Purification, characterization and gene analysis of N-acetylglucosaminidase from Enterobacter sp G-1

Enterobacter sp. G-1 is a bacterium isolated previously as a chitinase-prod ucing bacterium. We found this bacterium also produced N-acetylglucosaminid ase and characterized that in this study. Extracellular N-acetyiglucosamini dase of 92.0 kDa was purified near homogeneity by 8,57-foId from Enterobact er sp. G-1. The optimum temperature and the optimum pH of the purified N-ac etylglucosaminidase was 45 degrees C and 6.0, respectively. The N-terminal amino acid sequence of 23 residues of N-acetylglucosaminidase was identifie d. Based on the N-terminal sequence, we amplified pieces of the DNA fragmen ts by PCR. Using these PCR products as probes, we screened the genomic libr ary and successfully isolated the entire N-acetylglucosaminidase gene (desi gnated nag1) from Enterobacter sp. G-1. The nucleotide sequence of the nag1 gene was found to consist of 2,655 bp encoding a protein of 885 amino acid residues. Comparison of the deduced amino acid sequence from the nag1 gene found 97.3% identity with chitobiase from Serratia marcescens, 54.4% ident ity with N,N'-diacetylchitobiase from Vibrio harveyi, and 42.7% identity wi th N-acetylglucosaminidase (ExoI) from Vibrio furnissii. Enzymatic activity assay of N-acetylglucosaminidase indicated stronger activity toward PNP-Gl cNAc than PNP-(GIcNAc)(2) or PNP-(GlcNAc)(3).