CMP-sialate synthetase from Neisseria meningitidis - Overexpression and application to the synthesis of oligosaccharides containing modified sialic acids

The gene siaB encoding the CMP-sialate synthetase [EC 2.7.7.43] from Neisse ria meningitidis serogroup B was subcloned for overexpression in Escherichi a coli K12 using the expression vector pKK223-3. With the recombinant strai n, 7500 U of synthetase could be produced per liter of cell culture on a 10 L-scale (1350 U/g cells; 20 U/mg protein). Purified enzyme was obtained in high yields (> 85%) and with a high specific activity (greater than or equ al to 134 U/mg protein) by an efficient, two-step scheme consisting of DEAE anion-exchange chromatography and ammonium sulfate precipitation. In contr ast to known bacterial CMP-sialate synthetases, this enzyme was found to ex hibit a broad substrate tolerance, particularly by accepting C5-modified Ne u5Ac derivatives as substrates. This included neuraminic acid N-carbamoylat ed with typical protective groups of different length and bulkiness, an uns aturated acrylamide derivative and the corresponding saturated moiety, as w ell as the deaminated KDN analogue. Also, the latter structure call be vari ed by deoxygenation, epimerization at C5 or at the terminal chain, and by s hortening the chain length to an octulosonic acid. The high expressivity of the recombinant production clone, the high catalytic efficiency or the enz yme, and its broad substrate tolerance make this synthetase from A meningit idis the preferred catalyst for the enzymatic synthesis of CMP-Neu5Ac and o f derivatives modified in the sialic acid moiety. Several CMP-conjugates ma de available by this procedure could be transferred effectively onto the ac ceptor N-acetyllactosamine using the alpha -2,6-sialyl-transferase from rat liver to generate the corresponding trisaccharides.