Fig. 9
GH16B β-agarase-catalyzed complete hydrolysis of agarose to NA4 and NA6, and purification of NA4 and NA6 from the hydrolysate via Sephadex G-15 column chromatography. a After agarose at various concentrations (5.0%[w/v], 7.0%[w/v], or 9.0%[w/v]) in 50 mM Mcllvaine buffer (pH 7.0) was autoclaved, the melted agarose was treated with the purified enzyme (1.6 µg/mL) under optimum reaction conditions (5 mM MnCl2, 10 mM TCEP, 50 mM Mcllvaine buffer, pH 7.0) and continuous magnetic stirring at 50 °C for 14 h, the reaction mixtures were subjected to TLC to identify NA4 and NA6 produced from agarose. b For purification of NA4 and NA6 from the enzymatic hydrolysate of agarose, 9.0%[w/v] agarose (20 mL) was treated with the enzyme (1.6 µg/mL) at 50 °C for 14 h and then freeze–dried. The dried sample was dissolved in DI water and subjected to Sephadex G-15 column chromatography. Fractions containing NA4 and NA6 were identified using TLC. Representative results are shown; two additional experiments yielded similar results