Enhanced Thermal Stability Of Polyphosphate-Dependent Kinase

Our group has heterologously expressed polyphosphate-dependent kinase(PPGMK)in E.coli,to synthesize mannose-6-phosphate(M6P)at a low price and efficiently use polyphosphate(Hexametaphosphate)instead of expensive ATP.However,the thermal stability and enzymatic activity of PPGMK are poor,and the enzyme is intended to be evolved.In this research,synthesize the L7 gene and construct heterologous expression vector p ET28a(+)-L7,the enzyme is modified by combining error-prone PCR(EP-PCR)and site-directed saturation mutagenesis.Two mutants LM4(H92L;A138V)and LM5(E119V)were screened from the random mutation library,the effect of each site on thermal stability was assessedand.Use these three mutation sites as hotspots and site-specific saturation mutations were performed at each site.Combining the most suitable amino acids at each site,the optimal amino acids for each site are:H92K、E119R and A138 R,the mutant LM6(H92K;E119R)was finally determined to be the best mutant.The thermal stability of the LM6 enzyme has been increased by 5.4times,the half-life at 50°C has reached 243 minutes,and the enzyme activity has increased by 2 times,the decrease in K_m value indicates that the affinity of PPGMK for mannose has been improved.After 100 ns analysis of molecular dynamics simulation results,the reason is that Lys-92 can form hydrogen bond interaction with Gln-87,and the hydrogen bond distance is shorter than before.And the carbonyl oxygen of Gln-87 and the nitrogen atom of Lys-92 form a salt bridge;the guanidino group of Arg-119 can form a salt bridge with the carboxyl group of Glu-258,and this salt bridge can firmly fix α-helix and β-sheet.The average RMSD of LM6 at a high temperature of 360 K is also much smaller than that of WT,which also shows the improvement of thermal stability of LM6.The improvement of thermal stability of PPGMK greatly facilitates the application of PPGMK in actual industrial production,which not only reduces costs,but also improves production efficiency.