Purification and characterization of intracellular alpha-galactosidase from Aspergillus sp.(96/S)
No Thumbnail Available
Date
1997
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Colombo: UC(MED).
Abstract
The intracellular alpaha-galactosidase (alpha-D-galactosidase EC 3.2.1.22) producing fungi were isolated from soil and air by enrichment culture technique, using raffinose as the inducer. Seventeen fungi species were isolated from enrichment culture method based on their morphological characteristics The intracellular ?-galactosidase (pellet) activity of the isolated fungi species varied from 405 Mu/G to 2Mu/G after 120 hours of cultivation. The supernatant activity varied from 108mU/ml to 3mU/ml. These fungi species were cultivated at pH 4.5, in raffinose enrichment culture medium and their ?-galactosidase enzyme activities in pellets as well as in supernatants were measured at Ph values of 5,6, and 7 McLLvaine buffer. The maximum ?-galactosidase activity was observed at pH 5, in both supernatant and pellet. Four fungi species having ?-galactosidase activity in the pellet from 3645Mu/g were used for further studies. Highest intracellular(pellet) activity of 3645Mu/g was obtained at 96 hours of cultivation in fungi Aspergillus species 96/S. ?-galactosidase produced by fungi aspergills species 96/S was purified by centrifugation, Ammonium sulphate fractionation, DEAE-A-25 ion exchange chromatography and affinity chromatography on sepharose 4B-lysine gala cturonate gel. Only one enzyme activity peak was observed in ion exchange chromatography, including that this fungi species does not contain isoenzymes of ?-galactosidase. The enzyme was purified 50.6 folds with a 8 percent recovery. Purified enzyme shown to be pure by polyacrylamide gel electrophoresis. The kinetic properties of the purified ?-galactosidase enzyme were studied using p-nitrophenyl ?-D-galactopyranoside as the substrate. Michaelis-Menten constant (Km) and maximum reaction velocity (Vmax) of ?-galactosidase enzyme obtained was 3.3x10-4 M AND 2.26 X 10-2 ?moles/ming/g(protein) respectively. Studies on effect of pH and pH stability of ?-galactosidase showed that the purified enzyme had a pH optima of pH 3.5 and 5. Maximum activity was observed at pH 3.5. The enzyme was observed to be stable over a broad at 500C and enzyme had high activity at 600C. The tempature stability of the enzyme was from 200C to 600C. Aspergillus species 96/S mycelium (3g) when kept in 1 percent raffinose solution with gentle stirring at 300C, raffinose content decreased by 12 percent after 4 hours. Aspergillusa species 96/S mycelium (3g) immobilized on 5 [percent polyacrylamide gel packed in to column and eluted with 1 percent raffinose solution at a flow rate of 60ml/h at 300C. After 4 hours the raffinose content in the 1 percent raffinose solution was decreased by 51 percent.
Description
Dissertation: M.Sc., University of Colombo: UC(MED), 1997.
Keywords
Aspergillus
Citation
JAYADEVA, HS, Purification and characterization of intracellular alpha-galactosidase from Aspergillus sp.(96/S), University of Colombo UC(MED), 1997: iv, 126p.