固定化β-葡萄糖醛酸酶反应器的制备及其在4-甲基亚硝胺基-1-(3-吡啶)-1-丁醇的O-糖苷化合物分析中的应用  

Preparation of immobilized β-glucuronidase enzyme reactor for the analysis of O-glucuronides of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol

作　　者：王婷 姚二民[2] 邓楠[1] 边阳阳[3] 刘萍萍[1] 张柯[1] 陈千思[1] 李斌[1] WANG Ting;YAO Ermin;DENG Nan;BIAN Yangyang;LIU Pingping;ZHANG Ke;CHEN Qiansi;LI Bin(Zhengzhou Tobacco Research Institute of China National Tobacco Corporation,Zhengzhou 450001,China;Medical Research Centre,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Zhengzhou University of Light Industry,Zhengzhou 450001,China)

机构地区：[1]中国烟草总公司郑州烟草研究院,河南郑州450001 [2]郑州轻工业学院,河南郑州450001 [3]郑州大学第一附属医院医学研究中心,河南郑州450052

出　　处：《色谱》2018年第3期245-252,共8页Chinese Journal of Chromatography

基　　金：国家自然科学基金项目(21505158,81600046).

摘　　要：采用溶胶-凝胶法,以丙烯酰胺作为单体,制备了基于有机-硅胶杂化整体柱的β-葡萄糖醛酸酶反应器。优化了硅酸甲酯和γ-(甲基丙烯酰氧)丙基三甲氧基硅的物质的量的比、丙烯酰胺和聚乙二醇的用量,以及水浴温度等制备条件,获得了孔隙均匀、通透性良好、机械强度高的有机-硅胶杂化整体柱。采用光学显微镜和扫描电镜表征杂化整体柱。进一步将β-葡萄糖醛酸酶共价键合在整体柱上,以4-甲基亚硝胺基-1-(3-吡啶)-1-丁醇(NNAL)的O-糖苷化合物(NNAL-O-Gluc)为底物研究酶反应器的水解效果,实验结果证明酶反应器在室温条件下的水解效率大大提高,实现了NNAL-O-Gluc高效水解与分析,解决了目前NNAL-O-Gluc分析中前处理水解效率低的问题。An immobilized 6-glucuronidase enzyme reactor(IMER)based on organic-silica gel hybridized capillary column was prepared by the sol-gel method with acrylamide(AAM)as the organic functional monomer.The effects of the amounts of tetramethyl orthosilicate(TMOS),3-glycidoxypropyltrimethoxysilane(y-MAPS),acrylamide(AAM),polyethylene glycol(PEG),and the water bath temperature on the quality of the microscopic structure were studied.After optimization,the monolithic column with good permeability and high mechanical strength was obtained.Then 6-glucuronidase was covalently immobilized,and the hydrolysis efficiency of the 6-glucuronidase IMER was investigated.As the substrate,0-glucuronides of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL-0-Gluc)were hydrolyzed at room temperature,indicating the high hydrolysis efficiency of the 6-glucuronidase IMER,which demonstrated its high potential for the high throughput analysis of tobacco metabolites in the future.

关 键 词：有机-硅胶杂化整体柱 6-葡萄糖醛酸酶 固定化酶反应器 4-甲基亚硝胺基-1-(3-吡啶)-1-丁醇的0-糖苷化合物 

分 类 号：O658[理学—分析化学]