CLONING AND GENETIC-ANALYSIS OF THE VIBRIO-CHOLERAE AMINOPEPTIDASE GENE

The structural gene for the Vibrio cholerae leucine aminopeptidase (la p) was cloned and sequenced. The cloned DNA fragment contained a 1,503 -bp open reading frame potentially encoding a 501-amino-acid polypepti de with a calculated molecular mass of 54,442 Da. The deduced amino ac id sequence of the entire protein showed high homology with the sequen ce of Vibrio proteolyticus leucine aminopeptidase. The residues potent ially involved in binding the zinc ions were completely conserved in t he V. cholerae aminopeptidase as well as the V. proteolyticus aminopep tidase. The recombinant protein was partially purified and characteriz ed. The molecular mass was estimated by sodium dodecyl sulfate-polyacr ylamide gel electrophoresis to be 34 kDa, suggesting a processing of t he protein to acquire the mature form. The protease showed maximum act ivity at pH 9.0 and was thermostable at 70 degrees C . The substrate l eucyl-p-nitroanilide was cleaved by the protease, and its activity was inhibited by EDTA and bestatin. These results suggested that the prot ein was a leucine aminopeptidase. The PCR analysis of lap gene distrib ution showed that it was widely distributed among the V. cholerae stra ins. It was not present in the other species examined.