Purification and Characterization of ?-N-Acetylgalactosaminidase from <i>Hilsha ilisha</i>
Keywords:Hilsha ilisha, ?-N-Acetylgalactosaminidase, ?-Galactosidase, Purification.
The objective of this study was to purify and characterize the alpha-N-acetylgalactosaminidase (?-GalNAcase) from hilsha fish, Hilsha ilisha. Digestive organ of hilsha fish was found to contain a large amount of ?-GalNAcase in compared with the other tissues examined. ?-GalNAcase was purified from the crude extract of hilsha fish by ammonium sulphate precipitation, Sephadex G-200 gel filtration, and SP-Sephadex C-50 column chromatography. The purified enzyme gave a single band on sodium dodecylsulphate-polyacrylamide gel electrophoresis and exhibited a molecular mass of 48 kDa. The final preparation of ?-GalNAcase showed 3.02% ?-galactosidase activity. The enzyme had an optimum pH of 4.0 and was found to be quiet heat stable at 37°C. Inhibition studies with metal ions demonstrated that the enzyme was highly inhibited by silver and mercury ions. Both N-acetylgalactosamine and galactose affect the enzyme activity. Kinetic studies with the enzyme showed that the KM value for p-nitrophenyl-?-N-acetylgalactosaminide substrate was 3.31 mM and the Vmax value was 35.04 unit/mg.
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