Kirita Masanobu, Tanaka Yoshihisa, Tagashira Motoyuki, Kanda Tomomasa, Maeda-Yamamoto Mari
a Research & Development-Production Headquarters, Asahi Breweries Limited , Moriya-shi , Ibaraki , Japan.
Biosci Biotechnol Biochem. 2015;79(7):1111-8. doi: 10.1080/09168451.2015.1015955. Epub 2015 Mar 10.
A novel O-methyltransferase gene was isolated from Flammulina velutipes. The isolated full-length cDNA was composed of a 690-nucleotide open reading frame encoding 230 amino acids. A database search revealed that the deduced amino acid sequence was similar to those of other O-methyltransferases; the highest identity was only 61.8% with Laccaria bicolor. The recombinant enzyme was expressed by Escherichia coli. BL21 (DE3) was assessed for its ability to methylate (-)-epigallocatechin-3-O-gallate (EGCG). LC-TOF-MS and NMR revealed that the enzyme produced five kinds of O-methylated EGCGs: (-)-epigallocatechin-3-O-(3-O-methyl)gallate, (-)-epigallocatechin-3-O-(4-O-methyl)gallate, (-)-epigallocatechin-3-O-(3,4-O-dimethyl)gallate, (-)-epigallocatechin-3-O-(3,5-O-dimethyl)gallate, and (-)-4'-O-methylepigallocatechin-3-O-(3,5-O-dimethyl)gallate. The substrate specificity of the enzyme for 20 kinds of polyphenols was assessed using the crude recombinant enzyme of O-methyltransferase. This enzyme introduced methyl group(s) into polyphenols with pyrocatechol and pyrogallol structures.
从金针菇中分离出一个新的O -甲基转移酶基因。分离得到的全长cDNA由一个690个核苷酸的开放阅读框组成，编码230个氨基酸。数据库搜索显示，推导的氨基酸序列与其他O -甲基转移酶的序列相似；与双色蜡蘑的最高同源性仅为61.8%。该重组酶由大肠杆菌BL21(DE3)表达，并对其甲基化(-)-表没食子儿茶素-3-O-没食子酸酯(EGCG)的能力进行了评估。液相色谱-飞行时间质谱(LC-TOF-MS)和核磁共振(NMR)显示，该酶产生了五种O -甲基化的EGCG：(-)-表没食子儿茶素-3-O-(3-O-甲基)没食子酸酯、(-)-表没食子儿茶素-3-O-(4-O-甲基)没食子酸酯、(-)-表没食子儿茶素-3-O-(3,4-O-二甲基)没食子酸酯、(-)-表没食子儿茶素-3-O-(3,5-O-二甲基)没食子酸酯和(-)-4'-O-甲基表没食子儿茶素-3-O-(3,5-O-二甲基)没食子酸酯。使用O -甲基转移酶的粗重组酶评估了该酶对20种多酚的底物特异性。该酶能将甲基引入具有邻苯二酚和邻苯三酚结构的多酚中。
