An online immobilized α-glucosidase microreactor for enzyme kinetics and inhibition assays | |
Department | 中科院西北特色植物资源化学重点实验室/甘肃省天然药物重点实验室 |
Liu DM(刘冬梅)1,2; Chen J(陈娟)1; Shi YP(师彦平)1; Chen J(陈娟); Shi YP(师彦平) | |
2015 | |
Source Publication | RSC Advances |
ISSN | 2046-2069 |
Volume | 5Pages:56841-56847 |
Abstract | In this work, a facile protocol for preparing an online immobilized a-glucosidase microreactor was described, and also the enzyme kinetics and inhibition kinetics of such an immobilized enzyme were studied. In this procedure, a-glucosidase was immobilized on the inner wall of the capillary treated with 3-aminopropyltriethoxysilane (3-APTES) and the homobifunctional linker glutaraldehyde (GA). With cross-linking technology, the immobilized a-glucosidase microreactor was fabricated. As the key point for studying the enzyme kinetics and inhibition kinetics of the immobilized a-glucosidase with the developed microreactor, pressure was applied as the driving force to push the reaction mixture to the detection window, and the detection wavelength was set to 405 nm at which only the formed p-nitrophenol (pNP) could be detected. More importantly, with such a detection wavelength it is unnecessary to separate the substrate from the reaction mixture by applying the alkaline inactivating background electrolyte (BGE, borate buffer, pH $ 9.0) which is favorable for the separation. So the incubation buffer (phosphate buffer, pH 7.0) could be used as the running buffer making the online study of the enzyme kinetics and inhibition kinetics of immobilized a-glucosidase come true. The inhibition assay was performed by using acarbose as a model inhibitor, and under the optimized conditions, the Michaelis–Menten constant (Km), inhibition constant (Ki), and half-maximal inhibitory concentration (IC50) for the immobilized a-glucosidase were determined as 0.43 mM, 30.65 mM and 99.48 mM, respectively. This study indicates, for the first time, that the immobilized a-glucosidase microreactor could be used for studying the enzyme kinetics and inhibition kinetics online. |
Subject Area | 分析化学与药物化学 |
DOI | 10.1039/c5ra07982a |
Funding Organization | the National Nature Science Foundation of China (no. 21105106;21375136);the Scholar Program of West Light Project, Chinese Academy of Sciences |
Indexed By | SCI |
If | 3.289 |
Language | 英语 |
Funding Project | 药物化学成分研究组 |
compositor | 第一作者单位 |
Citation statistics | |
Document Type | 期刊论文 |
Identifier | http://ir.licp.cn/handle/362003/18975 |
Collection | 中科院西北特色植物资源化学重点实验室/甘肃省天然药物重点实验室 |
Corresponding Author | Chen J(陈娟); Shi YP(师彦平) |
Affiliation | 1.Key Laboratory of Chemistry of Northwestern Plant Resources, Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People's Republic of China 2.University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100049, P. R. China |
Recommended Citation GB/T 7714 | Liu DM,Chen J,Shi YP,et al. An online immobilized α-glucosidase microreactor for enzyme kinetics and inhibition assays[J]. RSC Advances,2015,5:56841-56847. |
APA | Liu DM,Chen J,Shi YP,陈娟,&师彦平.(2015).An online immobilized α-glucosidase microreactor for enzyme kinetics and inhibition assays.RSC Advances,5,56841-56847. |
MLA | Liu DM,et al."An online immobilized α-glucosidase microreactor for enzyme kinetics and inhibition assays".RSC Advances 5(2015):56841-56847. |
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