[Rational design for improving the thermostability of β-glucosidase from Bacillus thermoamylovorans]

The β-glucosidase from Bacillus thermoamylovorans (Bgl52) has been identified as a thermophilic and acidophilic β-glucosidase, which constitutes the most critical component in the cellulolytic enzyme system for cellulose hydrolysis. Although Bgl52 is a thermophilic enzyme, its poor thermal stability at high temperatures restricts its long-term industrial application under elevated-temperature conditions. In this study, rational design strategies, including two free energy calculation programs (Fireprot and FoldX) and homologous sequence alignment, were integrated to perform single-point and multi-point combinatorial mutagenesis on Bgl52. The enzymatic kinetic stability assay and molecular dynamics simulation were conducted to further investigate the catalytic performance and structural variations of positive mutants. After screening, the optimal stacked mutant M5 exhibited increases in T_m, optimal temperature, and T₅₀ by 8.7, 10 and 8℃, respectively, compared with Bgl52 (wild type, WT), and its half-life t_1/2 at 80℃ was enhanced by 32.5 folds. The catalytic efficiency (K_cat/K_m) of M5 showed a slight decrease compared with that of the wild-type enzyme. Furthermore, M5 hydrolyzed native cellulose into glucose more efficiently at the high temperature of 85℃. Structural analysis and molecular dynamics simulation revealed that the improved thermostability was attributed to increased hydrogen bonds, the proline effect, additional salt bridges, and enhanced hydrophobic interactions. The results indicate that the multi-strategy integrated rational design approach demonstrates outstanding efficiency and reliability in enzyme engineering modification, enhancing the industrial application potential of β-glucosidase.