Trypsin purification
Trypsin from the viscera of S. basilisca was extracted and purified successively by the four-step procedure described in Section 2. In the first step, the crude enzyme extract was fractionated with ammonium sulphate. The precipitate formed at 40–80% (w/v) saturation showed higher specific activity (0.23 U/mg of protein), than the precipitate formed at 0–40% saturation (0.05 U/mg of protein). No activity was detected in the final supernatant. The 40–80% fraction, which gave the highest specific activity, was then subjected to Sephadex G-100 gel filtration. This procedure yielded one peak of protease activity (BAPNA) (Fig. 1a). Fractions showing trypsin activity were pooled and then loaded on a Mono
Q-Sepharose anion-exchange chromatography column that had been equilibrated with buffer C. Binding proteins were eluted with a linear gradient of NaCl (0–0.5 M). BAPNA activity appeared in a single peak (Fig. 1b). Finally the active fractions were concentrated by ultrafiltration. The results of the purification procedure are summarised in Table 1. After the final purification step, the trypsin was purified 4.2-fold, with a recovery of 12% and a specific activity of 0.5 U/mg of protein, using BAPNA as a substrate. The purified enzyme gave a single band on SDS–PAGE and its molecular weight was estimated to be 27 kDa (Fig. 2a), corresponding to that determined by gel filtration. Purity of the enzyme was also evaluated using zymogram activity staining. As shown in Fig. 2a (line 6), a unique clear band of casein hydrolysis was observed in the gel, indicating the homogeneity of the purified protease. Generally, fish trypsins have been reported to have molecular weights in the range of 22–28 kDa (Whitaker, 1994). The molecular weight of S. basilisca trypsin was similar to those from other fish species, such as striped seabream (Lithognathus mormyrus)
Trypsin purificationTrypsin from the viscera of S. basilisca was extracted and purified successively by the four-step procedure described in Section 2. In the first step, the crude enzyme extract was fractionated with ammonium sulphate. The precipitate formed at 40–80% (w/v) saturation showed higher specific activity (0.23 U/mg of protein), than the precipitate formed at 0–40% saturation (0.05 U/mg of protein). No activity was detected in the final supernatant. The 40–80% fraction, which gave the highest specific activity, was then subjected to Sephadex G-100 gel filtration. This procedure yielded one peak of protease activity (BAPNA) (Fig. 1a). Fractions showing trypsin activity were pooled and then loaded on a Mono Q-Sepharose anion-exchange chromatography column that had been equilibrated with buffer C. Binding proteins were eluted with a linear gradient of NaCl (0–0.5 M). BAPNA activity appeared in a single peak (Fig. 1b). Finally the active fractions were concentrated by ultrafiltration. The results of the purification procedure are summarised in Table 1. After the final purification step, the trypsin was purified 4.2-fold, with a recovery of 12% and a specific activity of 0.5 U/mg of protein, using BAPNA as a substrate. The purified enzyme gave a single band on SDS–PAGE and its molecular weight was estimated to be 27 kDa (Fig. 2a), corresponding to that determined by gel filtration. Purity of the enzyme was also evaluated using zymogram activity staining. As shown in Fig. 2a (line 6), a unique clear band of casein hydrolysis was observed in the gel, indicating the homogeneity of the purified protease. Generally, fish trypsins have been reported to have molecular weights in the range of 22–28 kDa (Whitaker, 1994). The molecular weight of S. basilisca trypsin was similar to those from other fish species, such as striped seabream (Lithognathus mormyrus)
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