利用DNS法快速分析及优化柚皮苷的酶解过程

Rapid analysis and optimization of naringin enzymolysis by means of DNS method

摘要:

【目的】建立采用3,5-二硝基水杨酸(DNS)法快速测定柚皮苷水解率的方法，并利用该方法对柚苷酶催化水解柚皮苷生成柚皮素的反应过程进行优化研究。【方法】利用棘孢曲霉JMUdb058发酵得到的柚苷酶催化水解柚皮苷，采用DNS法对柚皮苷酶解过程中还原糖的生成量进行分析，经过换算得到柚皮苷的水解率，并在此基础上通过单因素实验优化柚皮苷的酶解过程。【结果】在柚皮苷的水解过程中，还原糖的生成量与柚皮苷的水解量及柚皮素的生成量均呈现出良好的线性关系，因此可利用DNS法测定体系中还原糖的生成量，并通过换算得到柚皮素的生成量。利用该方法优化柚皮苷的酶解过程得到柚苷酶转化柚皮苷的最适温度为50 °C、pH为5.0、酶用量为8 U/mL、底物浓度为0.2?g/100?mL。在此条件下，柚皮苷酶解150 min后可达到平衡，此时其水解率为85%。通过Lineweaver-Burk双倒数作图法测得Km为413.44 mg/L，Vmax为0.022 g/(min·L)。【结论】采用DNS法分析柚苷酶酶解柚皮苷的过程，具有方便快捷的特点，为柚皮苷的生物转化生成柚皮素的研究提供了新方法。

Abstract:

[Objective] 3,5-Dinitrosalicylic acid (DNS) assay, a rapid detection method of naringin conversion rate established and used to analyze the dynamic process of naringin conversion into naringenin catalyzed by naringinase. [Methods] Naringinase was obtained from Aspergillus aculeatus JMUdb058 under solid-state fermentation. The 3,5-dinitrosalicylic acid (DNS) method was used to determine the yields of reducing sugars to calculate the conversion rate of naringin via a certain way. Then the conditions of naringin enzymolysis were optimized by traditional “one-variable-at-a-time” strategy. [Results] Good linear relationships were showed among the yields of reducing sugars and hydrolysis of naringin as well as the yields of naringenin in the process of naringin enzymolysis. Therefore, the DNS method that was used to determine the yields of reducing sugars can be applied to analyze the process of naringin bioconversion into naringenin by naringinase. The optimum operating conditions obtained from the “one-variable-at-a-time” design were temperature of 50 °C, pH value of 5.0, enzyme dosage of 8 U/mL, and substrate concentration of 0.2 g/100 mL. Under the optimal condition, the conversion rate of naringin reached 85% after 150 min. Then Km=413.44 mg/L and Vmax=0.022 g/(min·L) were got by the double reciprocal plot of Lineweaver-Burk. [Conclusion] The DNS assay can be used to analyze the process of naringin bioconversion into naringenin by naringinase, which provided a new convenient and simple method for study on naringin enzymolysis into naringenin.