北京棒杆菌Corynebacterium pekinense天冬氨酸激酶P184M酶动力学分析及酶学性质表征

Enzyme kinetics analysis and enzymatic properties of aspartate kinase P184M from Corynebacterium pekinense

目的研究北京棒杆菌天冬氨酸激酶(aspartate kinase,AK)184位点脯氨酸(proline,P)突变为蛋氨酸(DL-methionine,M)对酶动力学及酶学性质的影响。方法对北京棒杆菌AK家族同源序列比对,选择出高度保守且远离底物结合位点的Pro184位点,对其进行饱和定点突变,成功构建突变体P184M。结果动力学和酶学性质研究表明:P184M AK Vmax比WT(wide type)提高了5.06倍;n值为0.19,突变体由WT的正协同性变为负协同性,同时Km(mol/L)值增大;突变体AK最适pH为6.5,低于野生型最适pH 7.0;最适反应温度28℃,低于WT的30℃,适宜温度区间变窄;半衰期由WT的2.1 h降为1.2 h;金属离子、有机溶剂和抑制剂Thr+Met、Lys+Met、Thr+Lys+Met对突变株AK均表现出激活作用。结论突变体天冬氨酸激酶P184M的酶动力学性质和酶学性质改变显著,为构建高产蛋氨酸菌株提供了一定的参考依据。

Objective To investigate the effects of site 184 proline （P） of aspartate kinases （AK） mutationed to DL-methionine （M） on enzyme kinetics and enzymatic properties.Methods Mutant P184M was successfully constructed by saturation site-directed mutagenesis of highly conserved amino acid and far away from inhibitors binding site P184 which was selected by sequence homology analysis of aspartate kinase family.Results The kinetics and enzymatic results showed that the Vmax of P 184M AK was 5.06 times higher than that of wide type （WT）.The n value was 0.19 indicating that mutant changed to negative synergistic reaction from synergistic reaction and Km value increased simultaneously.The optimum pH of P184M was 6.5,which was lower than that of WT （pH 7.0）.The optimum temperature of P 184M was 28 ℃,which was lower than that of WT （30 ℃）.The half-life period decreased from 2.1 h to 1.2 h.Furthermore,P184M AK activated significantly by metal ions,organic solvent and inhibitors （Thr＋Met,Lys＋Met,Thr＋Lys＋Met）.Conclusion The kinetics and enzymatic properties of mutant AK P184M are significantly changed,which provides a reference for the construction of high yield methionine strains.