Reactive Brown 10-attached Polyamide Hollow Fiber for Reversible Amyloglucosidase Immobilization

Authors : Fatma Yilmaz, Erkut Yilmaz, Lokman Uzun, Adil Denizli

Pages : 149-155

View : 18 | Download : 7

Publication Date : 2007-04-01

Article Type : Other

Abstract :Amyloglucosidase AMG [α-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 polyamidehollow 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 μmol/ghollow fiber. Affecting factors, pH and initial AMG concentration, on the immobilization was investigated. MaximumAMG immobilization was achieved at pH 4.0, and adsorption capacity of hollow fibers increased by increasing initialAMG 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 hasoptimal pH at its pI value, pH 4.5. Optimal temperature for immobilized enzyme was determined as 65°C, which ishigher 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 effectsof 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 immobilizationwithout causing any significant conformational changes. KM and Vmax values of immobilized and free enzymes werefound to be 1.08 and 1.31 mg/mL and 4.53 and 1.94 µmol/mg.min, respectively. The KM value of the immobilized AMGwas 1.2-fold higher than that of the free one. The Vmax value of the free AMG was found to be higher than that of theimmobilized AMG. These results showed that Reactive Brown 10-attached hollow fiber can be used for reversible immobilization of AMG in biotechnological applications
Keywords : Hollow fibers, Reactive Brown 10, amyloglucosidase, polyamide, reversible immobilization