*3.5. Determination of Hydrolysate Product Profiles*

The data presented above indicates that FAE5 and FAE6 could release hydroxycinnamic acids in the presence of Xyn11 while improving the production of reducing sugars from WAX, untreated and pre-treated CC. To gain a deeper insight into these synergistic interactions, the product patterns of individual enzymes and their combinations were evaluated for the types of XOS generated. Figure 6 shows the TLC analysis of XOS released from the hydrolysis of WAX (a), untreated (b), hydrothermal treated (c) and acid-treated CC (d) by single and combinations of enzymes. It is important to note that the dark-yellow colored spots on the plates represent glycerol which was used as a stabilizer during storage of the purified FAE5 and FAE6. In the case of WAX hydrolysis (Figure 6a), individual enzymes (lane 2) and combinations (lane 3 and 4) generated xylobiose, xylotetraose, xylopentaose, and xylohexaose. The hydrolysis product patterns of CC (Figure 6b–d) appeared to consist of xylobiose (a dominant product) and small quantities of xylotetraose. The quantities of xylobiose seem to increase for the treated CC, most especially for enzyme combinations (lane 3 and 4). Also, for acid pre-treated CC (Figure 6d), Xyn11 alone (lane 2) produced very small quantities of XOS, this was also observed in Figure 3b during the quantification of reducing sugars.

We then further attempted to quantify the XOS generated using HPLC-RID. It is noteworthy that the HPLC-RID system used in this study couldn't detect XOS of less than 0.05 mg/mL. Figure 7 shows that the quantity of xylobiose produced by the combination of Xyn11 and FAE5 or FAE6 was enhanced compared to Xyn11 alone, this pattern was more pronounced on untreated and pre-treated CC. High quantities of xylotetraose (0.24 mg/mL) were observed for WAX (Figure 7a), but there was no significant increase between individual enzymes and their combinations. The highest quantities of xylobiose were produced by enzyme combinations for hydrothermal pre-treated CC (0.28 mg/mL for xyn11: FAE5 and 0.40 mg/mL for Xyn11: FAE6) and acid-treated CC (0.34 mg/mL for Xyn11: FAE5 and 0.43 mg/mL for Xyn11: FAE6). This improvement in xylobiose production was significant (*p* < 0.05) when compared to single enzyme incubations, which resulted in 0.14 mg/mL and 0.12 mg/mL for hydrothermal and acid-treated CC, respectively. From the results presented above, it appears that Xyn11 produced XOS which become substrates to FAEs for the removal of FA or *p*-CA, and this allows Xyn11 to further hydrolyze these long XOS into the shorter xylobiose.

**Figure 6.** Thin-layer chromatography (TLC) analysis of hydrolysis of 0.5% WAX (**a**), 1% untreated (**b**), hydrothermal pre-treated (**c**) and acid pre-treated CC (**d**) by (2) Xyn11 alone, (3) a combination of 66% Xyn11: 33% FAE5, and (4) a combination of 66% Xyn11: 33% FAE6. Substrate without enzyme was used as a control (1). A mixture of xylo-oligosaccharides (X1-X6) was used as a standard. Arrows indicated observed bands, albeit feint in some instances.

**Figure 7.** Xylo-oligosaccharide content measured from the hydrolysis of 0.5% WAX (**a**), 1% untreated (**b**), hydrothermal pre-treated (**c**) and acid pre-treated CC (**d**) after incubation with Xyn11 alone or a combination of 66% Xyn11: 33% FAE5 or FAE6. Statistical analysis was conducted using *t*-test for improvement of hydrolysis with respect to xylo-oligosaccharides (XOS) by the enzyme combinations compared to single enzyme (Xyn11), key: \* (*p* value < 0.05).
