Purification and characterization of neuraminidase from Clostridium perfringens.
Neuraminidase (EC 3.2.1.18) has been purified from the culture medium of Clostridium perfringens ATCC 10543, through steps of gel filtration on Sephadex G-75 column, DEAE-cellulose DE 23 anion exchange chromatography, and isochromatofocusing. A homogeneous enzyme was obtained with a 7552-fold increase in specific activity to 295 units/mg protein. The yield was about 25%. The enzyme consists of a single polypeptide with a molecular weight of 69,000 as determined by SDS-polyacrylamide gel electrophoresis. Kinetic studies showed that Km is 1.5 mM for sialyllactose and Vmax is 0.41 mumole/min/ml at the enzyme concentration of 0.14 microgram/ml. The enzyme is stable at pH 5.2-8.0 with an optimal pH of 6.0. A concentrated solution of the purified enzyme was stable over one year at 4 degrees C. The purified enzyme hydrolyzed human alpha 1-acid glycoprotein completely; thus, it can be used in the clinical assay of N-acetylneuraminic acid in the serum.
Chien
C H
CH
Department of Biochemistry, National Yang-Ming Medical College, Taipei, Taiwan, R.O.C.
Chang
S C
SC
Wei
Y H
YH
D003016:Clostridium perfringens / Q000201:enzymology* / Q000254:growth & development; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009439:Neuraminidase / Q000302:isolation & purification*; D013379:Substrate Specificity
D009439:Neuraminidase