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 enzym...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2005-12, Vol.138 (6), p.701-710
Main Authors: Takahashi, Toshihiro, Ng, Kenneth K.-S, Oyama, Hiroshi, Oda, Kohei
Format: Article
Language:English
Subjects:
alcohol resistance
Bacillus thermoproteolyticus
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Cloning, Molecular
Escherichia coli
Genes, Bacterial
Metalloendopeptidases - genetics
Metalloproteases - genetics
metalloproteinase
Molecular Sequence Data
Mutation
Protein Stability
pseudolysin
Pseudomonas aeruginosa
random mutagenesis
Solvents
Vibrio
Vibrio - enzymology
Vibrio - genetics
Vibrio vulnificus
vimelysin
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Summary: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.
ISSN:0021-924X
1756-2651
DOI:10.1093/jb/mvi173