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李剑龙,杨媛,吴昊,汤宏赤,韦宇拓.大肠杆菌嗜盐α-淀粉酶基因的分子改造及其酶学特性[J].广西科学,2016,23(1):25-30. [点击复制]
- LI Jianlong,YANG Yuan,WU Hao,TANG Hongchi,WEI Yutuo.Escherichia coli Halophilic alpha Amylase Gene Molecular Modification and Enzymology Characteristics[J].Guangxi Sciences,2016,23(1):25-30. [点击复制]
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| 大肠杆菌嗜盐α-淀粉酶基因的分子改造及其酶学特性 |
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李剑龙1,2, 杨媛1,2, 吴昊1,2, 汤宏赤1,2, 韦宇拓1,2
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| (1.广西大学生命科学与技术学院, 广西南宁 530005;2.亚热带农业生物资源保护与利用国家重点实验室, 广西南宁 530005) |
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| 摘要: |
| [目的]对大肠杆菌Escherichia coli嗜盐α-淀粉酶基因进行改造,并探索嗜盐α-淀粉酶的嗜盐特性。[方法]从非嗜盐的大肠杆菌JM109中克隆到一个嗜盐α-淀粉酶基因k6并进行重组表达。通过同源建模,确定Na+结合位点上的氨基酸残基,并对相应位点进行定点突变。最后对突变酶的酶学性质进行研究。[结果]相对于野生酶,突变酶更加嗜盐,其最适NaCl浓度由2mol/L增加到3mol/L,最适pH值为7,最适温度为50℃,酶活力为4 831U/mg,提高近4倍。经HPLC检测,突变酶与2%(W/V)可溶性淀粉反应后的产物为葡萄糖、麦芽糖、麦芽三糖的混合物。[结论]嗜盐α-淀粉酶基因k6的嗜盐特性与Na+结合位点具有直接联系。 |
| 关键词: 非嗜盐菌 嗜盐淀粉酶 同源建模 定点突变 |
| DOI：10.13656/j.cnki.gxkx.20160315.005 |
| 投稿时间：2015-11-20修订日期：2016-01-31 |
| 基金项目:国家自然基金项目(31460437)和广西研究生教育创新计划项目(YCSZ2014050)资助。 |
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| Escherichia coli Halophilic alpha Amylase Gene Molecular Modification and Enzymology Characteristics |
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LI Jianlong1,2, YANG Yuan1,2, WU Hao1,2, TANG Hongchi1,2, WEI Yutuo1,2
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| (1.College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530005, China;2.State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Nanning, Guangxi, 530005, China) |
| Abstract: |
| [Objective] The modification of halophilic α-amylase gene -was conducted in Escherichia coli to explore halophilic properties of halophilic α-amylase.[Methods] Firstly, a halophilic amylase gene was cloned from the non-halophilic bacteria E.coli JM109, and the recombinant gene was successfully expressed.Secondly, the amino acid binding site of Na+ was determined by homology modeling, and site-directed mutagenesis was conducted at the corresponding sites.Finally, the enzymatic properties of the mutant enzymes were studied.[Results] The mutant enzyme was characterized by higher halophilic property than wild type, and the optimum concentration of NaCl increased from 2mol/L added to 3mol/L.The halophilic amylase had the optimum pH at 7and the optimum temperature at 50℃, under which its specific activity was 4 831 U/mg, revealing nearly fourfold increase than the original enzyme.HPLC testing showed that the products were mixed with glucose, maltose and maltotriose after the reaction with 2% (W/V)of soluble starch.[Conclusion] Halophilic characteristics of halophilic amylase gene k6 has direct relationship with Na+ binding site. |
| Key words: non-halophilic bacteria halophilic amylase homology modeling site-directed mutagenesis |
