Gene cloning, expression, and characterization of a second beta-N-acetylglucosaminidase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9.
A gene coding for a second beta-N-acetylglucosaminidase (nagB) was isolated from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9. The nagB open reading frame encoded a polypeptide of 618 amino acid residues with a molecular mass of 68.8 kDa. It did not contain a sequence characteristic of a signal peptide at the N-terminus. The deduced amino acid sequence showed high similarity to those of bacterial beta-N-acetylhexosaminidases classified into family 20 of glycosyl hydrolases. The nagB gene was successfully expressed in Escherichia coli, and the recombinant protein hydrolyzed N-acetylchitooligomers from dimer to hexamer and produced monomer as a final product. Reverse transcription-mediated PCR (RT-PCR) analysis revealed that nagB was transcribed when SUWA-9 cells were grown in the presence of colloidal chitin. In the upstream of the nagB gene, three genes, coding for putative N-acetylglucosamine kinase, beta-glucosidase, and ATP-binding protein of ABC-type transporter, were identified, and these genes likely to constitute an operon.
Lan
Xiqian
X
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan.
Zhang
Xin
X
Kodaira
Ritsuko
R
Zhou
Zeyang
Z
Shimosaka
Makoto
M
D000118:Acetylglucosaminidase / Q000235:genetics / Q000378:metabolism*; D016980:Aeromonas hydrophila / Q000201:enzymology*; D001483:Base Sequence; D002686:Chitin / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D002686:Chitin; D000118:Acetylglucosaminidase