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Streptomyces mobaraenesis transglutaminase has been widely used in food processing. We here significantly improved the catalytic properties of S2P-S23V-Y24N-S199A-K294L (TGm1), a highly stabilized variant of the transglutaminase. First, a virtual proline scan was performed based on folding free energy changes to obtain TGm1 variants with enhanced thermostability. Second, the residues within 15 Å of Cys64 in the enzyme–substrate complex of TGm1 were subjected to virtual saturation mutagenesis to generate the variants with reduced binding free energy and increased activity. After combining the favorable mutations, we obtained the variant FRAPD-TGm1-E28T-A265P-A287P (FRAPD-TGm2), exhibiting 66.9 min of half-life at 60 °C (t 1/2(60 °C)), 67.8 °C of melting temperature (T m), and 71.8 U/mg of specific activity, which are 2-fold, 2.6 °C, and 43.8% higher than those of FRAPD-TGm1, respectively. At last, to increase the surface negative net charge of FRAPD-TGm2, we introduced the mutations N96E-S144E-N163D-R183E-R208E-K325E, yielding FRAPD-TGm3. The latter’s t 1/2(60 °C), T m, and specific activity were 122.9 min, 68.6 °C, and 83.7 U/mg, which are 83.8%, 0.8 °C, and 16.6% higher than the former, respectively. FRAPD-TGm3 is thus a robust candidate for transglutaminase application.