Purification, Cloning, and Sequence Analysis of β-N-Acetylglucosaminidase from the Chitinolytic Bacterium Aeromonas hydrophila Strain SUWA-9

Abstract

A chitinolytic bacterium was isolated from Lake Suwa and identified as Aeromonas hydrophila strain SUWA-9. The strain grew well on a synthetic medium containing colloidal chitin as sole carbon source. Chitin-degrading activity was induced by colloidal chitin or N-acetylglucosamine (GlcNAc). Most of the activity, however, was not detected in culture fluid but was associated with cells. A β-N-acetylglucosaminidase was purified after it was solubilized from cells by sonication. The purified enzyme hydrolyzed N-acetylchitooligomers from dimer to pentamer and produced GlcNAc as a final product. The enzyme also hydrolyzed synthetic substrates such as p-nitrophenyl (pNP)-N-acetyl-β-D-glucosaminide and pNP-N-acetyl-β-D-galactosaminide. A gene coding for the purified β-N-acetylglucosaminidase was isolated. The ORF identified is 2,661 nucleotides long and encodes a precursor protein of 887 amino acids including a signal peptide of 22 amino acid residues. The amino acid sequence deduced showed a high similarity to those of bacterial β-N-acetylhexosaminidases classified in family 20 of glycosyl hydrolases.