Enhanced Biosynthesis Of ?-ketoisovalerate Via Molecular Engineering On L-amino Acid Deaminase

?-ketoisovalerate(KIV)is an important intermediate in the field of biosynthesis and chemical synthesis,which has been widely studied and applied in the field of medicine and chemical synthesis.Compared with the traditional chemical synthesis methods,biotransformation is highly efficient,highly specific and environmentally friendly,and has potential in the future production of ?-keto acid.Biotransformation by Whole-cell catalyst is beneficial to product separation and can maintain cell membrane enzyme stability.In this study,E.coli BL21(DE3)was used as the host strain,Proteus myxofaciens was selected as the source of L-amino acid deaminase to construct the expression vector.The molecular modeling of Lamino acid deaminase was carried out as the foundation of rational engineering.The molecular modification enhances the yield of whole cell-transformed L-valine(L-Val)production of KIV by L-amino acid deaminase.The main contents of the paper are as follows:(1)The recombinant E.coli BL21(ladf)was successfully constructed,which contained the L-amino acid deaminase gene from Proteus myxofaciens.To increase the utilization of the substrate: L-valine,the branched amino acid transporter gene livF and the amino acid transaminase gene ilvE on the cell membrane have been knocked out.(2)The conditions for the whole cell transformation of recombinant E.coli BL21(ladf)on shake flask were optimized.The optimum conditions for the whole cell transformation were illustrated as follows: 32?,pH6.5,100 mmol·L-1 substrate L-valine,10 g·L-1 DCW cells,25 mL reaction system in 250 m L triangular bottle and reaction continues for 12 hours.(3)The final concentration of the cells can reach 4.50 g · L-1 before the transformation of the target mutations.The optimal expression of recombinant L-amino acid deaminase was achieved by protein modification,optimization on inducer concentration and stage temperature control strategy.(4)Based on the results of sequence alignment,the structure of L-amino acid deaminase derived from Proteus myxofaciens was constructed by homology modeling,and L-amino acid deaminase and L-valine were flexivated docked by AutoDock4.0 Molecular docking,the key active sites were obtained.According to the results of molecular docking,four sites(N100,Q276,R316 and F318)were selected for the target saturation mutation,in which F318 T showed the highest catalytic efficiency.On the basis of F318 T,the transformation efficiency of N100 H,raised 28.03% and 25.62%,respectively,in order to further improve the efficiency of L-amino acid deaminase transformation and N100.In addition,the saturation mutations of Q276 were performed on complex mutants of F318 T and N100 H.The yield of complex mutants(F318T,N100 H and Q276E)was 8.197 g L-1.