Enhanced Thermostability and Catalytic Activity of Streptomyces mobaraenesis Transglutaminase by Rationally Engineering Its Flexible Regions.

Streptomyces mobaraenesis transglutaminase can catalyze the cross-linking of proteins, which has been widely used in food processing. In this study, we rationally modified flexible regions to further improve the thermostability of FRAPD-TGm2 (S2P-S23V-Y24N-E28T-S199A-A265P-A287P-K294L), a stable mutant of the transglutaminase constructed in our previous study. First, five flexible regions of FRAPD-TGm2 were identified by molecular dynamics simulations at 330 and 360 K. Second, a script based on Rosetta Cartesian_ddg was developed for virtual saturation mutagenesis within the flexible regions far from the substrate binding pocket, generating the top 18 mutants with remarkable decreases in folding free energy. Third, from the top 18 mutants, we identified two mutants (S116A and S179L) with increased thermostability and activity. Finally, the above favorable mutations were combined to obtain FRAPD-TGm2-S116A-S179L (FRAPD-TGm2A), exhibiting a half-life of 132.38 min at 60 °C (t1/2(60 °C)) and a specific activity of 79.15 U/mg, 84 and 21% higher than those of FRAPD-TGm2, respectively. Therefore, the current result may benefit the application of S. mobaraenesis transglutaminase at high temperatures.

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