Immobilization of ß-galactosidase in poly(vinyl alcohol)-calcium alginate, poly(n-isopropylacyrilamide)-calcium alginate

In this study, ß-galactosidase (E.C 3.2.1.23) was immobilized using entrapment method in poly(vinyl alcohol)-calcium alginate (CaAlj-PVA), poly(N-isopropylacrylamide)-calcium alginate (CaAlj-P(NIPA)), beads and also covalently immobilized by activating the beads with carbodiimide (CDI), N-hydroxysuccinimide (NHS), carbodiimide and Nhydroxysuccinimide (CDI-NHS). Michaelis-Menten constant (Km) and maximum reaction rate (Vmax) values were found as 0.343 mM and 0.0259 mM.min-1, for free enzyme respectively. Km and Vmax values were changed between 0.746 mM ? 4.97mM and 0.0049mM.min-1?0.7997 mM.min-1, for covalently bonded and entraped enzymes respectively. Optimum pH was determined as 5.0, 4.5 and 5.0 and optimum temprature was determined as 30oC, 35oC, 35oC for free ß- galactosidase, entrapment and covalently bonded enzymes respectively. At 45oC, inaktivation contstants were calculated as 0,0289 min-1 for free enzyme, 0,0100 min-1 - 0,0216 min-1 for covalently bounded enzymes and 0,0005 min-1 - 0,0135 min-1 for entrapped enzymes. After 30 days of storage at 4 oC free ß-galactosidase retained 10 % of its original activity. Also after 70 days of storage at 4 oC, entraped and covalently bonded enzymes were retained 45 % and 49 % of their original activities, respectively. It was observed that 20 ? 62.5 % of vii original activity were preserved when entraped ß-galactosidase were used repeatedly 5 to 10 times while covalently bonded enzymes were 56 to 60 times. Reuseability, storage stability and thermal stability of free enzyme were increased by immobilization.