Open AccessCharacterization of the enzyme activity of an extracellular metalloprotease (VMC) from Vibrio mimicus and its C‐terminal deletions
Author(s) -
Lee JongHee,
Ahn SunHee,
Lee EunMi,
Kim YoungOk,
Lee SangJun,
Kong InSoo
Publication year - 2003
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/s0378-1097(03)00401-4
Subject(s) - metalloproteinase , biochemistry , escherichia coli , biology , protease , amino acid , microbiology and biotechnology , enzyme , vibrio , bacteria , gene , genetics
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°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.