Significant improvement of the thermostability and catalytic efficiency of β-glucosidase Bgl59 via cysteine engineering and rational design.

β-glucosidase (BGL) is essential in high-temperature industrial processes, but naturally occurring enzymes often lack sufficient thermostability. Using the wild-type Bgl59 from Devosia psychrophilaas the template, we first constructed the double-cysteine mutant A153C-N213C via disulfide-bond design. Although no disulfide bond was detected, this mutant showed improved thermal stability, including a + 2.5 °C increase in melting temperature(Tₘ) and a 1.68-fold higher catalytic efficiency. Subsequently, starting again from the wild-type template, we rationally designed the mutant M3 (A20S-N213M-A308Y) without incorporating the prior cysteine mutations. M3 exhibited further enhanced thermostability, with a + 7.2 °C rise in melting temperature(Tₘ), a + 5 °C shift in optimal temperature, a 191.3-fold longer half-life(t₁/2) at 50 °C, and a 4.7-fold increase in catalytic efficiency. Molecular dynamics simulations revealed that the improved rigidity of M3 stemmed from strengthened hydrogen bonds, optimized hydrophobic interactions, and enhanced van der Waals contacts. These results demonstrate that structure-guided engineering of Bgl59 through both cysteine-based and multi-tiered protein engineering strategy can significantly enhance its thermal stability and catalytic performance, offering promising potential for industrial biocatalysis.