Unveiling Highly Active and Stable l-Glutaminase through Ancestral Sequence Reconstruction and Turnover Number Prediction

In our study, we employed ancestral sequence reconstruction and DLKcat analysis to engineer l-glutaminases with enhanced activity and thermal stability, using Bacillus subtilis 168 l-glutaminase (YbgJ) as the template. We identified two ancestral l-glutaminases, Anc165 and Anc194, with specific activities 730.6- and 203.5-fold higher than YbgJ, respectively. Anc165 retained 96% activity at 65 °C and 69% at 70 °C for 30 min, while Anc194 maintained over 40% activity at 70 °C, contrasting with YbgJ, which was inactivated above 55 °C. In a 15% NaCl solution, Anc165 and Anc194 retained 100 and 18% activity, respectively, compared to YbgJ's complete loss. Molecular dynamics simulations indicate that the enhanced thermal stability of Anc165 is due to its increased structural rigidity. The enhanced activity of Anc165 is due to a more stable enzyme-substrate complex with l-glutamine. In a simulated soy sauce fermentation system, Anc165 produced about 10% more glutamate than YbgJ. With high-thermal stability and activity, Anc165 could be a potential candidate for industrial applications.