An ORF encoding the gene (nahA)-related carbohydrate hydrolase was found in chromosomal DNA of the symbiotic thermophile, Symbiobacterium thermophilum. BLASTX results indicated that the product of this structural gene is P-glucosidase or beta-N-acetylhexosaminidase. To investigate details of the nahA gene product, cloning of the gene, overproduction of the gene product in Escherichia coli, and purification and characterization of the resulting protein were conducted. The nahA gene was amplified by PCR using fragmented chromosomal DNA of S. thermophilum as a template, sequenced, and then ligated into the BamHI and NdeI sites of plasmid pET-25b(+) to construct the expression vector pST-BNAH-A for overproduction of the gene product. Results of studies on the hydrolytic activity of cell-free extracts against pNP-beta-Glc, pNP-beta-GlcNAc and pNP-P-GalNAc, obtained by disruption of cultured E. coli cells harboring pST-BNAH-A, suggested that the nahA gene product was beta-N-acetylhexosaminidase. Comparison of the amino acid sequence of the recombinant protein with those of other beta-N-acetylhexosaminidases indicated that the beta-N-acetylhexosaminidase of S. thermophilum is a member of the 3-glycoside hydrolase family. The recombinant enzyme was purified to homogeneity from cell-free extract in an overall yield of 24%. This purified beta-N-acetylhexosaminidase possessed thermostability, was stable in alkaline solution, and exhibited greater hydrolytic activity against chitin oligosaccharides than against pNP-beta-GlcNAc. (c) 2005 Elsevier Inc. All rights reserved.