| Summary: | <p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>Penicillium funiculosum </it>produces a range of glycoside hydrolases (GH). The <it>XynD </it>gene, encoding the sole <it>P. funiculosum </it>GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast <it>Pichia pastoris</it>, in order to compare the results obtained with the <it>P. funiculosum </it>GH11 xylanases data.</p> <p>Results</p> <p>High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L<sup>-1</sup>. The protein was purified to homogeneity using one purification step. The apparent size on SDS-PAGE was around 64 kDa and was 46 kDa by mass spectrometry thus higher than the expected molecular mass of 41 kDa. The recombinant protein was N- and O-glycosylated, as demonstrated using glycoprotein staining and deglycosylation reactions, which explained the discrepancy in molecular mass. Enzyme-catalysed hydrolysis of low viscosity arabinoxylan (LVAX) was maximal at pH 5.0 with <it>K</it>m<sub>(app) </sub>and <it>k<sub>cat</sub></it>/<it>K</it>m<sub>(app) </sub>of 3.7 ± 0.2 (mg.mL<sup>-1</sup>) and 132 (s<sup>-1</sup>mg<sup>-1</sup>.mL), respectively. The activity of XynD was optimal at 80°C and the recombinant enzyme has shown an interesting high thermal stability at 70°C for at least 180 min without loss of activity. The enzyme had an endo-mode of action on xylan forming mainly xylobiose and short-chain xylooligosaccharides (XOS). The initial rate data from the hydrolysis of short XOS indicated that the catalytic efficiency increased slightly with increasing their chain length with a small difference of the XynD catalytic efficiency against the different XOS.</p> <p>Conclusion</p> <p>Because of its attractive properties XynD might be considered for biotechnological applications. Moreover, XOS hydrolysis suggested that XynD possess four catalytic subsites with a high energy of interaction with the substrate and a fifth subsite with a small energy of interaction, according to the GH10 xylanase literature data.</p>
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