Design of ancestral protein-glutaminase with improved thermal stability

Protein-glutaminase (PG) from Chryseobacterium proteolyticum is an industrially valuable enzyme for protein deamidation; however, its application is limited because of insufficient thermal stability. Previous rational design studies have enhanced the thermostability of PG but often required labor-intensive, stepwise optimization. In this study, ancestral sequence reconstruction (ASR) was used to engineer thermostable PG variants in a single design step. Eight ancestral PGs were constructed, all of which exhibited enhanced thermal stability compared with that of the wild type. The most stable variant, AncPG1, had a melting temperature of 76.2°C, 13.6°C higher than that of the wild type. Mutational dissection identified five substitutions, N148G, N154D, L156Y, L168S, and D179S, that individually enhanced thermal stability and showed cumulative stabilizing effects when combined. These substitutions were predominantly located in surface-associated loop regions. Notably, although the ancestral variants showed reduced catalytic efficiency toward the small synthetic substrate Cbz-Gln-Gly, they retained reactivity toward macromolecular protein substrates when compared at equal activity units. Furthermore, AncPG1 showed greater absolute protein-substrate reactivity than WT CpPG under elevated temperature conditions. These findings provide further support for ASR as a strategy for generating thermostable enzyme variants with improved functional performance under elevated-temperature conditions.IMPORTANCEProtein-glutaminase from Chryseobacterium proteolyticum is used to improve functional properties of food proteins, but its thermal stability limits its use at elevated temperatures. In this study, ancestral sequence reconstruction generated PG variants with substantially increased melting temperatures while retaining reactivity toward protein substrates. AncPG1 also showed greater absolute reactivity than WT CpPG toward multiple protein substrates at 70°C when compared at the same protein concentration. Several substitutions that individually enhanced thermal stability were located in surface-associated loop regions. These results illustrate the utility of evolutionary sequence information for engineering thermostable protein-modifying enzymes.