© 2005 The Japanese Biochemical Society.
Regular Paper |
Molecular Cloning of the Gene Encoding Vibrio Metalloproteinase Vimelysin and Isolation of a Mutant with High Stability in Organic Solvents
1 Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585; 2 Department of Bio Science, Bio College Kyoto, Kyuken-cho, Kamigyo-ku, Kyoto 602-0851; and 3 Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada
* To whom correspondence should be addressed. E-mail: bika{at}ipc.kit.ac.jp
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.