Fig. 3. Purification and characterization of the xylanase obtained from S. viridodiastaticus MS9.
(A) Estimation of molecular weight and xylanase activity of the purified protein. The xylanase-active fraction, obtained from Resource-Q column chromatography through several stages of purification, was analyzed using 0.1% sodium dodecyl sulfate– 15% polyacrylamide gel electrophoresis (lane 1). The xylanase activity of the isolated protein (molecular weight ≈ 21 kDa) was confirmed by a zymogram, as indicated by an arrow (lane 2). M, molecular-weight standard. (B) Substrate specificity was measured using the DNS method on different substrates (0.5%, w/v), such as birchwood xylan, beechwood xylan, oat spelt xylan, carboxymethyl (CM)-cellulose, and avicel. The enzyme reaction was performed in 20 mM Tris-Cl (pH 7.0) buffer at 60°C for 10 min. The enzyme activity of oat-spelt xylan was considered to be 100%. (C) TLC of the xylan hydrolysate obtained using purified xylanase. The enzyme reaction was carried out at 60°C for 12 h in 20 mM Tris-Cl (pH 7.0) containing 0.2% (w/v) beechwood xylan and then separated on a Silica Gel 60 TLC plate. X1: xylose; X2: xylobiose; X4: xylotetraose. The spot corresponding to X2 is indicated by arrows.