Free energy–guided Q302P mutation in GH1 β -glucosidase from Aspergillus fumigatus improves enzyme function via catalytic tunnel perturbation

Beta-glucosidase (EC 3.2.1.21) is crucial to biorefinery industries because it is the bottleneck enzyme in the enzymatic conversion of cellobiose to glucose, a key component of the enzyme complex that converts lignocellulosic biomass into glucose. The enzyme is inhibited by its own product, glucose, which accumulates and blocks the catalytic tunnel or the product exit region. This is the main drawback of this enzyme. In this work, we engineered AfBgl1 to enhance its thermal stability. FireProt was used to generate a mutant library, and the mutants with the highest free energies (Q302P, S187W, and S301D) were selected for further studies. The comparative analyses revealed that the mutant protein Q302P showed increased substrate affinity (Km of Q302P = 0.3 mM and AfBgl1 = 1.42 mM), activity-based thermostability (up to 70°C), and increased glucose tolerance (Ki of Q302P = 188.65 mM and AfBgl1 = 83.96 mM). Interestingly, a mutation based on free energy gains these properties of glucose tolerance and catalytic efficiency, which is generally unconventional. The mutational effect has been justified and explained through structural analysis and molecular dynamics simulations, indicating that the mutation's position is crucial for triggering this robust effect. The study delineates the scope of free-energy-based protein engineering beyond thermostability and highlights the influence of local fluctuations on the dynamics of the catalytic tunnel.