| 田菁根瘤菌嗜盐α-淀粉酶的基因克隆表达与分子改造 |
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| 引用本文： | 杨媛,姚甜甜,熊海涛,杜丽琴,韦宇拓.田菁根瘤菌嗜盐α-淀粉酶的基因克隆表达与分子改造[J].广西科学,2017,24(2):201-205. |
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| 作者姓名： | 杨媛 姚甜甜 熊海涛 杜丽琴 韦宇拓 |
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| 作者单位： | 广西大学生命科学与技术学院,广西南宁 530005;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】克隆表达田菁根瘤菌嗜盐α-淀粉酶基因,并对其进行基因改造,为探索与嗜盐性相关的氨基酸位点提供参考。【方法】从田菁根瘤菌及周围土壤的宏基因组中克隆到一个命名为RSA的极端嗜盐淀粉酶。通过与同源性为75.33%的嗜盐α-淀粉酶K6的序列比对发现,RSA-D205位点与已报道的K6-N204(嗜盐相关的氨基酸位点)相似,从而对其进行定点饱和突变以研究相关酶学性质。【结果】共获得17个突变子,其最适NaCl浓度较RSA均有不同程度的降低。RSA-D205R、RSA-D205C酶反应温度高达80℃,拓宽了其应用范围。【结论】RSA-D205氨基酸残基位点与Na~+结合位点有一定的联系;另外,精氨酸(R)、半胱氨酸(C)对淀粉酶的高温改造具有参考价值。
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| 关 键 词： | 克隆表达 嗜盐机理 α-淀粉酶 定点突变 离子结合位点 |
| 收稿时间： | 2016/12/20 0:00:00 |
Rhizobium sesbania Halophilic Alpha-Amylase Gene Cloning and Enzymology Characteristics Research |
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| YANG Yuan,YAO Tiantian,XIONG Haitao,DU Liqin and WEI Yutuo.Rhizobium sesbania Halophilic Alpha-Amylase Gene Cloning and Enzymology Characteristics Research[J].Guangxi Sciences,2017,24(2):201-205. |
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| Authors: | YANG Yuan YAO Tiantian XIONG Haitao DU Liqin and WEI Yutuo |
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| Institution: | College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530005, China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-biore-sources, Nanning, Guangxi, 530005, China,College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530005, China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-biore-sources, Nanning, Guangxi, 530005, China,College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530005, China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-biore-sources, Nanning, Guangxi, 530005, China,College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530005, China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-biore-sources, Nanning, Guangxi, 530005, China and College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530005, China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-biore-sources, Nanning, Guangxi, 530005, China |
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| Abstract: | Objective]The study was to provide the reference for exploring the amino acid sites related to halophilic characteristics by cloning,expressing and genetically modifying the gene of Rhizobium sesbania.Methods]An extreme halophilic amylase named RSA was cloned from the metagenome of Rhizobium sesbania and surrounding soil.RSA-D205 site was found to have a similarity to the reported K6-N204 (amino acid site related to halophilic characteristics) at sequence homology level about 75.33% with halophilic amylase K6.In order to study the relevant enzymatic properties,a site directed saturation mutagenesis was performed.Results]A total of 17 mutants were obtained,and the optimum NaCl concentration was lower than that of RSA in various degree.However,the reaction temperature of RSA-D205R and RSA-D205C were both up to 80℃,which broadened their application.Conclusion]There is a certain relationship between the amino acid residues of RSA-D205 and Na+ binding sites,and the arginine (R) and cysteine (C) have the reference value for the modification of amylase. |
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| Keywords: | cloning and expression halophilic mechanism alpha-amylase site-saturation mutagenesis ion binding site |
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