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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。
The carbohydrate-binding module (CBM), a non-catalytic moiety on certain carbohydrate-active enzymes, is primarily responsible for the specific binding to substrates to increase the number of adjacent catalytic domain pairs Catalytic efficiency of insoluble substrates. Conservative aromatic amino acids in CBMs are often the substrate binding sites for these proteins and amino acid site-directed mutagenesis techniques can be used to find and validate the key amino acids at the binding site in the CBM. The previous work in this study found that CBM46200 is much weaker than the other members of the same family on substrate binding. Multiple sequence alignment revealed that some of the conserved aromatic amino acids in the family were replaced by non-aromatic amino acids at CBM46200 Is the reason why CBM46200 has a weak binding ability to the substrate. It is of great significance to explore these reasons for the extensive specificity of the new family CBM identified in the previous work. In this study, the use of amino acid site-directed mutagenesis, affinity gel electrophoresis and protein adsorption of insoluble polysaccharides on the previous validation to be verified. The results showed that CBM46200 site-directed mutants did not enhance the binding capacity of polysaccharide substrate, and some even weakened, indicating that the substitution of these non-aromatic amino acids in CBM46200 is not caused by CBM46200 polysaccharide binding capacity weaker than other family members Of the reasons, there may be other factors that affect the CBM46200 substrate binding capacity, the paper also speculated on these possible factors.