Analytical Chemistry Division, Chemistry Department, Lomonosov Moscow State University, 119234 Moscow, Russia.
Research Center of Biotechnology, A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Russia.
Molecules. 2020 Dec 18;25(24):5996. doi: 10.3390/molecules25245996.
In the present work crude extract (ABE) has been prepared and characterized by its tyrosinase activity, protein composition and substrate specificity. The presence of mushroom tyrosinase (PPO3) in ABE has been confirmed using two-dimensional electrophoresis, followed by MALDI TOF/TOF MS-based analysis. GH27 alpha-glucosidases, GH47 alpha-mannosidases, GH20 hexosaminidases, and alkaline phosphatases have been also detected in ABE. ABE substrate specificity has been studied using 19 phenolic compounds: polyphenols (catechol, gallic, caffeic, chlorogenic, and ferulic acids, quercetin, rutin, dihydroquercetin, l-dihydroxyphenylalanine, resorcinol, propyl gallate) and monophenols (l-tyrosine, phenol, -nitrophenol, -nitrophenol, guaiacol, -cresol, -cresol, -cresol). The comparison of ABE substrate specificity and affinity to the corresponding parameters of purified tyrosinase has revealed no major differences. The conditions for spectrophotometric determination have been chosen and the analytical procedures for determination of 1.4 × 10-1.0 × 10 M l-tyrosine, 3.1 × 10-1.0 × 10 M phenol, 5.4 × 10-1.0 × 10 M catechol, 8.5 × 10-1.0 × 10 M caffeic acid, 1.5 × 10-7.5 × 10 M chlorogenic acid, 6.8 × 10-1.0 × 10 M l-DOPA have been proposed. The procedures have been applied for the determination of l-tyrosine in food supplements, l-DOPA in synthetic serum, and phenol in waste water from the food manufacturing plant. Thus, we have demonstrated the possibility of using ABE as a substitute for tyrosinase in such analytical applications, as food supplements, medical and environmental analysis.
在本工作中，通过其酪氨酸酶活性、蛋白质组成和底物特异性来制备和表征粗提取物（ABE）。使用二维电泳，随后进行 MALDI TOF/TOF MS 分析，证实 ABE 中存在蘑菇酪氨酸酶（PPO3）。还在 ABE 中检测到 GH27α-葡萄糖苷酶、GH47α-甘露糖苷酶、GH20 己糖胺酶和碱性磷酸酶。使用 19 种酚类化合物研究了 ABE 的底物特异性：多酚（儿茶素、没食子酸、咖啡酸、绿原酸和阿魏酸、槲皮素、芦丁、二氢槲皮素、L-二羟基苯丙氨酸、间苯二酚、没食子酸丙酯）和单酚（L-酪氨酸、苯酚、-硝基苯酚、-硝基苯酚、愈创木酚、-间甲酚、-间甲酚、-间甲酚）。ABE 底物特异性和对相应的纯化酪氨酸酶参数的亲和力的比较表明没有主要差异。选择了分光光度法测定的条件，并提出了测定 1.4×10-1.0×10 M L-酪氨酸、3.1×10-1.0×10 M 苯酚、5.4×10-1.0×10 M 儿茶素、8.5×10-1.0×10 M 咖啡酸、1.5×10-7.5×10 M 绿原酸、6.8×10-1.0×10 M L-DOPA 的分析程序。该程序已应用于食品补充剂中 L-酪氨酸、合成血清中 L-DOPA 和食品制造工厂废水中苯酚的测定。因此，我们证明了在食品补充剂、医学和环境分析等分析应用中使用 ABE 替代酪氨酸酶的可能性。
