Molecular cloning of the gene encoding Vibrio metalloproteinase vimelysin and isolation of a mutant with high stability in organic solvents.
Vimelysin is a unique metalloproteinase from Vibrio sp. T1800 exhibiting high activity at low temperature and high stability in organic solvents such as ethanol. A 1,821 bp open reading frame of the vimelysin gene encoded 607 amino acid residues consisting of an N-terminal pro-region, a mature enzyme, and a C-terminal pro-region. The mature enzyme region showed 80%, 57% and 35% sequence identity with the mature forms of vibriolysin from V. vulnificus, pseudolysin from Pseudomonas aeruginosa, and thermolysin from Bacillus thermoproteolyticus, respectively. The catalytic residues and zinc-binding motifs of metalloproteinases are well conserved in vimelysin. The vimelysin gene was expressed in E. coli JM109 cells and the recombinant enzyme was purified as a 38-kDa mature form from cell-free extracts. The purified recombinant enzyme is indistinguishable from the enzyme purified directly from Vibrio. To obtain mutants exhibiting higher stability in organic solvents, random mutations were introduced by error-prone PCR and 600 transformants were screened. The N123D mutant exhibits two times higher stability in organic solvents than the wild-type enzyme. A plausible mechanism for the stability of the N123D mutant in organic solvents was discussed based on homology models of vimelysin and the N123D mutant.
Takahashi
Toshihiro
T
Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585.
Ng
Kenneth K-S
KK
Oyama
Hiroshi
H
Oda
Kohei
K
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008666:Metalloendopeptidases / Q000235:genetics*; D045726:Metalloproteases / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D055550:Protein Stability; D012997:Solvents; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D012997:Solvents; D045726:Metalloproteases; D008666:Metalloendopeptidases; C099068:vimelysin