A family 11 xylanase from Penicillium funiculosum is strongly inhibited by three wheat xylanase inhibitors

Steady-state kinetic approaches were used to investigate the binding of a novel Penicillium funiculosum xylanase, XYNC, with three known xylanase inhibitor proteins from wheat (Triticum aestivum). The xylanase gene (xynC) was cloned from a P. funiculosum genomic library and the deduced amino acid sequence of XYNC exhibited high sequence similarity with fungal family 11 xylanases. xynC was overexpressed in P. funiculosum and the product (XYNC: M_r=23.6 kDa; pI=3.7) purified and shown to efficiently degrade birchwood xylan [K_m=0.47% w/v, V_max=2540 μmol xylose min^-1 (mg protein)^-1 at pH 5.5 and 30°C] and soluble wheat arabinoxylans [K_m=1.45% w/v, V_max=7190 μmol xylose min^-1 mg protein)^-1 at pH 5.5 and 30°C]. The xylanase activity of XYNC was inhibited strongly by three xylanase inhibitor proteins from wheat; XIP-I, TAXI I and TAXI II. The inhibition for each was competitive, with very tight binding (K_i=3.4, 16 and 17 nM, respectively) equivalent to free energy changes (ΔG°) of -49, -45 and -45 kJ mol^-1. This is the first report describing a xylanase that is inhibited by all three wheat xylanase inhibitor proteins described to date.