Development of high performance liquid chromatography with immobilized enzyme onto magnetic nanospheres for screening enzyme inhibitor.
A novel-immobilized enzyme strategy created by magnetic nanospheres for monitoring enzyme activity and screening inhibitors followed by high performance liquid chromatography (HPLC) has been demonstrated. Through the reaction of the aldehyde groups with amine groups, alpha-glycosidase was simply and stably immobilized onto magnetic nanospheres by the cross-linking agent glutaraldehyde. In order to profiling the activity of the immobilized alpha-glucosidase, the natural substrate was hydrolyzed by it and the yield of product was determined by HPLC. Compared with traditional bioassay approach, the prepared immobilized alpha-glucosidase displays a high activity and stability which allows it to be easily reused for 10 times. Enzyme inhibition assays by known inhibitor glucobay and three candidate traditional Chinese medicines (TCMs) were then investigated using a similar methodology. This assay was able to readily detect the change of the immobilized enzyme activity based on measuring a decrease of product formation using HPLC. The approach is general and offers many attractive advantages including easy product isolation, inexpensive cost, and high efficiency in terms of reagent consumption.
Hu
Fengli
F
Department of Chemistry, Fudan University, Shanghai, China.
Deng
Chunhui
C
Zhang
Xiangmin
X
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D004800:Enzymes, Immobilized / Q000737:chemistry; D008280:Magnetics; D054118:Nanospheres / Q000737:chemistry*; D009596:Nitrophenols / Q000032:analysis; D000520:alpha-Glucosidases / Q000378:metabolism
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D009596:Nitrophenols; D000520:alpha-Glucosidases; C024836:4-nitrophenol