Molecular characterization of an intracellular beta-N-acetylglucosaminidase involved in the chitin degradation system of Streptomyces thermoviolaceus OPC-520.
We purified and characterized an intracellular beta-N-acetylglucosaminidase (NagC) from a cytoplasmic fraction of Streptomyces thermoviolaceus OPC-520. The molecular mass of NagC was estimated to be 60 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the enzyme were 6.0 and 50 degrees C respectively. Purified NagC hydrolyzed chitin oligosaccharides from N,N'-diacetylchitobiose (GlcNAc)(2) to chitopentaose (GlcNAc)(5), hydrolyzed N,N'-diacetylchitobiose especially rapidly, and showed a tendency to decrease with increases in the degree of polymerization. But, NagC didn't hydrolyze chitohexaose (GlcNAc)(6). The gene encoding NagC was cloned and sequenced. The open reading frame of nagC encoded a protein of 564 amino acids with a calculated molecular mass of 62,076 Da. The deduced amino acid sequence of NagC showed homology with several beta-N-acetylglucosaminidases belonging to glycosyl hydrolase family 20. The expression plasmid coding for NagC was constructed in Escherichia coli. The recombinant enzyme showed pH and temperature optima and substrate specificity similar to those of the native enzyme. The gene arrangement near the nagC gene of S. thermoviolaceus OPC-520 was compared with that of S. coelicolor A3(2). Three genes, which appear to constitute an ABC transport system for sugar, were missing in the vicinity of the nagC gene.
Kubota
Takahiro
T
Department of Microbiology, Osaka University of Pharmaceutical Sciences, Osaka, Japan.
Miyamoto
Katsushiro
K
Yasuda
Masahide
M
Inamori
Yoshihiko
Y
Tsujibo
Hiroshi
H
D000118:Acetylglucosaminidase / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature
D002686:Chitin; D000118:Acetylglucosaminidase