## 14 Structure and Functionality of the Newly Characterized Processive Endocellulases
(1987) further demonstrated that a cleft active site was also essential to have exo-mode hydrolysis of xyloglucans by an endocellase to engage the hydrolytic action. The double Glu residues as the proton donor in the putative catalytic acid/base and nucleophile are at Glu136, and are consistent with the strictly conserved residues within GH5 catalytic domain functions (186). Intriguingly, a long tunnel-like active site with multiple Trp residues is clearly visualized along the enzyme protein surface and is located from the C-terminus of the \(\beta\)-strands or the TIM barrel ([PERSON] et al., 2019). This predicted long tunnel-like active site topology for this novel GH5-p4818 Cel5_2A has provided additional evidence that this novel GH5-p4818 Cel5_2A has exo-acting mode, and thus is a processive GH5 endocellase ([PERSON] et al., 2019). All exocellulases are processive ([PERSON] et al., 2019). Exocellulases have a tunnel-like active site topology ([PERSON] et al., 2019; [PERSON] et al., 2019; [PERSON] et al., 2019). However, [PERSON] et al. (1985) shown that the processive GH9A (E4) endocellulase _Thermobifida fusca_ had a cleft and not a tunnel-like active site topology, and a CBM was essential for this enzyme's activity on crystalline celluloses. [PERSON] and [PERSON] (2019) discussed that substrate binding or the formation of cellulase-substrate complexes was essential to characterize cellulase structure and catalytic relationship. Giving the multi-functional properties of this novel GH5-p4818 Cel5_2A endocellulase, crystallographic ensembles to be derived from multiple substrate binding states should be generated and used to study its functional structural variations ([PERSON] et al., 2019). The structure and functionality of have been revealed in details for GH5-tCel5A1 ([PERSON] et al., 2019; [PERSON] et al., 2019). Thus, it is very likely that GH5-p4818 Cel5_2A will be unique with a combined cleft and a tunnel-like active site topology in its structure to support its superb multifunctionality, which needs to be resolved in our future studies. Further crystallization and structural characterization of this GH5-p4818 Cel5_2A processive cellulase will facilitate further rationale design-based mutagenesis to improve functionality of these enzymes, such as resistance to mammalian proteases. To facilitate further effective adoption of these two unique GH5-tCel5A1 and GH5-p4818 Cel5_2A processive endocellularases for industrial applications, the following three aspects of research need to be pursued. Firstly, further enhancement of these wild-type of both GH5-tCel5A1 and GH5-p4818 Cel5_2A endocellase specific activities on crystalline celluloses and hemicelluloses will need to be conducted. [PERSON] et al. (1987) articulated that innovations at a small-scale through enzyme discovery in the laboratory could exert large-scale impacts when rolled out in industrial applications as demonstrated in commercial ethanol production. [PERSON] et al. (2019) reviewed that enzyme and pretreatment expenses represent about up to 48% of the total cost of commercial lignocellulosic ethanol production. Directed evolution is a very powerful protein-engineering tool for improving enzyme properties such as specific activity and thermostability without in-depth immediate understanding of enzyme protein structure and enzyme-substrate interactions ([PERSON] et al., 2019). Given the fact that the GH9 _Clostridium Phytorementans_ processive endocellase had a large molecular weight (over 100 kDa) and was multi-modular, [PERSON] et al. (2019) demonstrated this by using _B. subtilis_ as a host and applying directed evolution for improving the processive GH9 endocellular activity by about two-fold from the _Clostidium Phytorementans_ on the solid cellulosic substrate. Efforts ought to be made to further enhance both GH5-tCel5A1 and GH5-p4818 Cel5_2A endocellular activity by directed evolution and using _B. subtilis_ as a host.