嗜热真菌多糖单加氧酶的克隆表达及氧化产物初步鉴定  

Cloning and expression of thermophilic fungal polysaccharide monooxygenase genes and preliminary identification of oxidative products

作　　者：陈铭 耿志刚 刘宁[1] 李多川[1] 

机构地区：[1]山东农业大学植物保护学院菌物学系,山东泰安271018

出　　处：《菌物学报》2017年第12期1616-1624,共9页Mycosystema

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

摘　　要：多糖单加氧酶(Polysaccharide monooxygenases,PMOs)是一类含铜氧化酶,在还原型辅因子(如维生素C)存在下可使纤维素分子氧化降解,在纤维素的酶解中起重要作用。本研究克隆5个嗜热真菌PMOs基因,构建p PICZαA-PMO表达载体,电转毕赤酵母GS115进行真核表达,经镍柱纯化获得重组蛋白后对其氧化活性进行探究。用5个PMOs分别处理磷酸膨胀纤维素(PASC),薄层层析法(TLC)鉴定酶解产物主要为纤维二糖到纤维六糖;飞行时间质谱法(MALDI-TOF-MS)鉴定结果显示:PMO0810为C4或C6氧化,PMO2033、PMO0154、PMO4983既有C1氧化也有C4或C6氧化,PMO3424未检测到相应的氧化裂解峰。Polysaccharide monooxygenases （PMOs） are a class of copper oxidases, which can directly cleave cellulose molecules n an oxidative mechanism in the presence of reduced cofactor （such as vitamin C）. In order to investigate the oxidative cleavage, five PMO genes from two thermophilic fungi were cloned and successfully expressed in Pichia pastoris. The five expressed PMOs were purified to SAD-PAGE homogeneity by nickel affinity chromatography. The five recombinant PMOs were used for digesting phosphoric acid-swollen cellulose （PASC）, resulting in the formation of oxidized and nonoxidized oligosaccharides. TLC analysis showed that the treatments of PASC with the five PMOs mainly produced cello-oligosaccharides with a degree of polymerization （DP） from DP2 to DP6. MALDI-TOF-MS results showed that PMO0810 oxidized the C4 or C6 carbon of glucose, and PMO2033, PMO0154 and PMO4983 oxidized the C1 and C4 or C6 carbon of glucose, while PMO3424 did not produce detectable peakcorresponding for oxidative cleavage.

关 键 词：多糖单加氧酶 嗜热真菌 氧化裂解 飞行时间质谱 

分 类 号：Q936[生物学—微生物学]