Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis
Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis
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DOI：
10.1016/j.pep.2003.12.016
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发表时间：
2004-04-01
影响因子：
1.6
通讯作者：
Cerniglia, CE
中科院分区：
文献类型：
--
作者：
Chen, HZ;Wang, RF;Cerniglia, CE
偶氮染料是食品和消费品中普遍存在的一类主要合成着色剂。粪肠球菌是人类胃肠道微生物群的主要成员。菌株 ATCC 19433 在偶氮染料存在下生长并将其代谢为无色产物。已在该菌株中鉴定出编码假定的 FMN 依赖性需氧还原酶的基因，该基因与大肠杆菌的偶氮还原酶 (AcpD) 具有 34% 的同一性。粪肠球菌中的基因被命名为 azoA，编码 208 个氨基酸的蛋白质，计算出的等电点为 4.8。 AzoA 在大肠杆菌中异源过表达，在 SDS-PAGE 上具有 23 kDa 的强条带。纯化的重组酶是分子量为 43 kDa 的同型二聚体，每个二聚体可能含有一个 FMN 分子。 AzoA 需要 FMN 和 NADH，但不需要 NADPH，作为其活性的首选电子供体。 NADH 和 2-[4-(二甲基氨基)苯基偶氮]苯甲酸（甲基红）底物的表观 K-m 值分别为 0.14 和 0.024 mM。表观 V-max 为 86.2 muM/min/mg 蛋白质。该酶不仅能够使甲基红脱色，还能够转化磺化偶氮染料 Orange II、Amaranth、Ponceau BS 和 Ponceau S。AzoA 是第一个从人类肠道革兰氏阳性菌中鉴定和表征的需氧偶氮还原酶。 (C) 2004 Elsevier Inc. 保留所有权利。
Azo dyes represent a major class of synthetic colorants that are ubiquitous in foods and consumer products. Enterococcus faecalis is a predominant member of the human gastrointestinal microflora. Strain ATCC 19433 grew in the presence of azo dyes and metabolized them to colorless products. A gene encoding a putative FMN-dependent aerobic azoreductase that shares 34% identity with azoreductase (AcpD) of Escherichia coli has been identified in this strain. The gene in E faecalis, designated as azoA, encoded a protein of 208 amino acids with a calculated isoelectric point of 4.8. AzoA was heterologously overexpressed in E. coli with a strong band of 23 kDa on SDS-PAGE. The purified recombinant enzyme was a homodimer with a molecular weight of 43 kDa, probably containing one molecule of FMN per dimer. AzoA required FMN and NADH, but not NADPH, as a preferred electron donor for its activity. The apparent K-m values for both NADH and 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl red) substrates were 0.14 and 0.024 mM, respectively. The apparent V-max was 86.2 muM/min/mg protein. The enzyme was not only able to decolorize Methyl red, but was also able to convert sulfonated azo dyes Orange II, Amaranth, Ponceau BS, and Ponceau S. AzoA is the first aerobic azoreductase to be identified and characterized from human intestinal gram-positive bacteria. (C) 2004 Elsevier Inc. All rights reserved.