**4. Discussion**

The SSB and CC biomass samples were pretreated before they were hydrolysed with the holocellulolytic enzyme cocktail formulated with MFEs and Exg-D to assess their hydrolysability. El-Naggar et al. [7] argued that one of the factors that hinders lignocellulosic hydrolysis are biomass structural or chemical factors, which include biomass crystallinity, cellulose degree of polymerisation, porosity, accessible surface area, particle size, lignin, hemicellulose and acetyl-group content. In addition, Álvarez et al. [25] reported that chemical pretreatments were essential to changes in the biomass composition. The pretreatment of the biomass results in the removal of lignin, which makes the biomass less recalcitrant to enzyme hydrolysis [6,25]. The current study demonstrated that NaOH pretreatment of the CC and SSB samples significantly removed the lignin that led to an increase in relative cellulose, hemicellulose and biomass porosity (Table 1; Figure 1). However, Ca(OH)<sup>2</sup> pretreatment did not remove all the lignin as shown in Table 1 and Figure 1. Beukes and Pletschke [10] also argued that the Ca(OH)<sup>2</sup> pretreatment of the sugar cane bagasse modified the lignin and did not remove most of it from the biomass. Alkaline pretreatments induce a saponification reaction, where free hydroxide ions break ester bonds that exist between the inner molecules of lignocellulose, which connect hemicellulose and other components (such as between lignin and other hemicelluloses) which results in an increase in the pore structures of lignocellulose due to disappearance of the connecting bonds [26].

The FTIR results validated the morphological and chemical changes of the biomass after pretreatment. FTIR results demonstrated that crystalline cellulose Iβ peaks were absent in the spectrum of the Ca(OH)<sup>2</sup> and NaOH pretreated SSB and CC biomass. The 1730 cm−<sup>1</sup> peak corresponded to C-O stretching vibration for the acetyl and ester linkages in lignin and hemicellulose, while the 1250 cm−<sup>1</sup> peak corresponded to C–O out of plane stretching due to the aryl group in lignin [27]. The presence of 1430, 1162 and 1111 cm−<sup>1</sup> peaks indicates a prevalence of crystalline cellulose II [15]. The alkaline pretreated biomass showed improved cellulose II, hemicellulose and amorphous content. However, the SEM, chemical composition and FITR results revealed that the NaOH pretreatment was more effective in removing lignin from the biomass than Ca(OH)<sup>2</sup> pretreatment.

The MFEs displayed activity on the CMC, various xylan and xyloglucan substrates as shown in Table 2. Malgas et al. [5] reported that xylan is a complex substrate with different side chains that can hinder enzyme optimal function, if it possess a catalytic active site that is not suited to the side chains. Xyloglucan is also a complex substrate with various side chains as described by Fry et al. [28] and Rashmi and Siddalingamurthy [29]. These diverse and complex properties of the CMC, various xylan and xyloglucan substrates demonstrate that the MFE-5E, MFE-5H and MFE-45 were indeed multifucntional enzymes capable of hydrolysing the β-1,4-glucosidic bonds of the backbone chain. Several multifunctional (or promiscuous) enzymes have been reported previously and were defined as enzymes that play multiple physiological roles in a cell, or enzymes that display catalytic activities on a range of substrates which are structurally and chemically different [30–32]. In addition, mulifunctional/promiscous enzymes change their hydrolytic activities under different reaction conditions, which include various solvents, extreme temperature, various pHs and a range of substrates specificities. Aspeborg et al. [33] emphasised the idea that GH family 5 contains some of the multifunctional enzymes, which could explain the observed multifunctional nature of MFE-5E and MFE-5H.

HEC-H was applied to the alkaline pretreated biomass and the untreated biomass was used as a benchmark for hydrolysis improvement due to pretreatment. It was evident from Figure 3 that HEC-H demonstrated higher activity on pretreated biomass compared to the untreated biomass. In addition, the HEC-H performed much better on the CC biomass compared to the SSB biomass. The specific activity demonstrated that the MFEs used to formulate the HEC-H were highly active on the hemicellulose substrate and amorphous cellulose. These findings explain why the theoretical holocellulosic conversion

yields for alkali pretreated biomass were between 35% and 50%. Takada et al. [3] also demonstrated that the chemical composition of CC biomass consisted mostly of hemicellulose. However, SSB biomass after alkaline pretreatment consisted mainly of cellulose as the major component. We believe that the HEC-H is the first enzyme cocktail formulated using GHs derived from a termite hindgut metagenome for the effective hydrolysis of hemicellulose and amorphous cellulosic components of agricultural feedstocks. Other studies have used two or three enzymes derived from termites, i.e., Feng et al. [34] reported on the synergy formed by two β-glucosidase (CfGlu1C and CfGlu1B) enzymes from a lower termite (*Coptotermes formosanus*). CfGlu1C and CfGlu 1B displayed synergy on lactose hydrolysis and significantly increased the rate of hydrolysis. The GH7 enzyme, endoglucanase and β-glucosidase from *Reticulitermes flavipes* worker termites also displayed synergism on pine sawdust [35]. The synergism between these enzymes significantly increased the amounts of glucose released during the hydrolysis of the pine sawdust.
