Design of ancestral protein-glutaminase with improved thermal stability.

Ancestral sequence reconstruction produced protein-glutaminase variants with up to 13.6°C higher melting temperature than wild type, enhancing thermal stability in a single step.

• Why it matters: Improving the thermal stability of protein-glutaminase is crucial for expanding its industrial applications, but previous methods required labor-intensive optimization. This work addresses the need for efficient strategies to generate thermostable enzymes.
• What they did: Eight ancestral PG variants were engineered using ASR, with mutational analysis identifying five substitutions that individually increased stability; the most stable, AncPG1, had a melting temperature of 76.2°C.
• The result: AncPG1 retained reactivity toward protein substrates at elevated temperatures and showed greater absolute reactivity than wild type at 70°C, demonstrating the potential of ASR to produce thermostable enzymes with improved functional performance.