β-Galactosidase Immobilization on Chitosan-Hydroxyapatite Complex: Effects of Immobilization Conditions

Abstract

In this study, β-galactosidase from Kluyvermoyces lactis was immobilized onto chiosan-hydroxyapatite. Hydroxyapatite (HAP) is a nontoxic ceramic biomaterial generally that has been used for dental applications, drug delivery, immobilization of metals, enzymes and recently some yeast cells. Effects of immobilization parameters including medium temperature and pH, enzyme concentration and coupling time was evaluated to obtain the best reaction conditions. The optimal immobilization pH was determined as pH7.5 with the highest enzyme activity of 1337.2Ug-1 chitosan-HAP beads and highest immobilization efficiency of 81.4%. Furthermore, the immobilized protein amount showed increase with increasing enzyme concentration as expected. However, highest specific activity (365.2mg-1 protein) was achieved at low enzyme concentrations. Characterization of immobilized enzyme was tested using o-nitrophenol-β-D-galactopyranoside (ONPG) as a substrate. Immobilized enzyme exhibited better thermostability and higher stability at lower (pH 5.0) and higher (pH 9.0) pH values. Immobilization also resulted in a shift of 0.5units in pH optimum for maximum enzyme activity. In contrast to optimum pH value, after immobilization no shift occurred in optimum temperature. Furthermore enzyme retained 81.8% of its initial activity during consecutive eight cycles. After 15days of storage, immobilized enzyme obtained 82.6% of its original activity, whereas free enzyme showed a significant decrease.