Distinct Roles of Internal Cavity Volume and Shape in Modulating Thermostability and Catalytic Activity of Microbial...

Microbial transglutaminase (MTG) is widely used to improve protein cross-linking and textural properties in meat, dairy, and plant-based products, yet its catalytic efficiency under industrial conditions remains limited. Here, high-pressure molecular dynamics (HP-MD)-guided cavity engineering strategy was employed to elucidate the distinct roles of cavity volume and geometry in regulating MTG thermostability and activity. Representative conformations were obtained from HP-MD simulations via trajectory clustering, followed by inertia-matrix-based geometric characterization to guide targeted mutagenesis. Cavity-filling mutations reduced internal cavity volume and enhanced thermostability, showing a positive correlation with ΔT_m (r = 0.6158), whereas reshaping of the catalytic cavity toward an elongated geometry improved activity (r = 0.7070). The optimal variant N71M/R127L/S199C/S243M exhibited a 1 °C increase in melting temperature and ∼13-fold higher activity, mitigating the conventional stability-activity trade-off. These findings highlight cavity volume and geometry as complementary parameters for enzyme engineering.