Rational design strategy for thermostability enhancement of protein-glutaminase and investigation of the underlying mechanisms

Protein-Glutaminase (PG) with efficient deamidation ability has shown vital potential in food fields. Enzymes with high thermostability remain active in high-temperature environments, which can be applied to some steps requiring high temperature treatment in food processing, thereby greatly expanding their industrial application. In this study, an efficient comprehensive strategy based on consensus sequence and computer-aid analysis was proposed to develop a combinatorial mutant mPG-5M (A79S/T97V/S108P/N154D/L156Y), exhibiting a 55.1-fold increase in t_1/2 at 60 °C (1132.75 min) and a T_m value of 75.21 °C without loss of enzyme activity. Molecular dynamics simulation analysis insisted that the reduced flexibility, increased structural rigidity, and enhanced hydrogen bonding network observed in the mPG-5M all contribute to its superior stability, particularly under thermal stress. This study provided valuable strategy and comprehensive molecular mechanisms insights for protein engineering of the thermostability enhancement of PG, broadening its industrial applicability of food protein deamidation. These mutations collectively contributed to the enhanced thermal stability of the mPG-5M mutant.