Gene sequence, purification and characterization of N-acetyl-beta-glucosaminidase from a marine bacterium, Alteromonas sp. strain O-7.

The gene (cht60) encoding N-acetyl-beta-glucosaminidase (Cht; EC 3.2.1.30) from the marine bacterium Alteromonas sp. strain O-7 was cloned into pUC18 in Escherichia coli JM109. The nucleotide (nt) sequence of cht60 was determined. A 1797-bp open reading frame encoded a polypeptide of 598 amino acids (aa) (M(r) 64,535). The aa sequence of the cloned enzyme (Cht60) deduced from the nt sequence showed no significant sequence homologies with available aa sequences from databases. Cht60 was purified from the periplasmic fraction of E. coli cells carrying pCHT982. The enzyme was most active towards p-nitrophenyl-N-acetyl-beta-D-glucosaminide(PNP-beta-GlcNAc) and diacetylchitobiose. The optimum pH and temperature of the enzyme were pH 7.5 and 37 degrees C, respectively. The N-terminal 11 aa residues of Cht60 were sequenced, and the location of the signal peptide cleavage site was clarified.