SDS-PAGE (A) and zymography (B) of the native and recombinant β-1,3-xylanases. (A) SDS-PAGE. Proteins (20 μg) were stained with Coomassie brilliant blue R-250. (B) Zymography. Both purified enzymes were applied to an SDS-PAGE gel, followed by activity staining with glycol β-1,3-xylan as the substrate. Numbers on the left are molecular masses of the markers. Lanes: 1, molecular mass markers; 2 and 4, native β-1,3-xylanase; 3 and 5, recombinant β-1,3-xylanase.

Thin-layer chromatogram of hydrolysis products obtained from several polysaccharides with the recombinant β-1,3-xylanase. Fifty microliters of enzyme solution (0.05 U of β-1,3-xylanase) was added to 50 μl of 10 mM sodium acetate buffer (pH 6.0) containing 1% polysaccharide and incubated at 37°C overnight. The hydrolysis products were developed on a silica gel 60 plastic sheet in a solvent of n-butanol–acetic acid–water (10:5:1), and the oligosaccharides were visualized by spraying the plate with a diphenylamine-aniline-phosphate reagent (4). Lanes: 1 and 11, d-xylose; 2, β-1,3-xylanase; 3, β-1,3-xylan; 5, β-1,4-xylan; 7, curdlan; 9, carboxymethylcellulose; 4, 6, 8, and 10, the enzyme plus β-1,3-xylan, β-1,4-xylan, curdlan, and carboxymethylcellulose, respectively.