Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, 2699 Qianjin Street, Changchun, 130012, People's Republic of China.
Biotechnol Lett. 2011 Jun;33(6):1159-67. doi: 10.1007/s10529-011-0549-0. Epub 2011 Feb 4.
A novel 2,4-dichlorophenol hydroxylase (TfdB, EC 1.14.13.20) gene, designated as tfdB-JLU, was identified from a metagenome constructed from polychlorinated biphenyl-contaminated soil by functional screening and heterologously expressed in Escherichia coli. The deduced amino acid sequence of tfdB-JLU exhibited less than 48% homology with other known TfdBs. The enzyme exhibited a wider substrate spectrum than the previously reported TfdBs and higher relative activity towards ortho-substituted dichlorophenols, 2-chlorophenol, and 3-chlorophenol than towards 2,4-dichlorophenol, the preferred substrate of other known TfdBs. The enzyme had a K ( m ) of 5 μM for 2,4-dichlorophenol and 6 μM for NADPH. The optimal temperature and pH of the enzyme were 25°C and 7.5, respectively. Activity of the purified TfdB-JLU was slightly enhanced by Ca(2+), Mn(2+), Co(2+), and Fe(2+), and completely inhibited by Cu(2+), Hg(2+), and Zn(2+). This study is the first report to identify a novel TfdB from a metagenome.
从多氯联苯污染土壤的宏基因组中，通过功能筛选和异源表达，鉴定到一个新型 2,4-二氯苯酚羟化酶（TfdB，EC 1.14.13.20）基因，命名为 tfdB-JLU。tfdB-JLU 的推导氨基酸序列与其他已知的 TfdBs 的同源性小于 48%。该酶表现出比以前报道的 TfdBs 更广泛的底物谱，并且对邻位取代的二氯苯酚、2-氯苯酚和 3-氯苯酚的相对活性高于 2,4-二氯苯酚，2,4-二氯苯酚是其他已知 TfdBs 的首选底物。该酶对 2,4-二氯苯酚的 K ( m ) 为 5 μM，对 NADPH 的 K ( m ) 为 6 μM。酶的最适温度和 pH 分别为 25°C 和 7.5。纯化的 TfdB-JLU 的活性在 Ca(2+)、Mn(2+)、Co(2+)和 Fe(2+)的轻微促进下，完全被 Cu(2+)、Hg(2+)和 Zn(2+)抑制。本研究首次从宏基因组中鉴定到一种新型 TfdB。
