Immobilization of beta-galactosidase in poly (acrylonitrile-co-methyl methacrylate) poly (AN-co-MMA) Nanofibers was studied by electrospinning, and a spacer-arm i.e., (Polyethyleneimine (PEI)) was covalently attached by the reaction of carbonyl groups of Poly (AN-co-MMA) nanofibers. beta-galactosidase was then covalently immobilized through the spacer-arm of the Poly (AN-co-MMA) nanofibers by using glutaraldehyde (GA) as a coupling agent. Nanofibers mode of interaction was proven by FTIR and thermal gravimetric analysis and supported by morphological changes recognized through SEM examination. Factors affecting the modification process such as PEI concentration, reaction time, and reaction temperature have been studied. Its influence on the amount of coupled PEI was consequently correlated to the changes of the catalytic activity and the retained activity of immobilized enzyme, the main parameters judging the success of the immobilization process. Evidences of Poly (AN-co-MMA) nanofibers modification were extracted from morphological changes recognized through SEM examination. The maximum activity (V-max) and michaelis constant (K-m) of immobilized enzyme were found to be 8.8 mu mole/min mg protein and 236.7 mM, respectively. Stabilities of the immobilized beta-galactosidase were obviously improved. The optimum temperature for beta-galactosidase immobilized on the spacer-arm attached nanofiber was 5 degrees C higher than that of the free enzyme and was also significantly broader. The immobilized beta-galactosidase had better resistance to temperature inactivation than did the free form. (C) 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013