Ancestral Sequence Reconstruction for Novel Bifunctional Glutathione Synthetase with Enhanced Thermostability and Catalytic Efficiency

The bifunctional glutathione synthase (GshF) is able to catalyze glutathione synthesis and is favored for industrial application due to its lack of product inhibition. However, its practical use is limited by moderate catalytic efficiency and poor thermostability. Here, we applied ancestral sequence reconstruction (ASR) to engineer a more robust ancestral GshF (Anc427) with thermal denaturation temperature of 56.2 ± 0.2 °C, representing an increase of 10.8 ± 0.2 °C over the probe enzyme (St-GshF). Additionally, Anc427 exhibited a thermal half-life (t_1/2) of 3465.7 min at 40 °C, representing a 20-fold increase over that of St-GshF. Under optimal conditions (pH 7.0, 37 °C), Anc427 displayed a specific activity of 3.3 ± 0.02 U·mg^-1, representing a 20% enhancement compared to St-GshF. Structural modeling and molecular dynamics simulations indicated that the improved stability can be attributed to increased structural rigidity in Anc427. These findings demonstrate that ASR effectively enhances both thermostability and catalytic activity of GshF, significantly advancing its potential for industrial biocatalysis.