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Chitinase (Pa-Chi) and chitin oligosaccharide deacetylase (Pa-COD) are involved in the production of a heterodisaccharide, β-D-N-acetylglucosaminyl-(1,4)-D-glucosamine (GlcNAc-GlcN). These enzymes were recovered from the supernatant of Vibrio parahaemolyticus KN1699 cell culture and purified and characterized. For each enzyme, an ORF encoding gene and its signal sequence were cloned from genomic DNA of strain KN1699. In addition, the expression plasmid was constructed for each enzyme gene and inserted into Escherichia coli cells, and recombinant Pa-Chi and Pa-COD (Pa-rChi and Pa-rCOD) were secreted into the culture medium with the aid of signal peptides. Di-N-acetylchitobiose [(GlcNAc)2] was produced in 60% (w/w) yield by cultivating the Pa-rChi-secreting E. coli cells in 2% (w/v) β-chitin-containing medium. Moreover, GlcNAc-GlcN was produced in high yield by treating (GlcNAc)2 with the culture supernatant of Pa-rCOD-secreting E. coli cells.

Purification, Characterization and Cloning of Vibrio parahaemolyticus Chitinolytic Enzymes and Application to Oligosaccharide Production