Biochemical conditions for the production of polysialic acid by Pasteurella haemolytica A2

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a lin ear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. W hen the bacterium was grown at 37 degrees C for 90 h in 250 ml shake flasks at 200 rpm in Brain heart infusion broth (BHIB), it accumulated, attaining a level of 60 mu g/ml. Release of this polymer was strictly regulated by t he growth temperature, and above 40 degrees no production was detected. The pathway for the biosynthesis of this sialic acid capsular polymer was also examined in P. haemolytica A2 and was seen to involve the sequential prese nce of three enzymatic activities: Neu5Ac lyase activity, which synthesizes Neu5Ac by condensation of N-acetyl-D-mannosamine and pyruvate with apparen t Km values of 91 mM and 73 mM, respectively; a CMP-Neu5Ac synthetase, whic h catalyzes the production of CMP-Neu5Ac from Neu5Ac and CTP with apparent Km Values of 2 mM and 0.5 mM, respectively, and finally a membrane-associat ed polysialyltransferase, which catalyzes the incorporation of sialic acid from CMP-Neu5Ac into polymeric products with an apparent CMP-Neu5Ac Km of 2 50 mu M.