Molecular Cloning and Characterization of a New Cold-active Extradiol Dioxygenase from a Metagenomic Library Derived from Polychlorinated Biphenyl-contaminated Soil
Molecular Cloning and Characterization of a New Cold-active Extradiol Dioxygenase from a Metagenomic Library Derived from Polychlorinated Biphenyl-contaminated Soil
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多氯联苯污染土壤宏基因组文库中新型冷活性外二醇双加氧酶的分子克隆和表征
DOI：
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发表时间：
2012-07
影响因子：
3.1
通讯作者：
任何军
中科院分区：
文献类型：
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作者：
任何军
为了寻找新的外源双加氧酶(EDOS，EC 1.13.11.2)，从多氯联苯污染的土壤中构建了一个超基因组文库，并对一些具有芳香环裂解活性的双加氧酶进行了筛选。鉴定了一种新的EDO，命名为BPHC_A，并在大肠杆菌中异源表达。推导的BPHC_A氨基酸序列与其他已知EDO的同源性小于60%。BPHC_A的系统发育分析表明，该蛋白是EDO家族的一个新成员。该酶对3-甲基邻苯二酚的底物亲和力和催化效率高于对2，3-二羟基联苯或邻苯二酚的亲和力和催化效率，2，3-二羟基联苯或邻苯二酚是其他已知EDO的首选底物。纯化后的BPHC_A在pH=8.5和35℃时活性最高，5℃时BPHC_A活性为初始活性的40%以上。纯化后的BPHC_A活性受Mn~(2+)显著诱导，而Al~(3+)、Cu~(2+)和Zn~(2+)对活性略有降低。
To find new extradiol dioxygenases(EDOs, EC 1.13.11.2), a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity. A novel EDO, designated as BphC_A, was identified and heterologously expressed in Escherichia coli. The deduced amino acid sequence of BphC_A exhibited a homology of less than 60% with other known EDOs. Phylogenetic analysis of BphC_A suggests that the protein is a novel member of the EDO family. The enzyme exhibits higher substrate affinity and catalytic efficiency toward 3-methylcatechol than toward 2,3-dihydroxybiphenyl or catechol, the preferred substrate of other known EDOs. The optimum activity of purified BphC_A occurred at pH=8.5 and 35 °C, and BphC_A showed more than 40% of its initial activity at 5 °C. The activity of purified BphC_A was significantly induced by Mn^(2+) and slightly reduced by Al^(3+), Cu^(2+) and Zn^(2+).