多结构域双功能纤维素酶/木聚糖酶CbXyn10C/Cel48B底物特异性的分子机制研究

纤维素和木聚糖是两种主要的非淀粉多糖，使用双功能酶同时降解这两种多糖有利于降低饲用酶的成本，但对此类酶底物特异性分子机制研究不多，限制了分子改良。CbXyn10C/Cel48B是一个独特的多结构域双功能纤维素酶/木聚糖酶，其N端和C端分别为木聚糖酶和纤维素酶，间以结合纤维素的双CBM3bs。CbXyn10C是10家族酶中第一个能高效降解微晶纤维素的；而CbCel48B却又能降解木聚糖。由于CBM在催化中心的拓扑结构、酶和底物的相互作用等方面也影响着酶的底物特异性，推测CbXyn10C/Cel48B的底物特异性不仅受两端催化结构域决定，还受CBM3bs影响。将通过分子对接和动力学模拟分析酶-底物复合物结构，系统引入突变和新型免疫荧光技术，测定酶的两个催化结构域间的协同作用、野生型和突变体在模式底物和完整植物细胞壁上的作用方式，据此推定决定CbXyn10C/Cel48B底物特异性的分子机制。

Cellulose and xylan are two major non-starch polysaccharides. Using bifunctional enzymes to simultaneously deconstruct the two polysaccharides can reduce the cost of feed enzymes. However, there are few researches in the molecular mechanism underlying the substrate specificity of such kind of enzymes, which limits their molecular improvement. CbXyn10C/Cel48B is a unique multimodular bifunctional cellulase/xlyalanse, with its N- and C-termini being a xylanase and cellulase, respectively. Two cellulose-binding modules, i.e. CBM3bs, separate the two catalytic domains apart. CbXyn10C is the first GH10 xylanase which can efficiently hydrolyze crystalline cellulose. In addition, CbCel48B can degrade xylan. Since CBM affects the substrate specificity of enzymes in various aspects including the topology of the enzyme active center and the interaction between enzyme and substrates, it is postulated that the substrate specificity of CbXyn10C/Cel48B is not only determined by the two catalytic domains in the N- and C-termini, but also affected by the CBM3bs. By using molecular docking and dynamic simulation, the enzyme-substrate structures will be determined. A series of mutant enzymes will be constructed, while a novel immunofluorescence technique will be introduced. The synergy between the two catalytic domains and the biochemical characteristics of the wild-type and mutants of CbXyn10C/Cel48B on model substrates as well as intact plant cell walls will be determined. Based on the results from these experiments, the molecular mechanism underling the substrate specificity of CbXyn10C/Cel48B will be uncovered.