目的建立pNPG法测定马蓝叶中β-葡萄糖苷酶的活性, 研究马蓝叶中β-葡萄糖苷酶的基本酶学性质, 为进一步阐明青黛炮制原理提供依据。方法采用pNPG法考察马蓝叶中β-葡萄糖苷酶活性测定的最佳条件。结果最适反应条件为总反应体积1 mL, 反应时间45 min, 底物pNPG体积0.2 mL, 浓度25 mmol/L, 缓冲液pH值6, 反应温度55℃。在此条件下研究马蓝叶的粗酶液中β-葡萄糖苷酶的基本酶学性质, 结果表明该酶在30~60℃热稳定性较差。该酶在pH 5.0~7.0比较稳定, 而在酸性条件下酶活下降较快, 酶的pH值稳定性范围较窄。酶在4℃下的保存稳定性较好。结论确定了测定马蓝叶中β-葡萄糖苷酶活性的最佳条件, 探索了马蓝叶中β-葡萄糖苷酶的酶学性质, 该酶的热稳定性、pH值稳定性和保存稳定性的研究为青黛炮制原理的研究奠定了理论依据。

Objective To establish pNPG method for activity study of β-glucosidase in Baphicacanthus cusia and investigate the basic enzymatic properties,then provide accordance for further study on processing principle of Indigo Naturalis.Methods pNPG Method was adopted to investigate the optimum conditions for activity study of β-glucosidase.Results The optimum conditions were as following: the reaction volume was 1 mL,the reaction time was 45 min,the volume of substrate pNPG was 0.2 mL,the concentration was 25 mmol/L,the pH value of buffer solution was 6,and the reaction temperature was 55 ℃.Besides this,we investigated basic enzymatic properties of β-glucosidase in B.cusia.It indicated that β-glucosidase was unstable under 30-60 ℃,while it was stable under 4 ℃.It was stable under pH 5.0-7.0 and its activity decline rapidly acidic condition which manifested that the pH stability scope was narrow.Conclusion Through researching for optimum conditions of activity and enzymatic properties of β-glucosidase,we draw the conclusion of thermal stability,pH stability,and stability in storage.Then this lay the foundation of further study on processing principle of Indig Naturalus.

国家自然科学基金资助项目(30772784)