Molecular cloning, expression and properties of an alpha/beta-Galactoside alpha2,3-sialyltransferase from Vibrio sp. JT-FAJ-16.
We cloned, expressed and characterized a novel alpha/beta-galactoside alpha2,3-sialyltransferase from Vibrio sp. bacterium JT-FAJ-16. Using a alpha2,3-sialyltransferase gene from a marine bacterium as a probe, a DNA sequence encoding a 402-amino-acid protein was identified from the JT-FAJ-16 genomic library. The protein showed 27.3-64.7% identity to the bacterial sialyltransferases classified into glycosyltransferase family 80. The protein showed sialyltransferase activity when expressed in Escherichia coli. The N-terminal truncated form of the enzyme was amplified in E. coli and its recovered activity was 215.7 unit/l culture medium. It was purified as a single band on SDS-PAGE through the three chromatographic steps. The specific activity of the purified recombinant enzyme reached 57.5 unit/mg protein. The alpha2,3sialylation was confirmed by (1)H- and (13)C-NMR analyses of the reaction products. The enzyme was optimally active at pH 5.5 and at 20 degrees C. Interestingly, the enzyme used both the alpha- and beta-anomers of galactosides as acceptors, suggesting that it can be described as an alpha/beta-galactoside alpha2,3-sialyltransferase. The enzyme had a wide range of acceptor substrate specificities. It transferred N-acetylneuraminic acid (NeuAc) to various monosaccharides and various oligosaccharides, and both N-linked and O-linked asialo-glycoprotein. These results suggest that the enzyme can be used as a powerful tool for the study for glycotechnology.
Takakura
Yoshimitsu
Y
Glycotechnology Business Unit, Japan Tobacco Inc, 700 Higashibara, Iwata, Shizuoka, Japan. yoshimitsu.takakura@ims.jti.co.jp
Tsukamoto
Hiroshi
H
Yamamoto
Takeshi
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014733:Vibrio / Q000201:enzymology*
D017931:DNA Primers; D012799:Sialyltransferases