Molecular analysis of the gene encoding a novel transglycosylative enzyme from Alteromonas sp strain O-7 and its physiological role in the chitinolytic system

We purified from the culture supernatant of Alteromonas sp. strain O-7 and characterized a transglycosylating enzyme which synthesized beta-(1-->6)-(G lcNAc)(2), 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D glucopyranosyl)-2-de oxyglucopyranose from beta-(1-->4)-(GlcNAc)(2). The gene encoding a novel t ransglycosylating enzyme was cloned into Escherichia coli, and its nucleoti de sequence was determined. The molecular mass of the deduced amino acid se quence of the mature protein was determined to be 99,560 Da which correspon ds very closely with the molecular mass of the cloned enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular ma ss of the cloned enzyme was much larger than that of enzyme (70 kDa) purifi ed from the supernatant of this strain. These results suggest that the nati ve enzyme was the result of partial proteolysis occurring in the N-terminal region. The enzyme showed significant sequence homology with several bacte rial beta-N-acetylhexosaminidases which belong to family 20 glycosyl hydrol ases. However, this novel enzyme differs from all reported beta-N-acetylhex osaminidases in its substrate specificity. To clarify the role of the enzym e in the chitinolytic system of the strain, the effect of beta-(1-->6)-(Glc NAc)(2), on the induction of chitinase was investigated. B-(1-->6)-(GlcNAc) (2) induced a level of production of chitinase similar to that induced by t he medium containing chitin. On the other hand, GlcNAc, (GlcNAc)(2), and (G lcNAc)(3) conversely repressed the production of chitinase to below the bas al level of chitinase activity produced constitutively in medium without a carbon source.