GH1家族β-葡萄糖苷酶葡萄糖耐受机制研究：以Bgl1A和Bgl1B为例

β-glucosidases are enzymes that hydrolyze glycosidic bonds to release nonreducing terminal glucosyl residues from glycosides and oligosaccharides. The hydrolysis activity of β-glucosidases has significant application potential in industries, including bio-fuel produced from lignocellulose etc.. However, most of the β-glucosidases are feed-back inhibited by the product glucose, which restrict their application in industries. Elucidation of the glucose tolerance mechanism of β-glucosidases will be essential to enhance the glucose tolerance of β-glucosidases by genetic engineering technology, and thus contribute to improve their application in the biotechniques such as ethanol producing. However, the researches concerning the glucose tolerance of β-glucosidases focused mainly on the property characterization, rare of which dealt with the tolerance mechanism. By studying two β-glucosidases of GH1 family with high sequence similarity but significant difference in glucose tolerance property, the research is to investigate the potential glucose toletance mechanism of β-glucosidase, which will survey the role of different regions and sites in sequence and structure by rational design and directed resolution, and resolve the three-dimension structure of the two enzymes and their complex with glucose by X-ray crystallography. The results might contribute a lot to elucidating the glucose tolerance mechanism, and thereafter to improving the performance of β-glucosidases in industral application.

β-葡萄糖苷酶可水解糖苷和寡糖中的β-1,4-糖苷键，释放末端非还原性的葡萄糖或其它糖苷配基，其水解活性在木质纤维素炼制生物能源等方面有重要应用价值。然而，β-葡萄糖苷酶易受其催化产物葡萄糖的反馈抑制，限制了其在工业中的应用。阐明β-葡萄糖苷酶的葡萄糖耐受机制，并以此为基础通过基因工程改造提升酶的葡萄糖耐受性，对于改善纤维素乙醇发酵工艺等具有重要意义。本项目拟以实验室前期报道的两个序列高度相似而葡萄糖耐受性迥异的GH1家族β-葡萄糖苷酶Bgl1A和Bgl1B为研究对象，结合应用合理设计和定向进化的策略，研究其一级序列和空间结构上相关区段和位点在酶的葡萄糖耐受性中扮演的角色，并通过解析酶蛋白及其与葡萄糖复合物的晶体结构，阐明酶的葡萄糖耐受机制，为改善β-葡萄糖苷酶的工业应用性能奠定理论和技术基础。

β-葡萄糖苷酶可水解糖苷和寡糖中的β-1,4-糖苷键，释放末端非还原性的葡萄糖或其它糖苷配基，其水解活性在木质纤维素炼制生物能源等方面有重要应用价值。然而，β-葡萄糖苷酶易受其催化产物葡萄糖的反馈抑制，限制了其在工业中的应用。阐明β-葡萄糖苷酶的葡萄糖耐受机制，并以此为基础通过基因工程改造提升酶的葡萄糖耐受性，对于改善酶的应用性能具有重要意义。本项目以序列一致性较高而葡萄糖耐受性差异显著的β-葡萄糖苷酶Bgl1A（葡萄糖促活）和Bgl1B（葡萄糖抑制）为研究对象，初步探讨了GH1家族β-葡萄糖苷酶可能的葡萄糖耐受机制。研究中，依据Bgl1A、Bgl1B氨基酸序列差异和同源建模结果，将Bgl1B底物通道口及其周边的差异位点残基分别定点突变成Bgl1A上的对应残基，明确了Bgl1B葡萄糖耐受性发生本质改变的关键位点，以及转糖苷活性对于酶葡萄糖耐受的重要作用。采用分子对接模拟酶与底物、葡萄糖的结合，获取Bgl1A和Bgl1B及其突变体与葡萄糖和底物的结合位点和亲和力等信息。另一方面，对Bgl1A和Bgl1B的非同源区段进行体外重组，获得同时具有两者不同区段的片段替换重组体蛋白。通过葡萄糖耐受性测定、动力学分析和分子对接研究，明晰Bgl1A酶蛋白中影响其葡萄糖耐受性的关键氨基酸区段。依据以上研究，对GH1家族β-葡萄糖苷酶的葡萄糖耐受机制提出如下假说：葡萄糖与β-葡萄糖苷酶在活性中心以外，还存在多个结合位点，比如底物通道中部和外部的某些位点；不同位点与葡萄糖的结合能力不同，决定了葡萄糖对酶活影响的差异。本研究为进一步阐明β-葡萄糖苷酶的葡萄糖耐受机制，并以此为基础通过基因工程改造提升酶的葡萄糖耐受性奠定了基础。