Two simple and easily reproducible methods for the immobilization of beta-galactosidase (beta-gal) from Aspergillus oryzae on electrospun gelatin nanofiber mats (GFM) were developed. The process was optimized regarding the electrospinning solvent system and the subsequent cross-linking of GFM in order to increase their stability in water. beta-Gal was covalently immobilized on activated gelatin nanofiber mats with hexamethylenediamine (HMDA) as a bifunctional linker and secondly via entrapment into the gelatin nanofibers during the electrospinning process (suspension electrospinning). Optimal immobilization parameters for covalent immobilization were determined to be at pH 7.5, 40 degrees C, beta-gal concentration of 1 mg/mL and immobilization time of 24.5 h. For suspension electrospinning, the optimal immobilization parameters were identified at pH 4.5 and beta-gal concentration of 0.027 wt.% in the electrospinning solution. The pH and temperature optima of immobilized beta-gal shifted from 30 degrees C, pH 4.5 (free enzyme) to pH 3.5, 50 degrees C (covalent immobilization) and pH 3.5, 40 degrees C (suspension electrospinning). Striking differences in the Michaelis constant (K-M) of immobilized beta-gal compared with free enzyme were observed with a reduction of K-M up to 50% for immobilized enzyme. The maximum velocity (v(max)) of immobilization by suspension electrospinning was almost 20 times higher than that of covalent immobilization. The maximum GOS yield for free beta-gal was found to be 27.7% and 31% for immobilized beta-gal.