A novel β-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose

Abstract.

A β-N-acetylglucosaminidase gene (nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli. The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da. Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain. Nag3A was purified from recombinant E. coli and characterized. The enzyme hydrolyzed chitooligomers such as di-N-acetylchitobiose, tri-N-acetylchitotriose, tetra-N-acetylchitotetraose, penta-N-acetylchitopentaose, hexa-N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl β-D-glucosaminide [4-MU-(GlcNAc)], but had no activity at all against p-nitrophenyl-β-D-glucoside, p-nitrophenyl-β-D-xyloside, or p-nitrophenyl-β-D-galactosamine. The enzyme was optimally active at 50°C and pH 7.0, and the apparent K _m and V _max values for 4-MU-(GlcNAc) were 7.9 µM and 21.8 µmol min^–1 mg protein^–1, respectively. SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin.