| 北京棒杆菌天冬氨酸激酶突变体Q316P的酶学性质 |
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| 引用本文： | 张芷睿,陈晨,韩彩静,高云娜,刘春雷,闵伟红. 北京棒杆菌天冬氨酸激酶突变体Q316P的酶学性质[J]. 微生物学报, 2018, 58(5): 842-850 |
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| 作者姓名： | 张芷睿 陈晨 韩彩静 高云娜 刘春雷 闵伟红 |
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| 作者单位： | 吉林农业大学食品科学与工程学院小麦和玉米深加工国家工程实验室 |
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| 基金项目： | 吉林省玉米生物高效转化及精深加工创新团队（20150519012JH） |
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| 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°C升高至30°C;最适p H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。
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| 关 键 词： | 北京棒杆菌 天冬氨酸激酶 定点突变 动力学 酶学性质 |
| 收稿时间： | 2017-06-23 |
| 修稿时间： | 2017-07-29 |
Characterization of Q316P mutant of aspartate kinase from Corynebacterium pekinense |
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| Zhirui Zhang,Chen Chen,Caijing Han,Yunna Gao,Chunlei Liu and Weihong Min. Characterization of Q316P mutant of aspartate kinase from Corynebacterium pekinense[J]. Acta microbiologica Sinica, 2018, 58(5): 842-850 |
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| Authors: | Zhirui Zhang Chen Chen Caijing Han Yunna Gao Chunlei Liu Weihong Min |
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| Affiliation: | National Engineering Laboratory on Wheat and Corn Further Processing, College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, Jilin Province, China,National Engineering Laboratory on Wheat and Corn Further Processing, College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, Jilin Province, China,National Engineering Laboratory on Wheat and Corn Further Processing, College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, Jilin Province, China,National Engineering Laboratory on Wheat and Corn Further Processing, College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, Jilin Province, China,National Engineering Laboratory on Wheat and Corn Further Processing, College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, Jilin Province, China and National Engineering Laboratory on Wheat and Corn Further Processing, College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, Jilin Province, China |
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| Abstract: | |
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| Keywords: | Corynebacterium pekinense aspartate kinase site-directed mutagenesis kinetics characterization |
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