北京棒杆菌AS1.299高丝氨酸脱氢酶突变体L200F/D215K的异源表达及酶学性质

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

摘要:

摘要:【目的】对北京棒杆菌Corynebacterium pekinense高丝氨酸脱氢酶(homoserine dehydrogenase，HSD)进行空间结构改造从而获得优良性能新酶。【方法】利用定点突变技术构建HSD 双突变体L200F/D215A、L200F/D215E、L200F/D215G和L200F/D215K，并将其转入大肠杆菌E．coli BL21中进行高效表达，选取催化效率最高的双突变体L200F/D215K 与双突变前的L200F进行动力学和酶学性质比较。【结果】HSD双突变体L200F/D215K的Vmax为36.92 U/mg，较L200F提高1.24倍;最适反应温度为37℃，较L200F提高2℃;最适反应pH为7.5，与L200F经验值相同;最适温度下的半衰期为4.16 h，较L200F提高1.12倍;L200F/D215K和L200F对有机溶剂和金属离子均表现出较好的抗性。【结论】HSD通过空间结构改造活力得到提高，并且其酶学性质得到优化。本研究有助于认识HSD 突变体的酶学性质，为其新酶的研发利用提供有力依据。

关键词:关键词:北京棒杆菌，高丝氨酸脱氢酶，异源表达，酶学性质

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．

Key words:Keywords: Corynebacterium pekinense，homoserine dehydrogenase，heterologous expression，characterization