*3.4. Release of Hydroxycinnamic Acids from Substrates by Enzymatic Hydrolysis*

To further understand the synergism between Xyn11 and FAEs, the release of hydroxycinnamic acids from substrates by individual enzymes and their combinations was evaluated. Figure 4 shows the release of FA from WAX (a), untreated (b), hydrothermal pre-treated (c), and acid pre-treated treated CC (d). In the case of WAX (Figure 4a), a slight increase in FA release by FAE5 or FAE6 alone was observed. The combination of Xyn11 and FAE5 or FAE6 significantly improved (*p* < 0.05) the release of FA from 0.64 µg/mL and 0.65 µg/mL (individual enzyme) to 1.92 µg/mL and 2.68 µg/mL, respectively. A similar pattern was observed for the hydrolysis of untreated CC, although a combination of Xyn11: FAE5 released more FA compared to Xyn11: FAE6 (Figure 4b). With respect to pre-treated CC, the amount of

FA released from the substrate without enzymatic hydrolysis was enhanced (Figure 4c,d). This could be attributed to the disruption of the close inter-component associations between major constituents of lignocellulose during the pre-treatment step. Only Xyn11: FAE6 could significantly improve (*p* < 0.05) the release of FA from both pre-treated samples. The release of *p*-CA from CC was also observed and is shown in Figure 5. Similar to the results presented in Figure 4c,d, the pre-treated samples already showed increased amounts of readily soluble *p*-CA before enzymatic hydrolysis. However, the combination of Xyn11 and FAE5 or FAE6 were able to release considerable quantities of *p*-CA compared to individual enzymes and those already present and soluble in substrate controls. Interestingly, contrary to the FA release from pre-treated samples, both Xyn11: FAE5 and Xyn11: FAE6 could release comparable quantities of *p*-CA (Figure 5b,c). However, the yields obtained (no more than 10%) were much less when compared to release efficiencies (more than 70%) reported in some studies in the literature [27,28]. These results indicate that FAEs acted synergistically with Xyn11 in the co-production of XOS, FA and *p*-CA from the CC substrates.

**Figure 4.** Release of ferulic acid during the degradation of 0.5% WAX (**a**), 1% untreated (**b**), hydrothermal 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 Ferulic acid release by the enzyme combinations compared to single enzyme (FAE5/6), key: \* (*p* value < 0.05).

**Figure 5.** Release of *p*-coumaric acid during the degradation of 1% untreated (**a**), hydrothermal pre-treated (**b**) and acid pre-treated CC (**c**) by individual enzymes or a combination of 66% Xyn11: 33% FAE5 or FAE6. Note, WAX was not assessed as there was no *p*-coumaric acid detected in the substrate. Sub represents substrate control. Statistical analysis was conducted using *t*-test for improvement of *p*-coumaric acid release by the enzyme combinations compared to single enzyme (FAE5/6), key: \* (*p* value < 0.05).
