夏东慧;刘宁;郭秀娜;李多川;

• 1:山东农业大学植物保护学院

摘要(Abstract)：

以嗜热毛壳菌Chaetomium thermophilum多糖单加氧酶CtPMO1为研究对象，利用薄层层析色谱法(TLC)、高效液相色谱-示差折光检测法(HPLC-RID)和飞行时间质谱(MALDI-TOF-MS)检测CtPMO1的酶活性，并根据定点突变的原理，将CtPMO1第1位的组氨酸(His1)和第166位的谷氨酰胺(Gln166)突变为H1A、Q166A和Q166E，研究两个突变位点是否参与CtPMO1的氧化作用；另外，采用3,5-二硝基水杨酸(DNS)法检测CtPMO1与纤维素酶(EGⅡ、BGLⅠ和CBHⅠ)的协同效应。研究发现CtPMO1在温度为50℃、pH为5.0的条件下降解磷酸膨胀纤维素(PASC)，其酶解产物中不仅存在纤维二糖至纤维五糖，还存在C1氧化寡糖和C4氧化寡糖；此外，发现突变酶H1A完全丧失了酶活，Q166A丧失了C1和C4氧化活性，而Q166E保留了部分C1氧化活性；通过对CtPMO1与纤维素酶协同作用的探究，发现利用CtPMO1预处理磷酸膨胀纤维素(PASC)，分别添加EGⅡ、BGLⅠ和CBHⅠ，使还原糖产量分别提高2.10倍、2.08倍和2.16倍，协同度分别是1.022、0.799和0.875。研究结果表明CtPMO1对底物具有C1和C4氧化的功能，其反应的最适温度为50℃、最适pH为5.0;CtPMO1活性中心氨基酸His1和平坦表面氨基酸Gln166均是关键性位点；CtPMO1预处理PASC，使纤维素酶的降解效率发生不同程度的提高。

关键词(KeyWords)： 多糖单加氧酶;纤维素酶;C1/C4位氧化;定点突变

Abstract:

Keywords:

基金项目(Foundation): 国家科技支撑计划(2015BAD15B05);; 国家自然科学基金(31571949);; 国家高技术研究发展计划(2012AA10180402)~~

作者(Authors): 夏东慧;刘宁;郭秀娜;李多川;

DOI: 10.13346/j.mycosystema.210363

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