Cui Xiao-Dong, Wang Zhuan-Hua
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, PR China.
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, PR China.
Food Chem. 2012 May 1;132(1):60-6. doi: 10.1016/j.foodchem.2011.10.032. Epub 2011 Oct 18.
A rutin hydrolyzing enzyme (RHE) was isolated from Fagopyrum tataricum Moench seeds by using ammonium sulphate fractionation, anion exchange and size exclusion chromatography. The purified RHE has an apparent molecular weight of about 70kDa determined by SDS-PAGE, with an isoelectric point (pI) (determined by isoelectric focusing) of 6.7. RHE has a specific catalytic activity toward rutin when incubated together with rutin at 37°C for 30min in the presence of 20% ethanol, and its Km value for rutin is 1.04×10(-3)M. The RHE catalytic product analyzed by HPLC displayed high similarity with quercetin and this is confirmed by (1)H NMR spectroscopy and LC-ESI-MS/MS, suggesting that the RHE hydrolysis product is quercetin. These results suggest that the RHE from tartary buckwheat seeds is a specific rutin-hydrolyzing enzyme, providing a new enzymatic preparation method for quercetin.
通过硫酸铵分级沉淀、阴离子交换和尺寸排阻色谱法从苦荞麦种子中分离出一种芦丁水解酶（RHE）。经SDS-PAGE测定，纯化后的RHE表观分子量约为70kDa，通过等电聚焦测定其等电点（pI）为6.7。在20%乙醇存在下，RHE与芦丁在37℃孵育30分钟时对芦丁具有特定的催化活性，其对芦丁的Km值为1.04×10（-3）M。通过HPLC分析的RHE催化产物与槲皮素高度相似，这通过（1）H NMR光谱和LC-ESI-MS/MS得到证实，表明RHE水解产物为槲皮素。这些结果表明，苦荞麦种子中的RHE是一种特定的芦丁水解酶，为槲皮素提供了一种新的酶法制备方法。
