Heterologous expression and characterization of L200F/D215K mutant of homoserine dehydrogenase from Corynebacterium pekinense AS1.299

Abstract:

Abstract:［Objective］To obtain a new homoserine dehydrogenase with better properties from Corynebacterium pekinense by the spatial structure transfromation．［Methods］Double mutants L200F/D215A，L200F/D215E，L200F/D215G and L200F/D215K were constructed by site-directed mutagenesis and expressed in E．coli BL21．L200F /D215K was characterized for its highest catalytic efficiency and compared with that of L200F．［Results］The Vmax of L200F/D215K was 36.92 U/mg，1.24 times as that of L200F．The optimum reaction temperature of L200F/D215K was 37℃，2℃ higher than that of L200F．The optimum pH of L200F/D215K was 7.5，the same as that of L200F．The half-life time of L200F/D215K under optimum temperature was 4.16 h and was 1.12 times as that of L200F．Both L200F/D215K and L200F had good resistance to organic solvents and metal ions．［Conclusion］Through the spatial structure transformation，the enzymatic activity was increased，and the enzymology properties was optimized．