Department of Pharmaceutical and Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu, 610065, China.
Department of Pharmaceutical and Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu, 610065, China.
Enzyme Microb Technol. 2021 Oct;150:109884. doi: 10.1016/j.enzmictec.2021.109884. Epub 2021 Jul 27.
Tyrosinase plays an essential role in melanin biosynthesis and inherently exhibits both monophenolase and diphenolase activity. A first derivative synchronous fluorometric assay was established for directly monitoring monophenolase activity. The zero-crossing point at 322 nm for the first-derivative under synchronous fluorescence with Δλ = 67 nm was utilized to selectively quantify tyrosine in the presence of the reaction product dihydroxyphenylalanine (DOPA). The limit of detection (LOD) for tyrosine was 0.54 μM. The fluorescence intensity of tyrosine was monitored at intervals of 30 s to establish the time course of tyrosine consumption. The LOD for the monophenolase activity was 0.0706 U⋅ mL. The Michaelis-Menten e constant and maximum speed were 21.83 μM and 1.12 μM min, respectively. Zinc ions competitively inhibited the monophenolase activity, with an IC value of 14.36 μM. This assay is easily and rapidly executed and is of great significance for analyzing the kinetics of enzymatic reactions and in fundamental research on monophenolase. This approach has potential applications in the discovery of tyrosinase inhibitors for medicine and cosmetics, as well as in the industrial synthesis of substituted o-diphenol intermediates.
酪氨酸酶在黑色素生物合成中起着至关重要的作用，并且天然具有单酚酶和二酚酶活性。建立了一种一阶导数同步荧光法来直接监测单酚酶活性。在存在反应产物二羟基苯丙氨酸（DOPA）的情况下，利用 67nm 同步荧光的一阶导数的零交叉点 322nm 来选择性地定量酪氨酸。酪氨酸的检测限（LOD）为 0.54 μM。每隔 30s 监测酪氨酸的荧光强度，以建立酪氨酸消耗的时间过程。单酚酶活性的 LOD 为 0.0706U⋅mL。米氏常数和最大速度分别为 21.83 μM 和 1.12 μM min。锌离子竞争性抑制单酚酶活性，IC 值为 14.36 μM。该测定方法易于快速执行，对于分析酶反应动力学以及单酚酶的基础研究具有重要意义。该方法在医学和化妆品中寻找酪氨酸酶抑制剂以及工业合成取代邻二酚中间体方面具有潜在的应用。
