苦荞芦丁水解酶的最适作用条件及Cu^2＋对酶活性的抑制

Optimum Reaction Conditions for Rutin-Hydrolysis Enzyme (RHE) from Tartary Buckwheat Seeds and Inhibitory Effect of Cu^(2+) on Its Activity

干燥的苦荞麦种子粉碎后经20mmol/L的醋酸盐缓冲液抽提,Resource Q和Superdex G75 HR 10/300凝胶柱分离纯化,得到一种芦丁水解酶,测定该酶的最适作用条件,研究Cu2+对该酶的抑制作用。结果表明:苦荞芦丁水解酶的分子质量约70kD,最适作用pH值为5.0。反应体系选择20%乙醇时,可保留酶的活性不被抑制,当乙醇体积分数大于50%时,对酶的活性抑制率达90%以上,表明可选用低体积分数乙醇作为芦丁的溶剂,用于芦丁水解酶测活体系及后续槲皮素制备研究。荧光光谱表明,Cu2+与芦丁水解酶以物质的量比1:1的形成稳定的复合物,可以使酶的荧光发生猝灭,水解芦丁的活性降低。表明Cu2+对芦丁水解酶有抑制作用。

A rutin-hydrolysis enzyme （RHE） was isolated from Fagopyrum tataricum （tartary buckwheat） seeds by extraction with 20 mmol/L acetate buffer acetate buffer, ammonium acetate fractionation, anion exchange chromatography and size exclusion chromatography. The molecular weight of RHE, as determined by SDS-PAGE, was 70 kD. The optimum hydrolysis pH was 5.0. The activity was not inhibited by an ethanol concentration of 20% in the reaction system, but an inhibitory rate above 90% was observed when ethanol concentration was higher than 50%. Fluorescence spectroscopic analysis indicated that Cu2~ could be bound to RHE at a molar ratio of I： 1 to generate a stable complex without fluorescence. Meanwhile, Cu^2＋ revealed an obvious inhibitory effect on the activity of RHE. These investigations provided a theoretical basis for future enzymatic preparation of quercetin.