Trypsin was successfully entrapped in situ into nanofibers of poly(E-caprolactone) (PCL) prepared by electrospinning. The spinning dope was an emulsion consisting of an aqueous phase with the solubilized enzyme in a pH buffer plus an oil phase of the polymer solubilized in chloroform (CF)/dimethylformamide (DMF). The optimized materials were composed by random arrays of bead-free fibers with outer diameters in the range 110-180 nm without showing core-shell structure. The fiber size and morphology, membrane porosity and surface properties were shown to be influenced by the polymer concentration and the composition ratio of the solvent mixture, and also by the presence of the enzyme. The activity of the immobilized trypsin was studied toward both a low-molecular weight synthetic substrate (BAPNA) and a protein (casein). Fluorescence microscopy, the increasing hydrophilicity of the fibrous membrane and the observed catalytic activity confirmed the entrapment of the enzyme into the PCL nanofibers. The best activity retention (similar to 66% toward BAPNA) was achieved using 0.20 glmL PCL in CF/DMF [75:25], with trypsin in an aqueous buffer at pH 7.1 in the presence of benzamidine and Span80. The immobilized enzyme showed satisfactory operational stability retaining similar to 59% of its initial activity after five reaction cycles. Compared with the free enzyme, the storage (at 4 degrees C) and thermal stability of the immobilized enzyme were highly improved. The retained catalytic activity and the observed reusability can be explained by a heterogeneous distribution of the enzyme within the polymer fiber influenced by the electrostatic field during the electrospinning process, enabling a preferential location near the fiber surface but simultaneously assuring minimal leaching out during operations. Results suggest that trypsin-PCL fibrous membranes may be useful for concomitant proteolytic and separation commercial applications. (C) 2015 Elsevier Inc. All rights reserved.