Immobilization of alpha-galactosidase from coconut endosperm
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Date
1988
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University of Colombo: UC(MED).
Abstract
Alpha-galactosidase was purified from coconut endosperm by hydrophobic chromatography. The preliminary purification included extraction, acidification, centrifugation and ammonium sulphate fractionation. Centrifugation was done at 7 500 g at room temperature (30§)C). The recoveries from acidification and ammonium sulphate fractionation were 94 percent and 60 percent respectively. Hydrophobic gel Sepharose-4B-capranilide with a capacity of 0.63 mg/ml was used for the purification of Alpha-galactosidase. The purified enzyme had a specific activity of 24.5 units/mg protein and the recovery was 58 percent. The purified enzyme was shown to be homogeneous by polyacrylamide gel electrophoresis and isolectric focusing.Studies on inhibition of Alpha-galactosidase by carbohydrates showed that the enzyme is no inhibited by sucrose and glucose and only 29 percent inhibited by a 1 percent raffinose solution. This indicates that the enzyme can be used to hydrolyse raffinose present in soybean milk and beetsugar molasses. Purified Alpha-galactosidase (60 units) was immobilized on Sepharose-4B-capranilide gel (72 ml) in the presence of ammonium sulphate with 100 percent binding and 59 percent retained activity. The immobilized gel was used for the determination of the hydrolysis of a 1 percent raffinose solution at different flow rates. The results on the percentage hydrolysis of raffinose indicate that the immobilized enzyme has a very low efficiency when compared to the native enzyme. The percentage hydrolysis increased when the flow rate was reduced. Purified Alpha-galactosidase was immobilized in polyacrylamide gels by entrapment. Entrapment in the 15 percent polyacrylamide gel gave a percentage binding of 58 percent and a retained activity of 72 percent. When 7.75 units of Alpha-galactosidase was immobilized by entrapment in the K-carrageenan gel (20 ml) there was a percentage binding of 57 percent and a retained activity of 54 percent. Alpha-galactosidase was covalently bound to nylon pellets with a percentage binding of 14 percent and a retained activity of 49 percent. When Alpha-galactosidase was covalently bound to nylon powder there was a percentage binding of 19 percent and a retained activity of 64 percent. Alpha-galactosidasewas covalently bound to Sepharose-4B-lysine with a percentage binding of 36 percent and a retained activity of 2.25 percent. Alpha-galactosidase was also immobilized on Sepharose-4B-caproic acid with a percentage binding of 48 percent and a retained activity of 32 percent. Alpha-galactosidase immobilized in the 15 percent polyacrylamide gel, K-carrageenan gel, nylon pellets and nylon power were used to study the properties of the immobilized enzyme. The optimum pH for both native and immobilized enzymes were 5.5. When the pH was lower than 5.5 the activity of the immobilized enzyme was lower than the activity of the native enzyme. When the pH was greater than 5.5 the activities of bothe native and immobilized enzymes were similar. 40§C was the optimum temperature for both the native and immobilized Alpha-galactosidase . Within the temperatre range employed (i.e. 10§ - 60§C) enzymes immobilized on the polyacrylamide gel and on nylon pellets had a higher activity than the native enzyme while the enzymes immobilized in carrageenan and on nylon powder had a lower activity than the native enzyme. Further the thermal stability of immobilized enzymes were greater than that of the native enzyme within the temperature range 10§-60§
Description
Dissertation: M.Sc., University of Colombo: UC(MED), 1988.
Keywords
Enzymes
Citation
GUNARATNE, HDJG, Immobilization of alpha-galactosidase from coconut endosperm, University of Colombo UC(MED), 1988: ix,159p.