Thermostability Enhancement of <i>Streptomyces mobaraensis</i> Transglutaminase by Folding Free Energy-Guided Helix-Corner Motif Engineering

The industrial application of <i>Streptomyces mobaraensis</i> transglutaminase (smTG) is limited by its poor thermostability. To enhance its stability, we engineered a thermostable mutant, FRAPD-TGm2A1, using helix-corner design. Following the introduction of the known stabilizing mutation Y34W, Cartesian_ddG was used to virtually screen 56 helix-corner residue sites, from which 20 top-ranking mutations with reduced folding free energy were experimentally tested. Four single mutations (G157N, G157Q, G157W, and A160P) that notably improved thermostability were combined, generating FRAPD-TGm2A-Y34W-G157Q-A160P (FRAPD-TGm2A1) with the highest thermal stability. Compared with FRAPD-TGm2A, the half-life of FRAPD-TGm2A1 increased by 44.46% at 60 °C (176.32 min), 44.01% at 65 °C (14.66 min), and 108.21% at 70 °C (2.79 min), while the melting temperature rose by 1.81 °C. FRAPD-TGm2A1 also efficiently cross-linked β-casein at 75 °C. Importantly, FRAPD-TGm2A1 was successfully expressed in <i>S. mobaraensis</i>, exhibiting a half-life of 160.03 min at 60 °C, far exceeding that of wild-type (<2 min) and demonstrating promising potential for industrial application.