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氧化石墨烯基质毛细管电色谱胰蛋白酶微反应器的性能研究
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作者单位
刘佳,董旭,郭华,王嫚,申刚义 中央民族大学药学院,民族医药教育部重点实验室(中央民族大学) 
基金项目:国家自然科学基金(81573834);民族医药教育部重点实验室(中央民族大学)自主课题(KLEM-ZZ201808);中央民族大学青年学术团队引领计划成果(2019QNYL26)资助
中文摘要:基于石墨烯优良的物化性能,利用层层组装法将氧化石墨烯修饰于石英毛细管内壁,制备了氧化石墨烯基质的毛细管电色谱,通过电渗流、拉曼光谱等对其进行表征。在此基础上,基于离子键合法将胰蛋白酶固定于毛细管电色谱柱头,制备胰蛋白酶微反应器。两者结合构成毛细管电色谱胰蛋白酶微反应器。实验结果显示,氧化石墨烯作为基质既可提高样品的分离效率,还能促进胰蛋白酶的催化性能。氧化石墨烯修饰的毛细管电色谱对N-苯甲酰-L-精氨酸乙酯盐酸盐(BAEE)和N-苯甲酰-L-精氨酸(BA)混合物的分离度从裸毛细管的3.70提升至4.71,而其固定化酶活性(米氏常数Km=1.10 mmol/L,最大反应速率Vmax=0.32 mmol?L-1?s-1)也明显优于裸毛细管(Km=109.77 mmol/L,Vmax=0.000 46 mmol?L-1?s-1)。利用所制备的微反应器从10种中药材中筛选胰蛋白酶抑制剂活性成分的药材,结果发现三七和大黄中均存在胰蛋白酶抑制剂活性成分。
中文关键词:酶微反应器  氧化石墨烯  毛细管电色谱  胰蛋白酶  抑制剂
 
Study on Performance of a Capillary Electrochromatography Enzyme Immobilized Micro-reactor with Graphene Oxide as Carrier
Abstract:fabricated by a layer-by-layer assembly immobilizing the GO nanosheets onto capillary wall.The characterization on the colomn was carried out by measuring the electro osmotic flow(EOF) and Raman spectrum,etc.Based on this,an about 1 cm length of capillary electrochromatography trypsin micro-reactor was prepared by immobilizing the trypsin on end of capillary column through ion bonding.The performance of this technique was investigated.Results showed that the introduction of GO to the capillary column not only improved its separation efficiency on sample,but also promoted its enzyme catalytic performance.The separation degree of the GO-coated capillary on a mixture of N-benzuyl-L-arginine ethyl ester hydrochloride(BAEE) and N-benzuyl-L-arginine(BA) was increased from 3.70 to 4.71 compared with that of bare capillary.Meanwhile,the enzyme activity of immobilized enzyme with GO as carrier(Michaelis constant Km=1.10 mmol/L,the maximum reaction rate Vmax=0.32 mmol?L-1?s-1) was also enhanced significantly compared with that of the immobilized enzyme on unmodified capillary(Km=109.77 mmol/L,Vmax=0.000 46 mmol?L-1?s-1.The prepared trypsin micro-reactor was finally employed as a model for the screening of enzyme inhibitors from ten kinds of herbs in the crude extracts.Results showed that the Pseudo-ginseng and the Rheum officinale both contained trypsin inhibitor activity,which indicated that the prepared enzyme immobilized micro-reactor is a potential tool for rapid inhibitor screening.
Key Words:enzyme microreactor  graphene oxide  capillary electrochromatography  trypsin  inhibitor
引用本文:刘佳,董旭,郭华,王嫚,申刚义.氧化石墨烯基质毛细管电色谱胰蛋白酶微反应器的性能研究[J].分析测试学报,2019,(6):734-738.
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