substrate specificity and susceptibility to wheat inhibitor proteins of Penicillium funiculosum xylanases from a commercial enzyme preparation

Filamentous fungi produce a range of carbohydrate-degrading enzymes, in particular hemicellulases, that are often exploited by the baking, brewing and feed industries. Controlled xylan degradation is a key component in several industrial processes, in which the efficiency of xylanases (endo-1,4-beta-D-xylan hydrolases) depends on both their substrate specificity and their susceptibility to inhibitors present in the plant material. The three major xylanases present in a commercial hemicellulase product derived from a Penicillium funiculosum fermentation broth were characterized with respect to their substrate specificity and interaction with three known xylanase inhibitor proteins (XIP-I, TAXI I and TAXI II) present in wheat flour. XYNA (48 kDa) and XYNB (22 kDa) have significant sequence similarity with family 7 cellobiohydrolases and family 11 xylanases respectively (previous studies), while XYND (36 kDa) has similarity with family 10 xylanases (this paper). All three enzymes hydrolysed various heteroxylan substrates, including birchwood xylan and soluble wheat arabinoxylan, at 30 degrees C and pH 5.5. XYNA was strongly inhibited by XIP-I, TAXI I and TAXI II (K(i) = 106, 46 and 46 nM respectively); XYNB was only significantly inhibited by TAXI I (K(i) = 165 nM). In each case the inhibition of XYNA and XYNB was competitive. XYND was only inhibited by XIP-I but, in contrast to XYNA and XYNB, the apparent inhibition was dependent on the order that xylanase, inhibitor and substrate were added to the incubation mixture; the inhibition was weak when incubations were initiated by addition of xylanase (to a mixture of substrate and XIP-I), but was very strong when enzyme and XIP-I were pre-incubated before addition of substrate. These findings demonstrate that the susceptibility of individual xylanases to inhibitor proteins present in cereals is likely to be a critical factor in determining their efficiency in various commercial applications.