Computational approach for identification and characterization of a glucose-tolerant thermostable β-glucosidase from marine metagenome

Cellulase enzymes comprising endo-1,4-β-glucanase, exo-1,4-β-glucanase, and β-glucosidase mediate the degradation of cellulosic biomass and are frequently used in biofuel production from lignocellulose. β-glucosidases that convert cellobiose to glucose are sensitive to temperature and glucose concentration and thus often show limited catalytic efficiency. Several β-glucosidases having high temperature or glucose tolerance have been evaluated, but a potential candidate having high efficiency along with thermostability and glucose tolerance is yet to be identified. The present study focuses on marine metagenome investigation for the identification of high-potential β-glucosidase. Nine β-glucosidases of the GH 1 family having (β/α)₈ barrel domains were observed. Six β-glucosidases were predicted to have a Tm value higher than 65 ℃, including ECV39653.1 β-glucosidase. Molecular docking of all identified β-glucosidases with cellobiose and glucose revealed that ECV39653.1 β-glucosidase has the highest negative binding energy of - 7.4 kcal/mol for cellobiose at the active site, while having insignificant binding of glucose with binding energy of -5.4 kcal/mol at a site different from the active site. The structural analysis showed an effective similarity of ECV39653.1 β-glucosidase with known thermostable and glucose-tolerant β-glucosidases. The prediction of kinetic parameters gave k_cat/K_m value of 989.08163 sec^-1 mM^-1 for cellobiose. In-depth MD simulation and free binding energy analysis showed highly effective binding of cellobiose over the 100 ns trajectory with an average total binding energy of - 17.45 kcal/mol. The PCA and analysis of free energy landscape showed less variance and conformational changes in ECV39653.1 β-glucosidase cellobiose complex form in comparison to apo-form and disclosed attainment of global minima, thus proving the high ECV39653.1 β-glucosidase-cellobiose complex stability. The analysis of the simulation trajectory revealed that glucose left the binding cavity during simulation, thus disclosing weak binding and, hence, effective glucose tolerance. Therefore, the present in-silico investigation provides a promising high-efficiency, thermostable, and glucose-tolerant ECV39653.1 β-glucosidase. Further studies can provide scope for its utilization in the development of effective technologies for large-scale biofuel production.