Erkut YILMAZ, Fatma YILMAZ, Adil DENİZLİ, Lokman UZUN

Reactive brown 10-atteched polyamide hollow fiber for reversible amyloglucosidase immobilization

Amyloglucosidase (AMG) [$alpha$-1, 4-D-glucan glucohydrolase (E.C.3.2.1.3)] is an exo-enzyme, which is used in the hydrolysis of starch to glucose in industry. In this study,AMG was immobilized onto Reactive Brown 10-attached polyamide hollow fibers. Dye-attached hollow fibers were characterized by scanning electron microscopy. The amount of attached dye was determined by elementel analysis considering the sulphur stoichiometry. It was found as 18.4 $mu mol/g$ hollow fiber. Affecting factors, pH and initial AMG concentration, on the immobilization was investigated. Maximum AMG immobilization was achieved at pH 4.0, and adsorption capacity of hollow fibers increased by increasing initial AMG concentration and reached the plateau at 1.5 mg/mL. The effects of temperature and pH on the enzyme activity were also investigated. Immobilized enzyme has a wider pH dependence around pH 4.0-5.0, but free enzyme has optimal pH at its pI value, pH 4.5. Optimal temperature for immobilized enzyme was determined as 65°C, which is higher than free enzyme, 60°C. The immobilization process increased the thermal stability of AMG. In 180 min, although free enzyme lost the activity about 60% at 60°C, immobilized enzyme lost only 20% at same temperature. Significant amount of the immobilized AMG was desorbed using 1.0 M NaCl at pH 3.0. In order to determine the effects of adsorption and desorption conditions on possible conformational changes of AMG, fluorescence spect-rophotometry was employed. It was concluded that polyamide dye-affinity hollow fibers can be applied for AMG immobilization without causing any significant conformational changes. $K_M$ and Vmax values of immobilized and free enzymes were found to be 1.08 and 1.31 mg/mL and 4.53 and 1.94 $mu$ mol/mg.min, respectively. The $K_M$ value of the immobilized AMG was 1.2-fold higher than that of the free one. The $V_{max}$ value of the free AMG was found to be higher than that of the immobilized AMG. These results showed that Reactive Brown 10-attached hollow fiber can be used for reversible immobilization of AMG in biotechnological applications.

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