Characterization of levJ, a sucrase/fructanase-encoding gene from Actinomyces naeslundii T14V, and comparison of its product with other sucrose-cleaving enzymes.

A library of Actinomyces naeslundii T14V DNA was constructed in plasmid pUC18 and from this several sucrose-positive clones were isolated. Evidence was obtained that all these clones contained the same gene. One clone, which carried a plasmid that was named pPNG102, was chosen for further study. It was found that the enzyme specified by this plasmid hydrolyzed sucrose, raffinose, inulin and levan, but not dextran, and did not synthesize fructan or glucan from sucrose. The sequence of the insert in pPNG102 was determined and was found to contain a large ORF that specifies a polypeptide of 99,319 Da with similarity to other sucrases. This gene was named levJ. The deduced amino acid (aa) sequence contained both a potential signal sequence and potential C-terminal cell envelope attachment domain. Alignments revealed an internal 331-aa domain not present in other levanases and sucrases. A neighbour-joining tree showed that sucrases of eukaryotic origin form a cluster with eubacterial sucrase/fructanases, and this cluster does not include other eubacterial sucrases. It is postulated that certain eukaryotic sucrase-encoding genes are of eubacterial origin.