초록
<P><B>Background</B></P><P>The filamentous fungus <I>Penicillium funiculosum </I>produces a range of glycoside hydrolases (GH). The <I>XynD </I>gene, encoding the sole <I>P. funiculosum </I>GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast <I>Pichia pastoris</I>, in order to compare the results obtained with the <I>P. funiculosum </I>GH11 xylanases data.</P><P><B>Results</B></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 <I>K</I>m<SUB>(app) </SUB>and <I>k<SUB>cat</SUB></I>/<I>K</I>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><B>Conclusion</B></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>