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 0-7 was cloned i nto pUC18 in Escherichia coli JM109. The nucleotide (nt) sequence of c ht60 was determined. A 1797-bp open reading frame encoded a polypeptid e 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 seq uence homologies with available aa sequences from databases. Cht60 was purified from the periplasmic fraction of E. coli cells carrying pCHT 982. The enzyme was most active towards nyl-N-acetyl-beta-D-glucosamin ide(PNP-beta-GlcNAc) and diacetylchitobiose. The optimum pH and temper ature 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 t he signal peptide cleavage site was clarified.