北京棒杆菌E31天冬氨酸激酶突变体T361D酶学性质表征  

Enzymology Properties Characterization of Aspartate Kinase T361D from Corynebacterium pekinense E31

作　　者：陈晨 闵伟红[1] 张芷睿 方丽[1] CHEN Chen;MIN Wei-hong;ZHANG Zhi-rui;FANG Li(College of Food Science and Engineering,Jilin Agricultural University,Changchun 130118,China)

机构地区：[1]吉林农业大学食品科学与工程学院,吉林长春130118

出　　处：《食品研究与开发》2018年第17期153-159,共7页Food Research and Development

基　　金：长春市科技创新"双十工程"(17SS030)

摘　　要：对北京棒杆菌(Corynebacterium pekinense)中天冬氨酸激酶(aspartate kinase,AK)进行定点改造,为改善突变株的酶学性质,提高突变株的酶活力,削弱或解除AK的反馈抑制作用。对高度保守的T361位点进行定点突变及高通量筛选,以大肠杆菌BL21为载体,使突变体T361D在载体中实现高效表达,经分离及纯化后进行酶学性质和动力学的研究。结果表明:与野生型(wild type,WT)相比,突变体T361D Vmax提高7.63倍,最佳反应p H值降低为7.0,最佳反应温度升至30℃,半衰期延长1 h;突变体T361D在底物抑制剂赖氨酸(Lysine,Lys)存在下表现出激活作用,对金属离子Na^+、K^+和有机溶剂甲醇、异丙醇均表现出良好的抗性。试验为获得高产天冬氨酸族氨基酸菌株提供了参考和依据。To obtain the mutant with better properties, higher production and weaker feedback inhibition, AK from the Corynebacteriumpekinense was modified.The mutant was gained by site-directed mutagenesis of highly conserved T361 site and high throughput screening. The mutant was expressed in Escherichia coil BL21, the enzymatic kinetics and activities were determined after separation and purification. The results indicated that Vmax of T361D was increased by 7.63 times, comparing with wild type.The optimum pH was decreased to 7.0, the optimum temperature was increased to 30 ~ and the half-time period was extended for 1 hours. Within the experimental concentration range, substrate inhibitor Lysine or lysine-containing combination showed an activated function on the mutant. The T361D had better resistance to methanol, isopropyl alcohol, Na＋ and K＋. The research provided a theoretical basis for high-yield aspartic acid family engineering strain.

关 键 词：北京棒杆菌E31 天冬氨酸激酶 定点突变 酶动力学 酶学性质 

分 类 号：Q936[生物学—微生物学]