Modulation of the catalytic activity and thermostability of a highly thermostable GH7 endoglucanase by engineering the key loop B3

Abstract

Abstract Background The cellulases of glycoside hydrolase family 7 (GH7) are confined into two main types, endoglucanase and cellobiohydrolase, based on their subtle differences in loop structures. In the viewpoint of evolution, the loop regions of GH7 cellulases exhibit a more pronounced effect on enzyme properties. Results A thermophilic endoglucanase of GH7, TtCel7, having a long 18 amino acid loop B3 was identified in Thermothelomyces thermophilus ATCC 42464. It was successfully obtained with heterologous expression and then purified for activity assays. The recombinant TtCel7 was distinguished for the excellent thermostability at 90°C (> 30% residual activity after 1-h incubation). When truncated the loop B3 or mutated C220A to remove the disulfide bond on loop B3, both the TtCel7 variants showed decreased catalytic efficiency, but the ∆B3 showed improved thermostability, retaining higher residual activities (9–44%) at 70–90°C than the wild type. Based on the molecular dynamics (MD) simulation analysis, both the loops B1 and A3 of ∆B3 swing toward the catalytic center, which contributes to the reduced cleft space and more rigid structure; instead, the structural rigidity of C220A was decreased as an α-helix was introduced into the loop B3 due to the deletion of disulfide bond. Conclusions Two structural elements related to catalysis and thermostability of GH7 cellulases were identified in this study through structure-directed enzyme modulation. Of them, the loop B3 of TtCel7 possibly stretches the catalytic pocket, making the catalytic tunnel more open and the protein structure more flexible for efficient catalysis. Additionally, the disulfide bond in loop B3 stabilizes the loop structure and keeps it in a highly active and stable state. This strategy casts an insight into the engineering of GH7 endoglucanases for potential commercialization.