Photobacterium sp JT-ISH-224 produces two sialyltransferases, α-/β-galactoside α2,3-sialyltransferase and β-galactoside α2,6-sialyltransferase

A novel bacterium, Photobacterium sp. JT-ISH-224, that produces alpha-/beta-galactoside alpha 2,3-sialyltransferase and beta-galactoside alpha 2,6-sialyltransferase, was isolated from the gut of a Japanese barracuda. The genes that encode the enzymes were cloned from the genomic library of the bacterium using the genes encoding alpha-/beta-galactoside alpha 2,3-sialyltransferase from P. phosphoreum and beta-galactoside alpha 2,6-sialyltransferase from P. damselae as probes. The nucleotide sequences were determined, and open reading frames of 1,230 and 1,545bp for encoding an alpha 2,3-sialyltransferase and an alpha 2,6-sialyltransferase of 409- and 514-amino acid residues, respectively, were identified. The alpha 2,3-sialyltransferase had 92% amino acid sequence identity with the P. phosphoreum alpha 2,3-sialyltransferase, whereas the alpha 2,6-sialyltransferase had 54% amino acid sequence identity with the P. damselae alpha 2,6-sialyltransferase. For both enzymes, the DNA fragments that encoded the full-length protein and its truncated form lacking the putative signal peptide sequence were amplified by a polymerase chain reaction and cloned into an expression vector. Each gene was expressed in Escherichia coli, and the lysate from each strain had enzymatic activity. The alpha 2,3-sialyltransferase catalysed the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to lactose, alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside with low apparent K-m and the alpha 2,6-sialyltransferase catalysed the transfer of NeuAc from CMP-NeuAc to lactose with low apparent K-m.