title: 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.
authors.affiliation: Department of Biochemistry, National Yang-Ming Medical College, Shi-Pai, Taipei, Taiwan, Republic of China.
authors.forename: C H
authors.initials: CH
authors.lastname: Chien
authors.forename: Y H
authors.initials: YH
authors.lastname: Wei
authors.forename: S F
authors.initials: SF
authors.lastname: Yeh
authors.forename: C P
authors.initials: CP
authors.lastname: Li
mesh_terms: D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology; D004800:Enzymes, Immobilized; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009369:Neoplasms / Q000097:blood; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D011771:Pyruvate Oxidase / Q000378:metabolism; D012794:Sialic Acids / Q000097:blood*; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D012794:Sialic Acids; D009195:Peroxidase; D011771:Pyruvate Oxidase; D009439:Neuraminidase; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase