The inhibition specificity of recombinant Penicillium funiculosum xylanase B towards wheat proteinaceous inhibitors

The filamentous fungus Penicillium funiculosum produces a mixture of modular and non-modular xylanases belonging to different glycoside hydrolase (GH) families. In the present study, we heterologously expressed the cDNA encoding GH11 xylanase B (XYNB) and studied the enzymatic properties of the recombinant enzyme. Expression in Escherichia coli led to the partial purification of a glutathione fusion protein from the soluble fraction whereas the recombinant protein produced in Pichia pastoris was successfully purified using a one-step chromatography. Despite O-glycosylation heterogeneity, the purified enzyme efficiently degraded low viscosity xylan [ K m=40±3 g l −1, V max=16.1±0.8 μmol xylose min −1 and k cat=5405±150 s −1 at pH 4.2 and 45 °C] and medium viscosity xylan [ K m=34.5±3.2 g l −1, V max=14.9±1.0 μmol xylose min −1 k cat=4966±333 s −1 at pH 4.2 and 45 °C]. XYNB was further tested for its ability to interact with wheat xylanase inhibitors. The xylanase activity of XYNB produced in P. pastoris was strongly inhibited by both XIP-I and TAXI-I in a competitive manner, with a K i of 89.7±8.5 and 2.9±0.3 nM, respectively, whereas no inhibition was detected with TAXI-II. Physical interaction of both TAXI-I and XIP-I with XYNB was observed using titration curves across a pH range 3–9.