The substrate specificities of XynII were determined at standard condition by measuring the amount of reducing sugar equivalents released in reaction mixtures containing one of the following substrate solutions/suspensions: 0.5 % (w/v) beechwood xylan, or 1 % (w/v) birchwood xylan, insoluble and soluble oat spelt xylan, CMC, locust bean gum, and chitin. Specificity towards q-nitrophenyl-glyco- sides (PNP-glycoside) was determined by measuring the amount of q-nitrophenol released in reaction mixtures containing 20 mM PNP-glycoside according to the method of Ra¨tto¨ and Poutanen [26]. One unit (U) of xylanase activity for xylans or PNP-glycosides was defined as the amount of enzyme required to release 1 lmol of reducing sugar or q-nitrophenol, respectively, per minute under standard assay conditions. The kinetic constants (Vmax and Km) were determined at 50 or 60 C by reaction for 5 min
using beechwood xylan at concentrations 0.1–1.5 mg ml-1.
The Vmax and Km values were calculated by GraphPad Prism 5.0 software (http://www.graphpad.com/prism/) using nonlinear regression.