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J. Biol. Chem., Vol. 282, Issue 41, 29794-29802, October 12, 2007

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Purification, Cloning, and Expression of an /-Galactoside -2,3-Sialyltransferase from a Luminous Marine Bacterium, Photobacterium phosphoreum^*

From the Glycotechnology Business Unit, Japan Tobacco Incorporated, Higashibara, Iwata, Shizuoka 438-0802, Japan

A novel sialyltransferase, /-galactoside -2,3-sialyltransferase, was purified from the cell lysate of a luminous marine bacterium, Photobacterium phosphoreum JT-ISH-467, isolated from the Japanese common squid (Todarodes pacificus). The gene encoding the enzyme was cloned from the genomic library of the bacterium using probes derived from the NH₂-terminal and internal amino acid sequences. An open reading frame of 409 amino acids was identified, and the sequence had 32% identity with that of -galactoside -2,6-sialyltrasferase in Photobacterium damselae JT0160. DNA fragments that encoded the full-length protein and a protein that lacked the sequence between the 2nd and 24th residues at the NH₂ terminus were amplified by polymerase chain reactions and cloned into an expression vector. The full-length and truncated proteins were expressed in Escherichia coli, producing active enzymes of 0.25 and 305 milliunits, respectively, per milliliter of the medium in the lysate of E. coli. The truncated enzyme was much more soluble without detergent than the full-length enzyme. The enzyme catalyzed the transfer of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid to disaccharides, such as lactose and N-acetyllactosamine, with low apparent K_m and to monosaccharides, such as -methyl-galactopyranoside and -methyl-galactopyranoside, with much lower apparent K_m. Thus, this sialyltransferase is unique and should be very useful for achieving high productivity in E. coli with a wide substrate range.

Received for publication, March 5, 2007 , and in revised form, August 16, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB293982 and AB293983.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Fig. 1.

¹ To whom correspondence may be addressed. Tel.: 81-538-32-7337; Fax: 81-538-33-6046; E-mail: hiroshi.tsukamoto{at}ims.jti.co.jp. ²To whom correspondence may be addressed. Tel.: 81-538-32-7389; Fax: 81-538-33-6046; E-mail: takeshi.yamamoto{at}ims.jti.co.jp.

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