University of Colombo

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Browsing University of Colombo by Author "ARASARATNAM, V"

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    Immobilization and kinetic studies of alfa- amylase and glucoamylase
    (University of Colombo: UC(MED)., 1984) ARASARATNAM, V
    Alfa- amylase and glucoamylase were coupled to sepharose -4B which was activated by nucleophilic and electrophilic methods using cyanogen bromide. The activity of Alfa-amylase and glucoamylase immobilized to gel activated by nucleophilic method were 19.7 percent and 20.42 percent respectively (40 mg CNBr / g wet gel was used). The activity of Alfa-amylase and glucoamylase immobilized to gel activated by electrophilic method were 23.64 percent and 26 percent respectively. (25 mg CNBr/ g wet gel was used) CNBr concentration was directly proportional to the concentration of the enzyme protein coupled. When 120 mg CNBr / g wet gel was used for activation by electrophilic and nucleophilic mrhtods, the protein coupled was 70 percent and 19 percent respectively. The coupling was linear up to 10 min and reached maximum by 0.5 hr. When the enzyme concentration added for coupling was increased from 1 - 1000 mg then the percent activity of the immobilized enzyme was decreased and it was inversely proportional to the log of enzyme concentration added. The activity of both soluble and immobilized Alfa- amylase and glucoamylase was linear for 3 min and 10 min respectively. The apparent Km values for the immobilized Alfa- amylase and glucoamylase for starch were 1.33 percent and 0.72 percent respectively. The optimum pH for soluble Alfa- amylase compared with that of the immobilized enzyme shifted from 6.9 to 6.5 in 0.02M phosphate buffer. The shift in pH optima for immobilized glucoamylase with that of soluble glucoamylase was from 4.8 to 5.2 where 0.1M acetate buffer was used. There were no shift in ionic strength optima for both immobilized Alfa-amylase \& glucoamylase from that of their respective soluble anzymes and the optimal ionic strengths were 0.02M (phosphate buffer) and 0.01M (acetate buffer) for Alfa-amylase and glucoamylase respectively. The temperature optimum of immobilized Alfa- amylase compared with the soluble enzyme shifted from 45 0 C to 50 0 C and that for gluccamylase was from 55 0 C to 58 0 C. in the temperature range from 30 0 C to 60 0 C the immobilized Alfa- amylase was most stable at 45 0 C indicating that the immobilized enzyme has a slightly higher temperature stability. The glucoamylase, both soluble and immobilized were most stable at 4 0 C that at higher temperatures.