Characterization of two GH5 endoglucanases from termite microbiome using synthetic metagenomics

Abstract

Here, we characterize two novel GH5 endoglucanases (GH5CelA and GH5CelB) from an uncultured bacterium identified in termite gut microbiomes. Both genes were codon-optimized, synthetized, cloned, and expressed as recombinant proteins in Escherichia coli for subsequent purification. Both enzymes showed activity on the pNPC and barley β-glucan substrates, whereas GH5CelB also showed low activity on carboxymethyl cellulose. The optimum conditions for both enzymes were an acid pH (5) and moderate temperature (35 to 50 °C). The enzymes differed in the kinetic profiles and patterns of the generated hydrolysis products. A structural-based modeling analysis indicated that both enzymes possess a typical (β/α)8-barrel fold characteristic of GH5 family, with some differential features in the active site cleft. Also, GH5CelB presents a putative secondary binding site. Furthermore, adjacent to the active site of GH5CelA and GH5CelB, a whole subdomain rarely found in GH5 family may participate in substrate binding and thermal stability.Therefore, GH5CelA may be a good candidate for the production of cello-oligosaccharides of different degrees of polymerization applicable for feed and food industries, including prebiotics. On the other hand, GH5CelB could be useful in an enzymatic cocktail for the production of lignocellulosic bioethanol, because of the production of glucose as a hydrolysis product.

Biochemical characterization of a novel thermo‐halo‐tolerant GH5 endoglucanase from a thermal spring metagenome

“…Among the tested substrates, Cel M displayed the maximum specificity with β‐ d ‐glucan and the cellulose derivative, Na‐CMC (Table 1). This was in accordance with many previous reports on endoglucanases characterized from different sources (Guerrero et al, 2020; Ma et al, 2020; Wang et al, 2019; Yin et al, 2020). A limited activity was measured in the presence of galactomannan‐containing substrates, for example, Locust bean gum and Guar gum.…”

“…The kinetic analysis determined a fairly good substrate affinity of Cel M , which was comparable to that of the previously characterized GH5 endoglucanases from T. rubra YIM 77501 (Yin et al, 2020), B. subtilis 1AJ3 (Ma et al, 2020), and oil reservoir (Zhu et al, 2020). The turnover number and enzyme efficiency of Cel M were more or less similar to that of previously reported endoglucanases from termite microbiome (Guerrero et al, 2020) and cow rumen metagenome (Patel et al, 2020).…”

“…A limited activity was measured in the presence of galactomannancontaining substrates, for example, Locust bean gum and Guar gum. endoglucanases characterized from termite microbiome (Guerrero et al, 2020). Nonetheless, many endoglucanases had been reported to have the capabilities to hydrolyze both cellulose and xylan (Tan et al, 2018;Yin et al, 2020).…”

“…Endoglucanases have been reported from many glycoside hydrolase (GH) families, for example, GH5‐10, GH12, GH44‐45, GH48, GH51, GH74, GH124, and GH131 of Carbohydrate‐Active enZYme (CAZy) database (Garg et al, 2016; Yennamalli et al, 2011). Among these, GH5 is the largest family that contains not only endoglucanases, but also many other proteins exhibiting more than 20 catalytic activities, for example, xylanase, lichenase, mannosidase, so forth, propounding the significance of this family in multifarious applications (Guerrero et al, 2020). The first cellulase family was described from GH5; therefore, it is also known as “Cellulase family A.” GH5 endoglucanases have been reported from a plethora of eukaryotic and prokaryotic genera, mostly fungi and other microorganisms, residing in different ecological habitats.…”

“…(CAZy) database (Garg et al, 2016;Yennamalli et al, 2011). Among these, GH5 is the largest family that contains not only endoglucanases, but also many other proteins exhibiting more than 20 catalytic activities, for example, xylanase, lichenase, mannosidase, so forth, propounding the significance of this family in multifarious applications (Guerrero et al, 2020). The first cellulase family was described from GH5; therefore, it is also known as "Cellulase family A."…”