Screening and characterization of a novel fibrinolytic metalloprotease from a metagenomic library
Screening and characterization of a novel fibrinolytic metalloprotease from a metagenomic library
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DOI：
10.1007/s10529-006-9263-8
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发表时间：
2007-03-01
影响因子：
2.7
通讯作者：
Lee, Sang-Hyeon
中科院分区：
文献类型：
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作者：
Lee, Dong-Geun;Jeon, Jeong Ho;Lee, Sang-Hyeon
利用从韩国西海岸的泥浆中提取的总基因组 DNA 构建了宏基因组文库，并与 fosmid 载体 pCC1FOS 一起使用，以发现新的基因来源。从大约 30,000 个重组大肠杆菌克隆中鉴定出一个具有蛋白水解活性的克隆。蛋白水解酶的基因被亚克隆到 pUC19 中并进行测序，数据库中的同源性搜索表明它是一种锌依赖性金属蛋白酶。克隆的基因包括新型金属蛋白酶的完整编码基因及其自身的启动子。它包含一个1,080个碱基对的开放阅读框，编码39,490 Da的蛋白质，由359个氨基酸残基组成。在推导的该基因的氨基酸序列中发现了His-Glu-X-X-His序列，这是锌依赖性金属蛋白酶活性位点的保守序列，表明该酶是锌依赖性金属蛋白酶。纯化的酶在 50°C 1 小时和 pH 7.0 下显示出最佳活性。金属螯合剂（例如 EDTA、EGTA 和 1,10-菲咯啉）会抑制酶活性。该酶水解偶氮酪蛋白和纤维蛋白。因此，该酶可用作治疗血栓形成的治疗剂。
A metagenomic library was constructed using total genomic DNA extracted from the mud in the west coast of Korea and was used together with a fosmid vector, pCC1FOS in order to uncover novel gene sources. One clone from approximately 30,000 recombinant Escherichia coli clones was identified that showed proteolytic activity. The gene for the proteolytic enzyme was subcloned into pUC19 and sequenced, and a database search for homologies revealed it to be a zinc-dependent metalloprotease. The cloned gene included the intact coding gene for a novel metalloproteinase and its own promoter. It comprised an open reading frame of 1,080 base pairs, which encodes a protein of 39,490 Da consisting of 359 amino acid residues. A His-Glu-X-X-His sequence, which is a conserved sequence in the active site of zinc-dependent metalloproteases, was found in the deduced amino acid sequence of the gene, suggesting that the enzyme is a zinc-dependent metalloprotease. The purified enzyme showed optimal activity at 50 degrees C for 1 h and pH 7.0. The enzyme activity was inhibited by metal-chelating reagents, such as EDTA, EGTA and 1,10-phenanthroline. The enzyme hydrolyzed azocasein as well as fibrin. Thus, the enzyme could be useful as a therapeutic agent to treat thrombosis.