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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 assay for β-glucosidase activity in Malan leaves and to study the basic enzymatic properties of β-glucosidase in Malan leaves, which may provide the basis for further clarification of the processing principles of indigo plant. Methods The pNPG method was used to determine the optimal conditions for the determination of β-glucosidase activity in Malan leaves. Results The optimal reaction conditions were as follows: total volume of reaction 1 mL, reaction time 45 min, volume of substrate pNPG 0.2 mL, concentration 25 mmol / L, buffer pH 6, reaction temperature 55 ℃. Under these conditions, the basic enzymatic properties of β-glucosidase in the crude enzyme solution of horse blue leaf were studied. The results showed that the enzyme had poor thermal stability at 30 ~ 60 ℃. The enzyme is relatively stable at pH 5.0-7.0, while the enzyme activity decreases rapidly under acidic conditions and the pH stability of the enzyme is narrow. The enzyme has good storage stability at 4 ° C. Conclusion The optimal conditions for the determination of β-glucosidase activity in Malan leaves were determined, and the enzymatic properties of β-glucosidase in Malan leaves were explored. The thermostability, pH stability and storage stability The research laid the theoretical basis for the study of the principle of concoct processing.