A beta-N-acetylglucosaminidase gene (nag84A) was cloned from Clostridium paraputrificum M-21 in Escherichia coli. The nag84A gene consists of an open reading frame of 4647 bp encoding 1549 amino acids, with a deduced molecular weight of 174,311, which have a catalytic domain belonging to family 84 of the glycoside hydrolases. Nag84A was purified from a recombinant E. coli and characterized. Although Nag84A exhibited high homology to the hyaluronidase from Clostridium perfringens, it did not degrade hyluronic acid. The enzyme hydrolyzed chitooligomers such as di-, tri-, tetra-, penta- and hexa-N-acetylchitohexaose, and synthetic substrates such as 4-methylum-belliferyl N-acetyl beta-D-glucosaminide [4-MU-(GlcNAc)], but did not hydrolyze 4-MU-beta-D-glucoside, 4-MU-alpha-D-glucoside, 4-MU-alpha-D-GlcNAc, 4-MU-alpha-D-galactoside, 4-MU-beta-D-xyloside, PNP-beta-D-galactoside, and PNP-alpha-D-xyloside. The enzyme was optimally active at 50degreesC and pH 6.5, and the apparent K-m and V-max values for 4-MU-(GlcNAc) were 8.5 muM and 1.39 mumol/min/mg of protein, respectively. SDS-PAGE, zymogram, and immunological analyses suggested that Nag84A was inducible by ball-milled chitin. Since Nag84A has a high molecular weight with a family 84 catalytic domain with high homology to hyaluronidases but no hyaluronidase activity, the enzyme is a novel beta-N-acetylglucosaminidase different from others reported having low molecular weights and belonging to family 3 and family 18.