Purification and characterization of endo‐xylanases from Aspergillus niger. I. Two isozymes active on xylan backbones near branch points

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Abstract
Two endo‐xylanases (1,4‐β‐D‐xylan xylanohydrolase, EC 3.2.1.8) were purified to homogeneity from a crude Aspergillus niger pentosanase preparation by Ultrogel AcA 54 gel permeation chromatography, SP‐Sephadex C‐25 cation exchange chromatography at pH 4.5, Sephadex G‐50 gel permeation chromatography, and a second SP‐Sephadex C‐25 step, this one at pH 5.8. The two xylanases hydrolyzed soluble xylan more rapidly than insoluble branched xylan, but attacked each substance to an equal extent. Their low activity on a linear xylooligosaccharide mixture and absence of activity on insoluble xylan freed of branches suggest that the xylanases require a branch point nearby for significant attack. No xylose or L‐arabinose was produced, the major products of low molecular weight being tri‐ and pentasaccharides and smaller amounts of di‐, tetra‐, and hexasaccharides. There was low activity on untreated and crystalline cellulose and on carboxymethylcellulose and no activity on other polysaccharides tested. These two xylanases had molecular weights of ca. 1.3 × 104 and similar amino acid profiles, high in acidic and low in sulfur‐containing residues. Isoelectric points were 8.6 for I and 9.0 for II. Optimum pH values for activity were 6.0 and 5.5, respectively. In a 20‐min assay at pH 5.5, each was most active at 45°C, with activation energies up to 40°C of 30.4 and 38.8 kJ/ mol, respectively. Optimum pH levels for stability were 5.0 and 6.0, with half‐lives at 60°C and those pHs of 20 and 75 min, respectively.