Molecular and biochemical characterization of the recombinant endoglucanase GH8ErCel belonging to the cellulose synthesis system of Enterobacter sp. R1

ONTAÑON, ORNELLA; GHIO, SILVINA; MARRERO DÍAZ DE VILLEGAS, RUBÉN; PICCINNI, FLORENCIA E.; CAMPOS, ELEONORA

Florianopolis

Congreso; XIII Seminario Brasilero de Tecnologia Enzimática; 2018

ENZITEC

Endoglucanases are key enzymes for cellulose hydrolysis that can be used in multiple biotechnological processes, such as feed and food industries or ethanol biorefineries. In this study we characterized GH8ErCel, an endoglucanase belonging to the cellulose synthase system of Enterobacter sp. R1, which was isolated from a native cellulolytic consortium. GH8ErCel was cloned in pet28, expressed in the soluble fraction of E. coli system and purified to homogeneity at high yield. A molecular model was constructed with I-TAsser server, which suggested that GH8ErCel folds into an (α/α)6 barrel and comprises an additional β-motif that could participate in the substrate-binding cleft formation. Possible catalytic and oligosaccharide-binding residues were also identified by comparison with crystalized GH8 endoglucanases. The activity endo-β-1,4-glucanase of GH8ErCel was confirmed by zymografy and quantified in hydrolysis reactions using carboxymethylcellulose (CMC) as substrate. It presented a maximum activity at acidic pH and 30-60 °C, and remained active after prolonged incubation at 40-50 °C. The optimal reaction conditions were defined as pH4 and 40 °C. The CMC hydrolysis profile visualized by chromatography was similar to other GH8 endoglucanases, being celloligosaccharides (CE) of polymerization degree (DP)>3 the main reaction products. GH8ErCel was no active on Xylan, Avicel, PASC, filter paper, and the artificial substrates p-NPC and p-NPG, discarding xylanase, exoglucanase and beta-glucosidase side-activities. However, it was able to hydrolyze pre-treated agriculture residues to soluble sugars of DP>3.The results obtained suggest GH8ErCel as promising candidate for producing glucose free CE, compounds of great interests as possible prebiotics, from soluble cellulose and pre-treated biomass. Its combination with beta-glucosidases could also be studied as a possible strategy for the subsequent hydrolysis of these CE to fermentable glucose, desirable for the production of second generation bioethanol.