## 2 Experimental Section
### Xyl-O1 Purification  
Xyl-O1 was purified by ultrafiltration through a PM-10 ultrafiltration membrane, gel filtration chromatography on Sephacryl S-100 and Sephadex G-50 and ion exchange chromatography on Q-Sepharose. In the first step, a xylanase activity of only 0.294 IU\(\cdot\)mL\({}^{-1}\) was detected in the ultrafiltrate. Because this ultrafiltrate had low protein concentration, the solution was lyophilized prior to chromatography. The chromatographic profiles from the gel filtration columns (results not shown) initially showed that the enzyme sample subjected to chromatography on a Sephacryl S-100 column eluted in two peaks that had xylanase activity. The second peak was selected because it contained a xylanase activity that was approximately 3.5 times higher than the first peak and corresponded to the fractions with the greatest absorbance at 280 nm. The pooled fractions were applied onto a Sephadex G-50 column. The xylanase activity was eluted off this column in a single peak with a low absorbance at 280 nm. An additional peak was eluted with a greater absorbance at 280 nm and an unidentified colored substance. As shown in Figure 1, fractionation on a Q-Sepharose column revealed that Xyl-O1 eluted before the salt gradient at pH 7.0. According to [PERSON] _et al._[32], there is a conserved relationship between molecular weight and p\(I\) when xylanases from different species are compared. Low-molecular-weight xylanases tend to be basic, whereas high molecular weight is associated with a \begin{table}
\begin{tabular}{c c} \hline
**Enzymatic activity** & **IU\(\cdot\)mL\({}^{-1}\)** \\ \hline Xylanase & \(0.488\pm 0.011\) \\ Pecinase & \(0.258\pm 0.048\) \\ Mannanase & \(0.098\pm 0.006\) \\ CM-cellulose & \(0.095\pm 0.004\) \\ FPase & \(0.081\pm 0.015\) \\ Avicelase & \(0.009\pm 0.001\) \\ \hline \end{tabular}
\end{table} 
Table 1: Enzymatic activities of the filtered culture supernatant (FCS) from _Apergillus oryzae_ grown on pretreated dirty cotton residue (PDCR) for 6 days at 28 \({}^{\circ}\)C.  
more acidic p_I_. An exception to this relationship is the _Schizophyllum commune_ xylanase (21 kDa; p_I_ 4.5); however, the relationship is conserved even in yeast-like fungi that lack multiple xylanases, e.g., _Cryptococcus_ sp. As noted in Table 2, the xylanolytic activity remaining in the ultrafiltrate portion after lyophilization was considerably recovered during fractionation using gel filtration and ion exchange chromatography. In addition, Table 2 also shows Xyl-O1 purification and yield values of 27.1% and 16.7%, respectively. These values are consistent with other studies involving xylanase purification in \begin{table}
\begin{tabular}{c c c c c c c} \hline \multirow{2}{*}{**Purification steps**} & **Total volume** & **Total protein** & **Total activity** & **Specific activity** & **Purification (fold)** & **Yield (\%)** \\  & **(mL)** & **(mg)** & **(IU)** & **(IU\(\cdot\)mg\({}^{-1}\))*** & **(fold)** & **(\%)** \\ \hline Filtered culture supernatant & 110.00 & 5.830 & 53.350 & 9.151 & 1.000 & 100.00 \\ Ultrafiltrate & 100.00 & 1.500 & 29.400 & 19.600 & 2.142 & 55.11 \\ Lyophilization & 4.50 & 0.828 & 1.647 & 1.989 & 0.217 & 3.08 \\ Sephacryl S-100 & & & & & & \\ (fractions 58–64) & & & & & & \\ Sephadex G-50 & & & & & & \\ (fractions 71–84) & & & & & & \\ Q-Sepharose FF & & & & & & \\ (fractions 7–18) & 36.00 & 0.036 & 8.928 & 248.000 & 27.101 & 16.74 \\ \hline \end{tabular}  
* Specific activity (IU\(\cdot\)mg\({}^{-1}\)) was determined with soluble oat spelt xylan as substrate.  
\end{table} 
Table 2: Summary of purification of Xyl-O1. Figure 1: Ion exchange chromatography of Xyl-O1 on Q-Sepharose. Absorbance at 280 nm is represented by closed diamonds [––], xylanase activity is represented by thin Xs [ – –], and the linear gradient (0–1.0 mol\(\cdot\)L\({}^{-1}\) NaCl in equilibration buffer) is represented by a solid line [–]. which the recovery was low [33, 34, 35, 36]. The ultrafiltration procedure retained most of the \(\beta\)-xylanase activity in the concentrate.