A novel pullulanase partially purified from Fontibacillus sp. was covalently immobilized on FlorisilA (R) and nano-silica through both glutaraldehyde and (3-glycidyloxypropyl)trimethoxysilane spacer arms. The pullulanase immobilized on FlorisilA (R) and nano-silica through glutaraldehyde spacer arm showed 85 and 190 % activity of its free form, respectively, whereas no activity was observed when it was immobilized on the same supports through (3-glycidyloxypropyl)trimethoxysilane spacer arm. The maximum working pHs of both the immobilized pullulanases on FlorisilA (R) and nano-silica through glutaraldehyde spacer arm were determined as 5.0; however, the maximum working pH of the free pullulanase was pH 6.0. The maximum temperatures of all the pullulanase preparations were determined as 35 A degrees C. The apparent K (m) values were 1.49, 1.54, and 0.59 mg/mL pullunan, respectively, for the free and immobilized pullulanases on FlorisilA (R) and nano-silica. The corresponding apparent V (max) values were 0.59, 1.53, and 1.57 U mg prot.(-1) min.(-1). Thermal stability of pullulanases immobilized on FlorisilA (R) and nano-silica was enhanced 6.5- and 15.6-folds, respectively at 35 A degrees C and 6.6- and 16.0-folds, respectively, at 50 A degrees C. The pullulanases immobilized on FlorisilA (R) and nano-silica protected 71 and 90 % of their initial activities after 10 reuses.