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-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.
Matsuo
Y
Y
Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Japan.
Kurita
M
M
Park
J K
JK
Tanaka
K
K
Nakagawa
T
T
Kawamukai
M
M
Matsuda
H
H
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
D004269:DNA, Bacterial; D000118:Acetylglucosaminidase