## 2 Experimental Section
### Enzyme Characterization
NBS is an effective inhibitor of xylanases [44,45] and promotes the oxidation of tryptophan residues that are involved in enzymatic catalysis. Xyl-O1 was slightly inhibited by the alkylating reagent iodoacetamide, indicating the need for thiol groups in catalysis. DEPC and DTP reduced the Xyl-O1 activity by 15 and 12%, respectively (Table 3). The assays investigating the effect of incubation time on Xyl-O1 activity at pH 7.0 revealed that at 50 \({}^{\circ}\)C, the enzyme remained active for 5.0 h, retaining 60% of its activity. Conversely, at 55 \({}^{\circ}\)C, the activity decreased to less than 30% within the first hour of incubation. The thermostability of Xyl-O1 at 50 \({}^{\circ}\)C was greatly increased in the presence of 20 mmol\(\cdot\)L\({}^{-1}\) l-cysteine, and the activity remained close to 100% for an incubation period of 6.0 h (Figure 3(A)). At 55 \({}^{\circ}\)C, the xylanase activity was enhanced in the presence of l-cysteine and remained stable for 1.0 h, with the catalytic activity remaining greater than 75% (Figure 3(B)). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Modifying agents or amino** & & & **Concentration** \\
**acids** & **Activity (IU\(\cdot\)mL\({}^{-1}\))** & **Relative activity (\%)** & **(mmol\(\cdot\)L\({}^{-1}\))** \\ \hline H\({}_{2}\)O control & \(0.262\pm 0.021\) & 100.00 & — \\
4\% Ethanol control & \(0.285\pm 0.026\) & 100.00 & — \\ NBS & \(0.285\pm 0.079\) & 108.42 & 1 \\ DTNB * & \(0.399\pm 0.025\) & 140.02 & 2 \\ DTT & \(0.300\pm 0.037\) & 114.35 & 20 \\ l-Cysteine & \(0.367\pm 0.076\) & 140.04 & 20 \\ H\({}_{2}\)O control & \(0.290\pm 0.029\) & 100.00 & — \\
4\% Ethanol control & \(0.322\pm 0.006\) & 100.00 & — \\ DTP * & \(0.285\pm 0.025\) & 88.43 & 2 \\ l-Tryptophan & \(0.285\pm 0.012\) & 98.13 & 5 \\ Iodoacetamide & \(0.255\pm 0.046\) & 87.71 & 5 \\ DEPC & \(0.248\pm 0.037\) & 85.32 & 5 \\ EDC & \(0.291\pm 0.046\) & 100.08 & 5 \\ \(\beta\)-Mercaptoethanol & \(0.397\pm 0.026\) & 136.68 & 5 \\ H\({}_{2}\)O control & \(0.306\pm 0.031\) & 100.00 & — \\ SDS & \(0.225\pm 0.025\) & 73.56 & 20 \\ \hline \hline \end{tabular}  
* These compounds were diluted in 4% ethanol. Enzymatic activity: \(0.320\pm 0.014\) IU\(\cdot\)mL\({}^{-1}\).  
\end{table} 
Table 3: Effect of modifying agents and amino acids on the xylanolytic activity of purified Xyl-O1. The purified Xyl-O1 efficiently hydrolyzed the substrates that contained d-xylose residues, especially the soluble fraction of oat spelt and the birchwood and beechwood xylans (Table 4). These results suggest that Xyl-O1 is a potential candidate for the pulp bleaching processes, whereas the application of xylanases in the pulp and paper industry requires a cellulase-free activity [8, 11, 41, 46]. However, the enzyme activity was significantly reduced in the insoluble fractions of the same substrates, especially for oat spelt xylan, which had a relative activity of less than 1.0%. The enzyme also showed a hydrolysis efficiency of 49.87% when tested with 4-\(O\)-methyl-glucuron-d-xylan as the substrate. The relative activities for the birchwood and beechwood xylans were 85 and 84.4%, respectively (Table 4). The residual activity was less than 10% for filter paper, CM-cellulose, and the enzyme activity was significantly reduced in the insoluble fractions of the same substrates, especially for oat spelt xylan, which had a relative activity of less than 1.0%. laminarin, mannan and pectin, indicating that these substrates might have been contaminated with pentosan, which is susceptible to hydrolysis [14]. Furthermore, Xyl-O1 was completely inactive against the avicel and _p_-nitrophenyl glycoside substrates (Table 4). The specificity of Xyl-O1 was determined by analyzing the hydrolysis products of the soluble and insoluble birchwood xylan using the DNS method and HPLC on a Dionex system. The results presented in Table 5 show that the enzyme was more active on the soluble fraction, with a maximum hydrolysis of approximately 33% obtained after 12 h of incubation.