Screening and characterization of a novel fibrinolytic metalloprotease from a metagenomic library |
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Authors: | Dong-Geun Lee Jeong Ho Jeon Min Kyung Jang Nam Young Kim Jong Hyun Lee Jung-Hyun Lee Sang-Jin Kim Gun-Do Kim Sang-Hyeon Lee |
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Institution: | (1) Department of Pharmaceutical Engineering, College of Medical Life Science, Silla University, Busan, 617-736, Korea;(2) Marine Biotechnology Research Center, Korea Ocean Research and Development Institute, Ansan, Gyeonggido, 425-600, Korea;(3) Department of Microbiology, Pukyong National University, Busan, 608-737, Korea |
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Abstract: | 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°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.
The sequence reported in this paper has been deposited in the GenBank database (Accession number: EF100137). |
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Keywords: | Azocasein Fibrinolytic Metagenome Metalloprotease |
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