A fi-N-acetylglucosaminidase gene (nag84A) was cloned from Clostridium paraputr$cum 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-methylumbelliferyl N-acetyl fi-D-glucosaminide (4-MU-(GlcNAc)], but did not hydrolyze 4-MU-fl-D-glucoside,4-MU-a-D-glucoside, 4-MU-a-D-GlcNAc, 4-MU-a-D-galactoside, 4-MU-fl-D-xyloside, PNPP-D-galactoside, and PNP-a-D-xyloside. The enzyme was optimally active at 5O℃ and pH 6.5, and the apparent K, and Vm,, values for 4-MU-(GlcNAc) were 8.5pM and 1.39 ymol/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 j3-N-acetylglucosaminidase different from others reported having low molecular weights and belonging to family 3 and family 18.