*3.4. Holocellulolytic Enzyme Cocktail Formulation and Its Application on Pretreated Biomass*

The holocellulolytic enzyme cocktail (HEC-H) used to assess the effectiveness of the alkaline pretreatments in lowering biomass recalcitrance was formulated using a 60%: 20%: 10%: 10% combination of the MFE-5E, MFE-5H, MFE-45 and exoglucanase (Exg-D) enzymes. Exg-D was previously characterised by Mafa et al. [23] and its biochemical properties demonstrated that it can effectively be used in synergy with MFEs.

HEC-H hydrolysed the alkaline pretreated SSB and CC substrates better than their untreated biomasses (i.e., the controls) (Figure 3A). Also, HEC-H exhibited the highest activity on the CC biomass compared to SSB biomass. Higher activity (approximately 0.72 µmol/mL) was observed when HEC-H hydrolysed Ca(OH)<sup>2</sup> pretreated CC compared to SSB biomass, while an approximately 1.14 µmol/mL higher activity was recorded when the HEC-H hydrolysed NaOH-pretreated CC compared to SSB

biomass. We propose that the HEC-H hydrolysed the alkaline pretreated CC better than the SSB due to differences in their biomass composition; CC had a higher hemicellulose content compared to SSB. Furthermore, our results also demonstrated that the alkaline pretreatment removed lignin from the biomass and improved the activity of the HEC-H, while there was no significant difference between the CC and SSB biomass controls (Figure 3A).

**Figure 3.** Application of the holocellulolytic enzyme cocktail (HEC-H) formulated with multifunctional enzymes (MFEs) and exoglucanase (Exg-D) derived from a termite bacterial hind-gut metagenome on pretreated sweet sorghum bagasse (SSB) and corncob (CC). (**A**) Activity in µmol/min, and (**B**) holocellulose conversion yields (total reducing sugars produced by HEC-H divided by the sum of cellulose and hemicellulose content) of SSB and CC samples. \* Represents *p*-values < 0.01 and # represents *p*-values < 0.05. All experiments were performed in triplicate and the values represent means ± SD.

Figure 3B shows the theoretical holocellulosic hydrolytic yields of the HEC-H activity on the SSB and CC samples. The results suggest that the conversion rates of the Ca(OH)<sup>2</sup> pretreated CC samples was 48.30%, while the conversion rate of the NaOH pretreated CC was about 49.46%. The HEC-H only converted about 21.91% of the untreated CC samples, which illustrated that the enzyme cocktail displayed about 2.25-fold higher efficiency on the alkali pretreated biomass. In contrast, the HEC-H displayed an approximate 1.67-fold increase in the theoretical conversion rate of alkali pretreated SSB samples, compared to the untreated SSB (control). We suggest that the enzyme cocktail efficiency was higher on CC compared to SSB samples, because of the higher holocellulosic content in CC biomass (see Table 1).
