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Journal of Biochemistry Advance Access published online on August 2, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm147
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© 2007 The Japanese Biochemical Society

Molecular cloning, expression, and properties of an {alpha}/ß-galactoside {alpha}2,3-sialyltransferase from Vibrio sp. JT-FAJ-16

Yoshimitsu Takakura*, Hiroshi Tsukamoto and Takeshi Yamamoto*

Glycotechnology Business Unit, JAPAN TOBACCO INC. 700 Higashibara, Iwata, Shizuoka 438-0802, JAPAN

*To whom correspondence should be addressed: Glycotechnology Business Unit, Japan Tobacco Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, JAPAN. Tel.: +81-538-32-7389; FAX: +81-538-33-6046; E-mail: takeshi.yamamoto{at}ims.jti.co.jp, or Tel.: +81-538-32-8291; E-mail: yoshimitsu.takakura{at}ims.jti.co.jp

Received June 14, 2007; Accepted July 3, 2007


  Abstract

We cloned, expressed, and characterized a novel {alpha}/ß-galactoside {alpha}2,3-sialyltransferase from Vibrio sp. bacterium JT-FAJ-16. Using a {alpha}2,3-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 {alpha}2,3sialylation was confirmed by 1H- and 13C- NMR analyses of the reaction products. The enzyme was optimally active at pH 5.5 and at 20 °C. Interestingly, the enzyme used both the {alpha}- and ß-anomers of galactosides as acceptors, suggesting that it can be described as an {alpha}/ß-galactoside {alpha}2,3-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

Key Words: cloning, expression, sialic acids, sialyltransferase, Vibrio


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