*3.3. Release of XOS from Substrates by Enzymatic Hydrolysis*

In order to evaluate synergistic interactions between FAEs and Xyn11 on untreated and pre-treated CC (agricultural residue), the enzymes were tested for their ability to release XOS and FA from a model arabinoxylan substrate (WAX). A 0.5% WAX substrate loading was used for a better comparison with natural substrates since they have xylan contents of between 20–40%. Figure 3 shows the production of reducing sugars after enzymatic hydrolysis of WAX (a), untreated (b), hydrothermal treated (c) and acid-treated CC (d) by single or combinations of the enzymes. The trend in the production of reducing sugars was similar in all substrates, with the Xyn11 to FAE5/6 combinations releasing higher reducing sugars than those from the reactions containing individual enzymes. As expected, FAE5 or FAE6 alone were not able to release reducing sugars, this was also indicated in the specific activity determination study (Table 2). For hydrolysis of WAX (Figure 3a), both FAEs displayed synergy with Xyn11 by improving the release of reducing sugars. It was found that Xyn11 alone released 0.89 mg/mL, while co-incubation with FAE5 or FAE6 resulted in 1.18 mg/mL (25.61% increase) and 1.22 mg/mL reducing sugars (a 28.06% increase), respectively. The hydrolysis of untreated CC with xylanase alone and a combination of Xyn11 and FAE5 or FAE6 produced reducing sugars at concentrations of 0.94 mg/mL, 1.17 mg/mL, and 1.22 mg/mL, respectively (Figure 3b). The hydrothermal pre-treated sample exhibited a similar hydrolysis pattern to untreated CC, but the amounts of reducing sugars released in the co-incubation sets were higher (Figure 3c). However, the amount of reducing sugars was significantly less for acid pre-treatment when Xyn11 was applied alone (yield of 28.80%), but a combination of Xyn11 and FAE5 or FAE6 resulted in more reducing sugars (yield of 100% for both combinations) compared to untreated CC (Figure 3d). The hydrothermally pre-treated sample exhibited yields of 60.76% for Xyn11 alone, while co-incubation with FAE5 and FAE6 resulted in yields of 98.21% and 100%, respectively. These results indicate that FAEs play an important role in the synergistic hydrolysis of WAX, and the arabinoxylan contained in untreated and pre-treated CC samples.

**Figure 3.** Release of reducing sugars during hydrolysis of 0.5% wheat arabinoxylan (WAX) (**a**), 1% untreated (**b**), hydrothermally pre-treated (**c**) and acid pre-treated CC (**d**) by individual enzymes or a combination of 66% Xyn11: 33% FAE5 or FAE6. Sub represents substrate control. Statistical analysis was conducted using *t*-test for improvement of hydrolysis with respect to reducing sugar by the enzyme combinations compared to single enzyme (Xyn11), key: \* (*p* value < 0.05).


**Table 2.** Specific activities of the enzymes assessed in this study (U/mg protein).

<sup>a</sup> Analyzed by 3,5-dinitrosalicylic acid (DNS) method for xylanase activity and <sup>b</sup> analyzed by HPLC diode array detector (DAD) for feruloyl esterase activity. "Nd" = not detected.
