## 14 Structure and Functionality of the Newly Characterized Processive Endocellulases  
Unique functionality of enzyme proteins is supported by their unique structures. It has been well documented that substrate specificity and the mode of action of various glycosyl hydrolases are governed by exquisite details of their three-dimensional structures rather than their global fold with three active-site topologies of (i) a pocket or crater; and (ii) a cleft or grove; and (iii) a tunnel [184]. These unique GH5-tCel5A1 and GH5-p4818 Cel5_2A processive cellulases have three major unique biochemical properties within one enzyme molecule in comparison with other endocellulares reported in the literature. Firstly, GH5-tCel5A1 and GH5-p4818 Cel5_2A endocellulares are small monomodular enzyme proteins with a molecular weight (below 50 kDa) and relatively high activity on crystalline celluloses [45; 127]. Only two other reported studies in the literature have so far shown that an endocellulae with a single module and such a comparable small molecular weight and size could hydrolyze crystalline cellulose substrates, including the study by [PERSON] et al. (2005) [118] on the Cel5A from the brown rot basidomycete _Postia placenta_; the study by [PERSON] et al. (2011) [54] on a yak ruminal GH5 cellulase. Secondly, both GH5-tCel5A1 and GH5-p4818 Cel5_2A endocellulares have exocellulose activity on crystalline celluloses thus are a processive endocellulares. Only two other reported studies in the literature have shown that a processive endocellase without a CBM could hydrolyze crystalline cellulose substrates, including the study by [PERSON] et al. [118] on the Cel5A from the brown rot basidomycete _Postia placenta_, and the study by [PERSON] et al. (2011) [54] on a yak ruminal GH5 cellulase. Classic processive endocellulares are all primarily GH9 cellulases and they all contain a CBM, including the _Clostridium thermocellum_ noncellulosomal endoglucanase GH9B cellulose GH9B [131], the _Thermobifida fusca_ processive endoglucanase GH9A (E4) [185], and the _Clostridium Phytorementans_ processive endoglucanase Cel9 [135]. Structural analyses by [PERSON] et al. [185] show that the presence of a CBM was essential for the processive nature of these GH9 endocellases. However, [PERSON] et al. [133] reported that the presence of CBM was not essential for the processive property in the _Saccharomyces degradans_ GH5 processive endoglucanas. Third, apart from hydrolyzing crystalline celluloses, both GH5-tCel5A1 and GH5-p4818 Cel5_2A endocellases have been shown to have activities on other hemicelluloses, i.e., \(\beta\)-glucans, xylan, xyloglucans, mannan, glucomanan and galactomanan. To the best of our knowledge, no other cellulases have been reported with such wide substrate specificity in the literature. Sequence alignments of several different GH5 processive cellulases, including both GH5-tCel5A1 and GH5-p4818 Cel5_2A, had been previously illustrated in the supplemental Figure-S5 in the study by [PERSON] et al. (2019) [PERSON] et al. (2019). Attempts have been made to predict the structure of this GH5-p4818 Cel5_2A endocellular with the SWISS-MODEL online server by [PERSON] et al. (2019). This predicted TIM barrel fold of this cellulase GH5-p4818 Cel5_2A is consistent with the TIM barrel motif superfamily previously reported in the GH5 endocellase CeIA from _Clostridium cellulolyticum_[186]. A cleft or groove structure was apparently seen in this GH5-p4818 Cel5_2A endocellular as was shown in the _Clostridium cellulolyticum_ GH5 endocellase CeIA by [PERSON] et al. (1986) and reviewed by [PERSON] and [PERSON] (1984). High activity of GH5-p4818 Cel5_2A on the branched substrate xyloglucan and locust bean gum, as summarized in Table 1, would suggest the presence of a cleft active site in this cellulase structure, since branched substrates such as xyloglucans and locust bean gum could not effectively fit into a tunnel-like or a pocket active site topology (162). [PERSON] et al.