Molecular cloning of the gene encoding a novel beta-N-acetylhexosaminidase from a marine bacterium, Alteromonas sp. strain O-7, and characterization of the cloned enzyme.
We have reported that the chitinolytic system of Alteromonas sp. strain O-7 consists of chitinases (ChiA, ChiB, and ChiC), a chitinase-like enzyme (ChiD), beta-N-acetylglucosaminidases (GlcNAcasesA, GlcNAcaseB, and GlcNAcaseC), and a novel transglycosylative enzyme (Hex99). The gene encoding a beta-hexosaminidase with an unusual substrate specificity (hex86), located upstream of the hex99 gene, was cloned and sequenced. The gene encoded a protein of 761 amino acids with a calculated molecular mass of 86,758 Da. The deduced amino acid sequence of Hex86 showed sequence similarity with beta-hexosaminidases belonging to family 20. The hex86 gene was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme rapidly cleaved p-nitrophenyl-beta-N-acetyl-D-glucosaminide and slowly cleaved p-nitrophenyl-beta-N-acetyl-D-galactosaminide. Unexpectedly, the enzyme did not hydrolyzed chitin oligosaccharides under the assay conditions for synthetic glycosides. However, after prolonged incubation with excessive quantities of the enzyme, Hex86 hydrolyzed chitin oligosaccharides. These results indicate that Hex86 is a novel enzyme that prefers p-nitrophenyl-beta-N-acetyl-D-glucosaminide to chitin oligosaccharides as a substrate.
Tsujibo
Hiroshi
H
Osaka University of Pharmaceutical Sciences, Takatsuki, Japan. tsujibo@gly.oups.ac.jp
Miyamoto
Katsushiro
K
Yoshimura
Makiko
M
Takata
Miwa
M
Miyamoto
Junko
J
Inamori
Yoshihiko
Y
D020591:Alteromonas / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D001619:beta-N-Acetylhexosaminidases