title: Purification, characterization and gene analysis of N-acetylglucosaminidase from Enterobacter sp. G-1.
abstract: Enterobacter sp. G-1 is a bacterium isolated previously as a chitinase-producing bacterium. We found this bacterium also produced N-acetylglucosaminidase and characterized that in this study. Extracellular N-acetylglucosaminidase of 92.0 kDa was purified near homogeneity by 8.57-fold from Enterobacter sp. G-1. The optimum temperature and the optimum pH of the purified N-acetylglucosaminidase was 45 degrees C and 6.0, respectively. The N-terminal amino acid sequence of 23 residues of N-acetylglucosaminidase was identified. Based on the N-terminal sequence, we amplified pieces of the DNA fragments by PCR. Using these PCR products as probes, we screened the genomic library and successfully isolated the entire N-acetylglucosaminidase gene (designated 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% identity with N,N'-diacetylchitobiase from Vibrio harveyi, and 42.7% identity with N-acetylglucosaminidase (ExoI) from Vibrio furnissii. Enzymatic activity assay of N-acetylglucosaminidase indicated stronger activity toward PNP-GlcNAc than PNP-(GlcNAc)2 or PNP-(GlcNAc)3.
authors.affiliation: Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Matsuo
authors.forename: M
authors.initials: M
authors.lastname: Kurita
authors.forename: J K
authors.initials: JK
authors.lastname: Park
authors.forename: K
authors.initials: K
authors.lastname: Tanaka
authors.forename: T
authors.initials: T
authors.lastname: Nakagawa
authors.forename: M
authors.initials: M
authors.lastname: Kawamukai
authors.forename: H
authors.initials: H
authors.lastname: Matsuda
mesh_terms: D000118:Acetylglucosaminidase / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004754:Enterobacter / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D000118:Acetylglucosaminidase