separation followed by washing were conducted prior to hydrolysis. Among the resulting minces, that subjected to membrane separation with subsequent washing (MS/W) contained the lowest remaining myoglobin content, phospholipid content, heme iron and non-heme iron contents (p < 0.05) and showed the lowest TBARS values throughout 9 days of storage at 4 °C in the presence and absence of 0.15 mol L⁻¹ cupric acetate (p < 0.05). When hydrolysates from 1) mince, 2) MS/W and 3) protein isolate from MS/W (PI) with different degree of hydrolysis (DH) (20, 30 and 40%) were prepared using proteases from pyloric caeca of brownstripe red snapper, antioxidative activities determined by DPPH, ABTS radical scavenging activities, ferric reducing antioxidant power and metal chelating activity varied with hydrolysates and DH. Antioxidative activities increased with increasing DH up to 40% (p < 0.05). At all DH tested, hydrolysate prepared from MS/W exhibited the highest antioxidative activities determined by all assays, compared to those from mince and PI (p < 0.05). Hydrolysate from MS/W with 40% DH had the molecular weight lower than 6.5 kDa as determined by SDS-PAGE. In liposome oxidation system, the addition of hydrolysate from MS/W resulted in the lower TBARS, compared with the control throughout the incubation period of 48 h at room temperature (25-28 °C). Therefore, fish mince with membrane separation followed by washing was the most appropriate source for production of hydrolysate possessing antioxidative activity with the lowered amount of lipids and pro-oxidants.