Background
The filamentous fungus Penicillium funiculosum produces a range of glycoside hydrolases (GH). The XynD gene, encoding the sole P. funiculosum GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast Pichia pastoris, in order to compare the
Conclusion
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.
Results
High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L-1. 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 Km(app) and kcat/Km(app) of 3.7 ± 0.2 (mg.mL-1) and 132 (s-1mg-1.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.