Enhanced Thermal Stability of Polyphosphate-Dependent Glucomannokinase by Directed Evolution

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

Enhanced Thermal Stability of Polyphosphate-Dependent Glucomannokinase by Directed Evolution

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

Enhancing the Thermal Stability of Glutathione Bifunctional Synthase by B-Factor Strategy and Un/Folding Free Energy Calculation

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

Synthesis of 6‑Fluoroalkyl 6<i>H</i>‑Benzo[<i>c</i>]chromenes via Visible-Light-Promoted Radical Addition/Cyclization of Biaryl Vinyl Ethers

A novel visible-light-promoted cascade cyclization reaction for the synthesis of 6-fluoroalkyl 6<i>H</i>-benzo­[<i>c</i>]­chromenes has been successfully realized, which was initiated by intermolecular radical addition to biaryl vinyl ethers using easily available fluoroalkylated reagents BrCF₂CO₂Et or 2-bromo-2,2-difluoroamides as the sources of fluorinated radicals, followed by the cyclization onto an aromatic ring process. This protocol tolerated a wide range of functional groups and provided the desired products in moderate to good yields

Application of titanium gypsum as raw materials in cement-based self-leveling mortars

There are studies on titanium gypsum (TG) as a cement retarder, cementing material, or the production of lightweight wall materials and roadbed materials, but the application of TG is still limited, the utilization of TG is in the stage of exploration. This work studied the possibility of recycling TG as raw materials for the preparation of cement-based (Sulphate Aluminate Cement and Ordinary Portland Cement) self-leveling mortars. The effects of TG on mechanical strength, fluidity, shrinkage, and wear resistance were carried out. With the help of XRD, TG-DTG, and SEM, the microstructural characteristics were investigated to determine the mechanism of TG. It was concluded that the 1 d compressive and flexural strength reached 9.6 MPa and 3.5 MPa, respectively, and the 28 d compressive and flexural strength reached 4.3 MPa and 17.9 MPa, respectively, when the mass ratio of Sulphate Aluminate Cement (SAC) and Ordinary Portland Cement (OPC) was 7:3, the TG content was 45 %. TG could significantly improve the shrinkage of the mortars to 0.10 %, which was far better than the standard requirements of 0.15 %, but reduce the wear resistance to some extent. The primary component of calcium sulfate dihydrate (CaSO4·2H2O) in TG can further react with the aluminum phase in cement to grow more ettringite, which can make the microstructure denser, thereby improving the strength and the deformation of the cement-based self-leveling mortars