| 碳水化合物结合组件的点突变研究 |
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| 引用本文： | 冯玉亮,黄明月,王菲,曹启龙,冯家勋,段承杰.碳水化合物结合组件的点突变研究[J].广西农业生物科学,2014(4):808-814. |
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| 作者姓名： | 冯玉亮 黄明月 王菲 曹启龙 冯家勋 段承杰 |
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| 作者单位： | 广西大学生命科学与技术学院,亚热带农业生物资源保护与利用国家重点实验室,南宁530005 |
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| 基金项目： | 国家自然科学基金（31260211）; 广西自然科学基金（2013jjAA30150）; 广西教育厅重点项目（201202ZD001）共同资助 |
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| 摘 要： | 碳水化合物结合组件（carbohydrate-binding module,CBM）是某些碳水化合物活性酶（carbohydrate-active enzymes）上的非催化部分,主要负责与底物特异性结合以提高与之相邻的催化结构域对不可溶底物的催化效率。CBM中的保守芳香族氨基酸通常是这些蛋白的底物结合位点,利用氨基酸定点突变技术可用来寻找和验证CBM中的结合位点的关键氨基酸。本研究的前期工作发现CBM46200与其同家族的其它成员相比对底物结合的能力要弱很多,多重序列比对发现家族中某些保守的芳香族氨基酸在CBM46200被非芳香族氨基酸替代,这可能是CBM46200与底物结合能力弱的原因,探究这些原因对研究前期工作中所鉴定的新家族CBM的广泛特异性具有重要意义。在本研究中利用氨基酸定点突变技术、亲和凝胶电泳技术以及蛋白对不可溶多糖的吸附技术等对前面的推论予以验证。结果表明,CBM46200的定点突变体对多糖底物的结合能力并没有增强,有些甚至还减弱了,这说明这些非芳香族氨基酸在CBM46200中的替代不是造成CBM46200对多糖的结合能力弱于其它家族成员的原因,可能还存在其它的因素影响CBM46200对底物的结合能力,本文对这些可能的因素也做了推测。
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| 关 键 词： | 碳水化合物结合组件 定点突变 芳香族氨基酸 结合能力 |
The Site-directed Mutagenesis Study of Carbohydrate-binding Module |
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| Feng Yuliang,Huang Mingyue,Wang Fei,Cao Qilong,Feng Jiaxun,Duan Chengjie.The Site-directed Mutagenesis Study of Carbohydrate-binding Module[J].Journal of Guangxi Agricultural and Biological Science,2014(4):808-814. |
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| Authors: | Feng Yuliang Huang Mingyue Wang Fei Cao Qilong Feng Jiaxun Duan Chengjie |
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| Institution: | (State Key Laboratory for Conservation and Utilization of Subtropical, Agro-bioresources, College of Life Science and Technology, Guangxi University, Narming, 530004) |
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| Abstract: | Carbohydrate binding modules（CBMs） are the non-catalytic module of carbohydrate active enzymes（CAZymes） and their main function is to bind the sugar-ligand specifically and increase the hydrolysis efficiency of the appended catalytic module. Generally, the conservative aromatic amino acids in binding sites of CBMs play important roles in recognizing and binding ligands in known CBM families. Site-directed mutagenesis experiment was applied to identify key amino acids in ligand binding site. The previous work of this study found that CBM46200 had lower binding capacity compared with other members of the same family of CBMs. Multiple sequence alignment showed some conservative aromatic amino acids in the family of CBMs were replaced by other non-aromatic amino acids（T23, H25, A61） in CBM46200, which may be lead to CBM46200 have weak binding capacity against the substrates. Finding out the reason is important for characterization of the broad specificity of the novel family of CBM in the previous work of this study. In this study, several techniques such as site-directed mutagenesis, affinity gel electrophoresis and protein adsorption technology of insoluble polysaccharide were used to verify the hypothesis. The results showed that the site-directed mutations of CBM46200 did not enhance binding capacity compared with wild type to the substrates, even mutant A61 F has lower binding capacity, which indicated that the replacements of these non-aromatic amino acids in CBM46200 were not the reason for CBM46200 have weaker binding capacity against the substrates compared with other family members. It is possible that other factors affect binding capacity of CBM46200 to the substrate, the possible factors were discussed in this paper. |
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| Keywords: | Carbohydrate-binding modules（CBM） Site-directed mutagenesis Aromatic amino acids Binding capacity |
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