title: Screening and characterization of a novel fibrinolytic metalloprotease from a metagenomic library.
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 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.
authors.affiliation: Department of Pharmaceutical Engineering, College of Medical Life Science, Silla University, Busan, Korea.
authors.forename: Dong-Geun
authors.initials: DG
authors.lastname: Lee
authors.forename: Jeong Ho
authors.initials: JH
authors.lastname: Jeon
authors.forename: Min Kyung
authors.initials: MK
authors.lastname: Jang
authors.forename: Nam Young
authors.initials: NY
authors.lastname: Kim
authors.forename: Jong Hyun
authors.initials: JH
authors.lastname: Lee
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Lee
authors.forename: Sang-Jin
authors.initials: SJ
authors.lastname: Kim
authors.forename: Gun-Do
authors.initials: GD
authors.lastname: Kim
authors.forename: Sang-Hyeon
authors.initials: SH
authors.lastname: Lee
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D002874:Chromosome Mapping / Q000379:methods*; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry*; D019015:Geologic Sediments / Q000382:microbiology*; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics*; D019151:Peptide Library; D020543:Proteome / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D005343:Fibrinolytic Agents; D019151:Peptide Library; D020543:Proteome; D045726:Metalloproteases