## 2 Experimental Section
### Enzyme Characterization
A similar effect was observed with the insoluble fraction, but the hydrolysis reached only 17.5%. These results are consistent with the observations of [PERSON] _et al._[25], who also found higher rates of hydrolysis for soluble xylan. The preference of Xyl-O1 for the soluble fraction of the substrate may indicate a catalytic efficiency for the branched sites of _O_-acetyl-(4-_O_-methyl-d-glucuron) in the structure of the birchwood xylan. According to [PERSON] and colleagues, certain constituents in the structure of the xylan act as binding sites for the catalytic activity of some xylanases [11]. The residues that comprise these sites in the branching structure were partially removed during the preparative alkaline and acid extractions that were performed on the soluble and insoluble fractions of the substrate. However, as shown in Table 5, we observed that 18- and 24 h hydrolysis periods did not increase the amount of reducing sugar that was released. It is possible that Xyl-O1-promoted hydrolysis is more efficient after 12 h and represents a plateau of activity that remains constant for longer incubations. \begin{table}
\begin{tabular}{c c c c} \hline
**Substrate** & **Main chain linkage** & **Activity (IU\(\cdot\)mL\({}^{-1}\))** & **Purified Xyl-O1** \\  & & & **(\% RA)**\({}^{a}\) \\ \hline Oat spelt xylan (S) \({}^{b}\) & \(\beta\)-1,4 & \(0.659\pm 0.005\) & 100.00 \\ Oat spelt xylan (I) \({}^{c}\) & \(\beta\)-1,4 & \(0.003\pm 0.004\) & 0.39 \\ Birchwood xylan (S) & \(\beta\)-1,4 & \(0.560\pm 0.011\) & 85.03 \\ Birchwood xylan (I) & \(\beta\)-1,4 & \(0.228\pm 0.025\) & 34.60 \\ Beechwood xylan (S) & \(\beta\)-1,4 & \(0.556\pm 0.030\) & 84.38 \\ Beechwood xylan (I) & \(\beta\)-1,4 & \(0.024\pm 0.030\) & 3.58 \\
4-_O_-methyl-glucuron-d- xylan (I) & \(\beta\)-1,4 & \(0.328\pm 0.028\) & 49.87 \\ Filter paper & \(\beta\)-1,4 & \(0.010\pm 0.007\) & 1.46 \\ CM-cellulose & \(\beta\)-1,4 & \(0.045\pm 0.004\) & 6.82 \\ Avicel & \(\beta\)-1,4 & \(0.000\pm 0.002\) & 0.00 \\ Laminarin & \(\beta\)-1,3 & \(0.039\pm 0.018\) & 5.93 \\ Pectin & \(\beta\)-1,4 & \(0.024\pm 0.019\) & 3.62 \\ Mannan & \(\beta\)-1,4 & \(0.002\pm 0.003\) & 0.37 \\ PNPX & PNP-\(\beta\)-1,4 & \(0.000\pm 0.000\) & 0.00 \\ PNPA & PNP-\(\alpha\)-1,4 & \(0.000\pm 0.000\) & 0.00 \\ PNPG & PNP-\(\beta\)-1,4 & \(0.000\pm 0.000\) & 0.00 \\ PNPM & PNP-\(\beta\)-1,4 & \(0.000\pm 0.000\) & 0.00 \\ \hline \end{tabular} \({}^{a}\) Relative activity to oat spelt xylan; \({}^{b}\) (S): Soluble fractions; \({}^{c}\) (I): Insoluble fractions.  
\end{table} 
Table 4: Substrate specificity of purified _Aspergillus oryzae_ Xyl-O1. The identification and quantitative analysis of the soluble xylooligosaccharides that were released seem to indicate that the enzyme catalyzes the random cleavage of internal glycosidic linkages in the soluble and insoluble fractions of the birchwood xylan (Table 6). A similar activity has been described for other enzymes, including the endo-\(\beta\)-1,4-xylanas produced by _Penicillium capsulatum_[25], _Cephalosporium_ sp. RYM-202 strain [38], _A. fumigatus_ Fresenius [34] and _Acrophialophora nainiana_[33]. As shown in Table 6, the hydrolysis products X2-X6 predominated during the first 12 h of incubation. During the subsequent 12 h, either the X4-X6 xylooligomers predominated, or it was not possible to identify the products, as shown for the insoluble fraction after 24 h (Table 6). The decreased identification or quantification of the hydrolysis products by HPAEC-PAD indicated the accumulation of higher xylooligomers after the elution of the xylohexaose standard primarily during incubation periods of 18 and 24 h (results not shown). This suggests that the purified Xyl-O1 is involved in transglycosylation reactions at certain times. In this case, the chromatographic profile showed irregular peaks presenting more intense electrochemical signals and longer retention times than those of established xylooligomers patterns.