Abstract
A novel sialyltransferase, alpha/beta-galactoside alpha-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 NH(2)-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 beta-galactoside alpha-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 NH(2) 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 alpha-methyl-galactopyranoside and beta-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.
Highlights
Synthesis of various oligosaccharide chains [6, 7]
Many sialyltransferases have been cloned from mammals to date, it is not easy to produce these enzymes in large quantity, because mammalian glycosyltransferases are rarely expressed as active enzymes in Escherichia coli
Isolation and Identification of Marine Bacteria Producing ␣-2,3-Sialyltransferase—More than 3000 isolates of bacteria were examined for sialyltransferase activity, and 10 of these tested positive in the enzymatic assay
Summary
Screening of Bacteria—Samples of seawater, sand, mud, small animals, and seaweed were collected from coasts of various locations in Japan. Sialyltransferase Assay—The 30-l reaction mixture consisted of a sample of enzyme, 120 mM lactose, 2.3 mM CMPNeuAc (Nakarai), 4620 Bq of CMP-[4,5,6,7,8,9-14C]NeuAc (Amersham Biosciences), 20 mM bis-Tris buffer (pH 6.0), 0.5 M NaCl, and 0.03% Triton X-100. The mixture was applied to a PALPAK type R analytical column (0.46 ϫ 25 cm; Takara Bio) that was equilibrated with 100 mM acetic acid-triethylamine buffer (pH 5.0) that contained 0.15% n-butanol in a high pressure liquid chromatograph (LC10A; Shimadzu). Sialidase Activity—The 15-l reaction mixture consisted of a sample of enzyme, 1.7 M pyridylaminated 3Ј-sialyllactose, 15 mM bis-Tris buffer (pH 6.0), and 0.1% Triton X-100 and was incubated at 25 °C. The active fractions were pooled, diluted to three volumes with 20 mM bis-Tris buffer (pH 7.0) that contained 0.3% Triton X-100 (Buffer C), and applied to a column of Mono Q 10/100 GL equilibrated with Buffer C. Construction of Expression Cassettes—For amplification of the ␣-2,3-sialyltransferase gene, three primers, PPSTF1, PPSTF2, and PPSTR1 (Table 1), were designed to create PciI, NcoI, and BamHI restriction sites, respectively, at the ends of TABLE 1
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