北京棒杆菌天冬氨酸激酶突变体Q316P的酶学性质

Characterization of Q316P mutant of aspartate kinase from Corynebacterium pekinense

摘要:

[目的]对北京棒杆菌（Corynebacterium pekinense）天冬氨酸激酶（Aspartate kinase，AK）进行改造，期望获得具有较高酶活力且酶活性质改善的高产天冬氨酸族氨基酸的优良突变株，并削弱甚至解除Thr对AK的反馈抑制作用。[方法]利用定点突变技术对Gln（Q）316位点进行突变，高通量筛选获得活力提高明显的突变体，并将其在大肠杆菌BL21中高效表达，对野生型（Wild type，WT）和突变体Q316P AK用镍柱纯化，进行酶动力学及酶学性质研究。[结果]获得突变体Q316P，并在大肠杆菌BL21中成功表达。与野生型相比，突变体Q316P的V_max提高8.53倍，n值由2.15降低为1.29，正协同性减弱；最适温度由25℃升高至30℃；最适pH由8.0降低至7.5，半衰期由3.8 h延长至5.0 h；且在实验范围浓度内，底物抑制剂苏氨酸对突变体Q316P表现出激活作用；Q316P AK对金属离子K⁺和有机溶剂甲醇表现出良好抗性。[结论]获得酶活力提高、酶学性质改善的突变体，并一定程度上解除苏氨酸对AK的反馈抑制，为构建高产天冬氨酸族氨基酸工程菌提供参考。

Abstract:

[Objective] To obtain a new aspartate kinase (AK) with higher activity and better properties of highly produced aspartic acid family from Corynebacterium pekinense by spatial structure transformation, and attenuate or release the feedback inhibition of AK. [Methods] The Gln(Q)316 of AK was mutated by site-directed mutagenesis, and mutant strain with the highest activity was selected by high throughput screening. The mutant was expressed in Escherichia coli BL21. AK of the wild type and mutant strain was purified by Ni²⁺-NTA column and then the characterized. [Results] The mutant Q316P was constructed, and successfully expressed in E. coli BL21. Compared with the wild type, V_max of Q316P was increased by 8.53 times, and the n values was decreased from 2.15 to 1.29. The optimum temperature was increased from 25 ℃ to 30 ℃, the optimum pH was decreased from 8.0 to 7.5 and the half-time period was increased from 3.8 h to 5.0 h. The inhibition of the substrate inhibitor threonine was not only relieved but also threonine had an active effect on the activity of Q316P. Q316P had better resistance to methanol and K⁺. [Conclusion] Our findings provided a reference for the construction of high-yield aspartic acid family engineering strain.