A chitin oligosaccharide deacetylase from Vibrio sp. SN184: Gene cloning, overexpression, purification and characterization

The chitinolytic bacterial strain SN184, which produces chitin oligosaccharide deacetylase (COD), was identified a species of the genus Vibrio. The COD gene was cloned from the chromosomal DNA of SN184 and its nucleotide sequence was analyzed. The gene encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence of this protein showed high similarity to those of several bacterial CODs belonging to carbohydrate esterase family 4. An expression plasmid containing the COD gene of strain SN184 was constructed and inserted into Escherichia coli cells; the recombinant enzyme was secreted into the culture medium with the aid of the signal sequence. The recombinant enzyme was purified from the culture supernatant and its substrate specificity was investigated. These studies confirmed that the recombinant COD hydrolyzed N-acetyl groups of the second N-acetyl-D-glucosamine residue, from the nonreducing-ends of di-N-acetylchitobiose and tri-N-acetylchitotriose.