Immobilization of β-Galactosidase From Aspergillus oryzae on Electrospun Gelatin Nanofiber Mats for the Production of Galactooligosaccharides

Two simple and easily reproducible methods for the immobilization of β-galactosidase (β-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. β-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 °C, β-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 β-gal concentration of 0.027 wt.% in the electrospinning solution. The pH and temperature optima of immobilized β-gal shifted from 30 °C, pH 4.5 (free enzyme) to pH 3.5, 50 °C (covalent immobilization) and pH 3.5, 40 °C (suspension electrospinning). Striking differences in the Michaelis constant (K(M)) of immobilized β-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 β-gal was found to be 27.7% and 31% for immobilized β-gal.