Molecular characterization of a glycosyl hydrolase family 10 xylanase from Aspergillus niger

• First report about cloning and expression of a glycosyl hydrolase family 10 xylanase from Aspergillus niger in P. pastoris.
• A thorough study on physicochemical properties of the recombinant GH family 10 xylanase from A. niger.
• Stable at a broad pH range of 4.5–8.5 and resistant to detergents and acetone: potential for enzymatic reactions.
• Hydrolysis endproducts of birch wood xylan were xylotriose, xylotetraose, and xylopentose.

A gene coding for an endo-β-1,4-xylanase (XlnA) (glycosyl hydrolase family 10) from Aspergillus niger DSM 1957 was cloned and sequenced. The cDNA sequence (984 bp) and its putative endoxylanase (327 aa protein with a predicted molecular mass of 35.5 kDa and pI 6.23) showed 91.3–99.5% and 96.3–99.1% identities with cDNA sequences and their corresponding endoxylanases from A. niger strains from GenBank, respectively. The cDNA was expressed in Pichia pastoris GS115 under the control of AOX1 promoter at a level of 46.4 U/ml culture supernatant, after 144 h of growth at 30 °C in YP medium induced with 0.5% (v/v) of methanol. The molecular mass of the purified XlnA determined by SDS–PAGE was 35.5 kDa with a specific activity of 808.5 U/mg towards 1% (w/v) of birch wood xylan. Temperature and pH optimum were observed at 50 °C and pH 7.0, respectively. The enzyme was stable over a temperature range of 25–40 °C and at pH range of 4.5–8.5 and resistant to Tween 80 and acetone. The Km and Vmax value obtained for the purified xylanase were 25.5 mg/ml and 5000 μmol/min/mg protein with birch wood xylan as substrate, respectively. The xylanase was free of cellulase and mannanase activity but highly active towards birch wood xylan. The major products of the birch wood xylan hydrolysis were predicted as xylotriose, xylotetraose, and xylopentose. The biochemical characteristics suggested that the recombinant xylanase has a potential application, including use as a feed enzyme.