CMP - N - acetylneuraminic a id synthetase of Escherichia coliz high level expression , purification and use in

The gene encoding CIIP-A'-acet\ ' lneuraminic acid (CIIPNeuAc) s1'nthetase (EC 2.7.7.43) in Escherichia coli serot!'pe 07 Kl w'as isolated and overexpressed in E.coli W3ll0. \Iaximum expression of 8-1Ooh of the soluble E.coli protein rras achieved b1'placing the gene rr i th an engineered 5'-terminus and Shine-Dalgarno sequence into a pKK223 rector derivative behind the tac promoter. The overexpressed s1'nthetase l las purif ied to >95o/o homogeneitf in a single step b)'chromatographl'on high titre Orange A N{atrexrv d1'e resin. Enzl'me purified b1' this method ll'as used directll' for the sy'nthesis of C\IP-NeuAc and derivatives. The enzl'matic s1'nthesis of Cl\'IP-NeuAc u'as carried out on a multigram scale using equimolar CTP and A'-acet1'lneuraminic acid as substrates. The resultant CN{P-NeuAc, isolated as its disodium salt by ethanol precipitation, was prepared in an overaf l f ield of 94oh and n'as judged to be >95o/o pure b1' lH N\IR anall 'sis. A'-Carbomethoxl'neuraminic acid and .A/-carbobenzl'lox1'neuraminic acid were also found to be substrates of the enz]'me; S-azidoneuraminic acid n'as not a substrate of the enz)'me. AlCarbomethoxl'neuraminic acid n'as coupled to C\{P at a rate similar to that obsen'ed with NeuAc, n'hereas l/-carbobenzr'loxvneuraminic acid $'as coupled > 100-fold more slon'|1'. The high level of expression achieved with the E.coli s1'nthetase, together with the high degree of purity readily obtainable from crude cell extracts, make the recombinant bacterial enzvme the preferred catall'st for the enzl'matic s] nthesis of C\{P-A/-acet1'lneuraminic acid.