Processivity and enzymatic mechanism of a non-modular family 5 endoglucanase from Sporocytophaga sp. CX11 with potential applications in cellulose saccharification

In this study, a novel GH5 processive endoglucanase SmCel5A from Sporocytophaga sp. CX11 was functionally expressed in E. coli. It could rapidly decrease the viscosity of carboxymethyl cellulose (CMC-Na) solution by nearly 50 % within the initial 8 min of incubation and exhibited a significantly high specific activity towards CMC-Na of 9940 U/µmol. SmCel5A could also hydrolyze other cellulosic substrates such as RAC, Avicel, filter paper, β-glucan and the chromogenic substrate pNPC. When hydrolyzing filter paper, about 89.1 % of soluble reducing sugars were generated after 180 min of incubation, and the main products were cellobiose followed by cellotriose and glucose. The processive ratio of SmCel5A increased from 2.32 to 11.22 as the reaction time was extended from 5 min to 180 min, which is significantly higher than those of other known processive endoglucanases. Moreover, SmCel5A could hydrolyze cellodextrins with the degree of polymerization (DP) ≥ 3, but it was not active on cellobiose. In combination reaction with β-glucosidase, the maximum substrate conversion rate reached 73.2 %, showing that the synergistic reaction of the two enzymes could efficiently reduce the accumulation of cellobiose and greatly improve the hydrolysis efficiency of cellulose. The three-dimensional structure of SmCel5A was predicted by AlphaFold2 and showed to feature a classic GH5 family (β/α)₈-barrel structure, with a deep substrate-binding cleft formed by the amino acids at the C-terminus. Molecular docking results indicated that when hydrolyzing cellulosic substrates, SmCel5A exhibits a preference for the exo-type mechanism of action over the endo-type mechanism of action.