title: Characterization of the enzyme activity of an extracellular metalloprotease (VMC) from Vibrio mimicus and its C-terminal deletions.
abstract: To investigate the enzymatic properties of Vibrio mimicus metalloprotease, the mature metalloprotease gene (vmc) was overexpressed in Escherichia coli and the recombinant protein (rVMC61) was purified by metal affinity chromatography. rVMC61 showed maximum activity at about 37 degrees C, pH 8. The purified rVMC61 was very specific toward collagen substrates, such as gelatin, type I, II, and III collagens and synthetic peptides (Cbz-GPLGP and Cbz-GPGGPA). But it did not show degrading activity toward other biological proteins including lysozyme, lactoferrin and bovine serum albumin. rVMC61 also showed cytotoxicity against CHSE-214 fish cells. To examine the role of the C-terminal region of rVMC61, the 3' end of the metalloprotease gene (vmc) was digested serially with exonuclease III. The truncated vmc derivatives encoding 57-42 kDa of the protease were isolated and overexpressed in E. coli. The collagenase activities of truncated proteins were investigated using gelatin as substrate. Deletion of 100 amino acids from the C-terminus resulted in loss of gelatin degrading activity. However, deletion of 67 amino acids from the C-terminus did not affect its gelatin degrading activity.
authors.affiliation: Department of Biotechnology and Bioengineering, Pukyong National University, Busan 608-737, South Korea.
authors.forename: Jong-Hee
authors.initials: JH
authors.lastname: Lee
authors.forename: Sun-Hee
authors.initials: SH
authors.lastname: Ahn
authors.forename: Eun-Mi
authors.initials: EM
authors.lastname: Lee
authors.forename: Young-Ok
authors.initials: YO
authors.lastname: Kim
authors.forename: Sang-Jun
authors.initials: SJ
authors.lastname: Lee
authors.forename: In-Soo
authors.initials: IS
authors.lastname: Kong
mesh_terms: D000818:Animals; D002460:Cell Line; D004625:Embryo, Nonmammalian / Q000166:cytology; D004789:Enzyme Activation / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D017434:Protein Structure, Tertiary; D012474:Salmon; D013696:Temperature; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics*
chemical_list: D008666:Metalloendopeptidases