Abstract: Streptomyces mobaranensis transglutaminase TGase is an important food enzyme, which is widely used in protein-based food modification. In order to improve the thermostability of TGase, intramolecular disulfide bonds were introduced. Firstly, based on the crystal structure of wild-type TGase (PDB∶1iu4), the simulated structure of TGase thermostable mutant MS (S2P-S23V-Y24N-S199A-K294L) was constructed. Secondly, the disulfide bonds were predicted by Disulfide Design 2.0. According to the free energy of disulfide bonds formation, 12 disulfide bonds mutant were selected and expressed in Escherichia coli BL21(DE3). Finally, the enzymatic properties of the purified mutants were characterized, and the stabilization mechanism was analyzed. The results showed that the residual enzyme activities of D118C-K121C, P244C-E249C and P22C-Q328C were 77.39%, 71.58% and 91.06% higher than MS after treated at 60 ℃ for 20 min, respectively. Compared to MS, the t1/2 (60 ℃) and tm of MS-P22C-Q328C increased by 2.06 times and 1.06 ℃, respectively, but its specific activity was decreased by 13.2%. The simulated structure analysis showed that the new disulfide bonds might by reducing the rigidity of the two highly flexible loops in MS-P22C-Q328C to enhance the thermostability of TGase.
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2021, Vol. 47 
