Enhanced Thermostability of Streptomyces mobaraenesis Transglutaminase by Multirational Design Methods

Streptomyces mobaraenesis transglutaminase is widely employed in food processing. This study aimed to enhance the thermostability of FRAPD-TGm2, a previously engineered thermostable mutant of transglutaminase. Three independent strategies were implemented. First, virtual saturation mutagenesis screened by folding free energy (ΔG) analysis yielded the mutant N176L with an 8.0% increase in residual activity (60 °C/30 min). Second, consensus design combined with ΔG prediction identified the mutation K152A, which improved the residual activity by 6.9%. Third, disulfide bond engineering generated three stabilizing mutants: D3C-G283C, T7C-E58C, and A160C-G228C, which increased the residual activity by 29.8, 15.8, and 8.1%, respectively. These positive mutations were strategically combined with previously mutations (S179L and Y34W) to construct the mutant FRAPD-TGm2C. The combined mutant exhibited a 7.5-fold longer half-life at 60 and a 7.58 °C increase in T_m relative to FRAPD-TGm2. Notably, at 78 °C, FRAPD-TGm2C achieved complete cross-linking of β-casein within 5 min, demonstrating superior performance for high-temperature food applications.