Glutathione is of great significance in pharmaceutical and health fields, and one-step synthesis of reduced glutathione by glutathione bifunctional synthase has become a focus of research. The stability of glutathione bifunctional synthase is generally poor and urgently needs to be modified. The B-factor strategy and un/folding free energy calculation were both applied to enhance the thermal stability of glutathione bifunctional synthase from Streptococcus agalactiae (GshFSA). Based on the concept of B-factor strategy, we calculated the B-factor by molecular dynamics simulation to find flexible residues, performed point saturation mutations and high-throughput screening. At the same time, we also calculated the un/folding free energy of GshFSA and performed the point mutations. The optimal mutant from the B-factor strategy was R270S, which had a 2.62-fold increase in half-life period compared to the wild type, and the Q406M was the optimal mutant from the un/folding free energy calculation, with a 3.02-fold increase in half-life period. Both of them have provided a mechanistic explanation.

Polyphosphate-dependent glucomannokinase (PPGMK) is able to utilize inorganic polyphosphate to synthesize mannose-6-phosphate (M6P) instead of highly costly ATP. This enzyme was modified and designed by combining error-prone PCR (EP-PCR) and site-directed saturation mutagenesis. Two mutants, H92L/A138V and E119V, were screened out from the random mutation library, and we used site-specific saturation mutations to find the optimal amino acid at each site. Finally, we found the optimal combination mutant, H92K/E119R. The thermal stability of H92K/E119R increased by 5.4 times at 50 °C, and the half-life at 50 °C increased to 243 min. Moreover, the enzyme activity of H92K/E119R increased to 16.6 U/mg, and its enzyme activity is twice that of WT. We analyzed the structure of the mutant using molecular dynamics simulation. We found that the shortening of the hydrogen bond distance and the formation of salt bridges can firmly connect the α-helix and β-sheet and improve the stability of the PPGMK structure.