Study and rational engineering of the glucose tolerance and promotion of β-glucosidase

Abstract:

[Objective] In this study, we identified possibly critical sites for the higher glucose tolerance and promotion of Bgl2A through three ways, and checked their significance by mutation and characterized the mutants. Further, we investigated the mechanism of the critical sites affecting the glucose tolerance and promotion by molecular docking. Further, Bgl3A (Bgl2A:A22S/V224S), with low glucose tolerance, was rationally engineered to obtain mutants with better application potential. [Methods] Through sequence and structure comparison, statistical coupling analysis, and structural analysis, we selected the residues in the substrate tunnel and near the active center that may indirectly affect the glucose tolerance and promotion for mutations to different type amino acids, then prepared the mutants and tested their enzymatic properties. [Results] Some mutants with higher glucose tolerance and promotion, such as D322I, W325A, W126Y, F172N, C173I and N226V were obtained. Molecular docking showed that these mutations may change the energy of glucose binding to the active center by allosteric effect, thus influence the glucose tolerance and promotion. Based on the above results, corresponding mutants were then made for Bgl3A. Some mutants, such as N226V and F172N, with high glucose tolerance and promotion while considerable activity and stability remained, have higher application potential. [Conclusion] The results demonstrated that in addition to the sites where glucose directly binds to, there are other sites that do not interact directly with glucose can indirectly influence the glucose tolerance and promotion of β-glucosidase through long range effect, which provides a new clue for rational engineering β-glucosidases.