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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. [点击复制]
- YANG Yuan,YAO Tiantian,XIONG Haitao,DU Liqin,WEI Yutuo.Rhizobium sesbania Halophilic Alpha-Amylase Gene Cloning and Enzymology Characteristics Research[J].Guangxi Sciences,2017,24(2):201-205. [点击复制]
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| 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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杨媛1,2, 姚甜甜1,2, 熊海涛1,2, 杜丽琴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]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。 |
| 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 |
| DOI：10.13656/j.cnki.gxkx.20170119.005 |
| 投稿时间：2016-12-20 |
| 基金项目:国家自然基金项目(31460437)资助。 |
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| Rhizobium sesbania Halophilic Alpha-Amylase Gene Cloning and Enzymology Characteristics Research |
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YANG Yuan1,2, YAO Tiantian1,2, XIONG Haitao1,2, DU Liqin1,2, 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-biore-sources, Nanning, Guangxi, 530005, China) |
| 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. |
| Key words: cloning and expression halophilic mechanism alpha-amylase site-saturation mutagenesis ion binding site |
