Kitosan kaplı manyetik nanotaneciklere alfa-amilaz immobilizasyonu
Alpha-amylase immobilization onto chitosan coated magnetic nanoparticles
Yazar: SEHER MERAL
Danışman: PROF. DR. SERPİL AKSOY
Gazi Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı
Derece: Yüksek Lisans, 2013
Konu: Kimya = Chemistry

Özet:
In this study, Fe3O4 nanoparticles were coated with chitosan (CS) by reversed-phase suspension method. ?-Amylase from Bacillus subtilis was immobilized onto chitosan coated Fe3O4 (Fe3O4-CS) nanoparticles by adsorption method and by covalent binding onto Fe3O4-CS nanoparticles which were activated by N-(3-dimethylaminopropyl)-N'- ethyl-carbodiimide hydrochloride (EDAC) and cyanuric chloride (SC). The effect of pH, temperature, substrate concentration, storage time on the activities of free and immobilized ?-amylases (Fe3O4-CS-A, Fe3O4- CS-EDAC-A, Fe3O4-CS-SC-A) were investigated. The optimum pH and optimum temperature were determined as 6,9; 8,0; 6,5; 6,0 and as 40°C, 30°C, 40°C ve 40°C for free ?-amylase, Fe3O4-CS-A, Fe3O4-CS-EDAC-A, Fe3O4-CS-SC-A respectively. Retained activities for immobilized systems were found to be 83%-97% after 10 runs, and 59%-80% after 30 runs in a day. The kinetic parameters were searched for free and Fe3O4- CS-A, Fe3O4-CS-EDAC-A, Fe3O4-CS-SC-A and calculated as 4,14; 2,41; 2,3; 2,31 mg/mL for Km, 2,57; 6,06; 5,52; 4,43 U/mg for Vmax respectively. The amount of bound enzyme was calculated as 478-488 mg enzyme/g support. It was shown that Langmuir model is well suited for the adsorption of ?-amylase onto Fe3O4-CS nanoparticles between 25°C45°C and the maximum adsorption capacity were calculated as 488 mg/g at 25°C and pH= 6,0. Thermodynamics of adsorption were indicated that adsorption process is spontaneous. Adsorption kinetic of ?-amylase onto Fe3O4-CS was searched and the experimental data fitted well the pseudo-first-order kinetic. Maximum desorption ratio of 65% was obtained over one adsorption/desorption cycle.Key Words : Magnetic nanoparticles, chitosan, ?-amylase, enzyme immobilization, adsorption

Abstract:
In this study, Fe3O4 nanoparticles were coated with chitosan (CS) by reversed-phase suspension method. ?-Amylase from Bacillus subtilis was immobilized onto chitosan coated Fe3O4 (Fe3O4-CS) nanoparticles by adsorption method and by covalent binding onto Fe3O4-CS nanoparticles which were activated by N-(3-dimethylaminopropyl)-N'- ethyl-carbodiimide hydrochloride (EDAC) and cyanuric chloride (SC). The effect of pH, temperature, substrate concentration, storage time on the activities of free and immobilized ?-amylases (Fe3O4-CS-A, Fe3O4- CS-EDAC-A, Fe3O4-CS-SC-A) were investigated. The optimum pH and optimum temperature were determined as 6,9; 8,0; 6,5; 6,0 and as 40°C, 30°C, 40°C ve 40°C for free ?-amylase, Fe3O4-CS-A, Fe3O4-CS-EDAC-A, Fe3O4-CS-SC-A respectively. Retained activities for immobilized systems were found to be 83%-97% after 10 runs, and 59%-80% after 30 runs in a day. The kinetic parameters were searched for free and Fe3O4- CS-A, Fe3O4-CS-EDAC-A, Fe3O4-CS-SC-A and calculated as 4,14; 2,41 2,3; 2,31 mg/mL for Km, 2,57; 6,06; 5,52; 4,43 U/mg for Vmax respectively.Key Words : Magnetic nanoparticles, chitosan, ?-amylase, enzyme immobilization, adsorption