Purification and study of the kinetic of alpha galacto idase from coconut kernel
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Date
1980
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Publisher
University of Colombo: UC(MED).
Abstract
Two Alpha Galactosidase isoenzymes have been isolated from coconut kernel and characterized using sephadex column chromotography. The elution had been done using Mcilvaine buffer (pH 5.5) containing KC1 (Balasubramanium et al 1974). The purity of these enzymes have not been tested. In this study Alpha Galactosidase was eluted from the sephadex column using Mcllvaine bufer (pH 5.5) without KC1. All the enzyme was eluted in the low molecular isoenzyme form. Recovery from the coloumn was 23.6 percent of the original extract, the purity of the enzyme increasing 171 fold. The enzyme thus obtained was further purified by concentration using ultrfiltration and ultracentrifugation. At this stage 11.5 percent of the original extract was recovered while the purity increased up to 512 fold. This enzyme preparation when tested by polyacrylamide gel electrophoresis showed a single protein band which corresponded to the fluorecent band obtained by the action of Alpha Galactosidase on umbelliferyl Alpha D galactopyranoside. On cellulose acetate paper electrophoresis a single enzyme activity band was obtained. As the protein concentration was too low, the corresponding protein band could not be detected. Molecular weight of the purified enzyme was 18330. The enzyme was inhibited by a substrate concentration of para nitrophenyl Alpha D galactopyranoside of 6 X 10-4M. For the above substrate Km was 2.5 X 10-4M and Vmax was 25 uMol/min/mg. of protein. The enzyme is stable upto 450C. Activation energy was 6.83 Kcal per mole. Enzyme activity showed a broad pH optimum from 2.5 to 6 with a peak at pH 5.5. Enzyme was noncompetitively inhibited by mercuric ions and was competitively inhibited by D galactose.
Description
Dissertation: M.Sc., University of Colombo: UC(MED), 1980.
Keywords
Enzymes
Citation
MATHEW, CPDW, Purification and study of the kinetic of alpha galacto idase from coconut kernel, University of Colombo UC(MED), 1980: 88p.