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Citation: WANG Ting, YAO Ermin, DENG Nan, BIAN Yangyang, LIU Pingping, ZHANG Ke, CHEN Qiansi, LI Bin. Preparation of immobilized β-glucuronidase enzyme reactor for the analysis of O-glucuronides of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol[J]. Chinese Journal of Chromatography, ;2018, 36(3): 245-252. doi: 10.3724/SP.J.1123.2017.11050 shu

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

WANG Ting ^1,3, ,
YAO Ermin ³ ,
DENG Nan ¹ ,
BIAN Yangyang ² ,
LIU Pingping ¹ ,
ZHANG Ke ¹ ,
CHEN Qiansi ¹ ,
LI Bin ¹

1.
Zhengzhou Tobacco Research Institute of China National Tobacco Corporation, Zhengzhou 450001, China;
2.
Medical Research Centre, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China;
3.
Zhengzhou University of Light Industry, Zhengzhou 450001, China

Received Date: 11 December 2017

Fund Project: National Natural Science Foundation of China (Nos. 21505158, 81600046).

An immobilized β-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 (γ-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 β-glucuronidase was covalently immobilized, and the hydrolysis efficiency of the β-glucuronidase IMER was investigated. As the substrate, O-glucuronides of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL-O-Gluc) were hydrolyzed at room temperature, indicating the high hydrolysis efficiency of the β-glucuronidase IMER, which demonstrated its high potential for the high throughput analysis of tobacco metabolites in the future.
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