An immobilized-enzyme system for the determination of sialic acid using cloned neuraminidase from Clostridium perfringens A.T.C.C. 10543.

Abstract

Neuraminidase expressed by cloned nanH of Clostridium perfringens has been immobilized and employed to determine the concentration of sialic acid in human serum. Two enzyme pairs, cloned neuraminidase-N-acetylneuraminate (NANA) lyase and pyruvate oxidase-peroxidase, have been respectively co-immobilized on to 1,12-aminododecane-agarose with glutaraldehyde. The relative specific activities of the co-immobilized neuraminidase and NANA lyase were 61 and 77%, and those of pyruvate oxidase and peroxidase were 51 and 96% of the corresponding soluble enzymes respectively. The optimal reaction pH at 37% C for each of the co-immobilized enzymes was about 1 pH unit higher than that of the corresponding soluble enzyme. The optimal reaction temperature of peroxidase was increased as a result of immobilization. The thermostability of the immobilized cloned neuraminidase, NANA lyase, pyruvate oxidase and peroxidase were increased 80-, 83-, 115- and 147-fold at 45 degrees C over the soluble forms respectively. The results correlated satisfactorily with those obtained by using a soluble enzyme system. The system is thus a reliable assay method for sialic acid in serum.