鲜地黄中β-葡糖苷酶及α-半乳糖苷酶分离纯化及性质探究.docVIP

鲜地黄中β-葡糖苷酶及α-半乳糖苷酶分离纯化及性质探究摘要：目的从鲜地黄分离纯化β-葡糖苷酶和α-半乳糖苷酶，并研究其性质。方法通过磷酸盐缓冲液提取、两步硫酸铵盐析和两步凝胶柱层析，分离纯化鲜地黄中β-葡糖苷酶和α-半乳糖苷酶。以pNPG为底物，研究两种酶的最适反应温度及温度稳定性。结果与结论从鲜地黄中分离得到1种β-葡糖苷酶和3种α-半乳糖苷酶同工酶。研究发现β-葡糖苷酶热稳定性较差，在50 ℃即迅速失活；而α-半乳糖苷酶在50 ℃总酶活性最高，热稳定性较好，70 ℃以上时活性迅速下降。 关键词：地黄；β-葡糖苷酶；α-半乳糖苷酶；纯化；性质 中图分类号：R282.71文献标识码：A文章编号：1672-979X(2008)11-0012-04 Separation, Purification and Property Study of β- Glucosidase and α- Galactosidase from Fresh Roots of Rehmannia glutinosa ZHAO Yu, WEN Xue-sen, CUI Jing, WU Wei-hong (Institute of Pharmacognosy, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China) Abstract：Objective To separate and purify β-glucosidase and α-galactosidase from the fresh roots of Rehmannia glutinosa, and study the properties of them. Methods β-glucosidase and α-galactosidase were extracted from the fresh roots with phosphate buffer and preliminarily purified by two-step salting out with ammonium sulfate and two-step gel column chromatography. The optimal activity and thermal stability of the two enzymes were studied with pNPG as a substrate.Results and Conclusion In the fresh roots of Rehmannia glutinosa, β-glucosidase was composed of only one ingredient, while α-galactosidase was composed of three isozymes. β-glucosidase had a poor thermal stability and it was inactivated rapidly at 50 ℃, while α-galactosidase got the highest total activity at 50 ℃ and lost its activity at the temperature over 70 ℃ for a short time. Key words：Rehmannia glutinosa; β-glucosidase; α-galactosidase; purification; property 地黄（Rehmannia glutinosa Libosch.）为玄参科多年生植物，其块根以鲜地黄、生地黄和熟地黄等形式入药。由于鲜地黄不易保存，入药以生地和熟地为主。梓醇为鲜地黄的主要有效成分，现已证明，它具有降血糖、缓泻[1]、滋阴[2]、抗炎[3]和抗肝炎病毒[4]等多种生物活性。水苏糖也是鲜地黄的主要成分，在加工过程中它可能生成半乳糖[5]，半乳糖有诱导衰老的作用[6]，可能是地黄中的有害成分。在加工成生地后，梓醇和水苏糖含量均显著下降，导致这种变化的原因可能是鲜地黄产后的加工方法。目前，鲜地黄多采用烘干干燥，文献记载的烘干工艺有较大差异，主要差别是加热温度的控制。干燥模式不同导致了成品药材品质差别较大。 我们通过前期研究证明，鲜地黄中有β-葡糖苷酶（β-Glu）和α-半乳糖苷酶（α-Gal），这两种酶在鲜地黄的产后加工过程中可能导致有效成分梓醇的分解和有害成分半乳糖生成。鲜地黄产后加工的机制可能是加热使这两种酶灭活，从而避免梓醇分解和半乳糖生成；同