(582d) Enabling Multi-Enzyme Biocatalysis Using Coaxial Electrospun Hollow Nanofibers: Design and Applications

Highly efficient immobilization of multi-enzyme systems involving cofactor regeneration represents one of the greatest challenges in bioprocessing. Multi-enzyme systems that have been reported so far are almost exclusively in discrete spherical capsule formations with single or multicompartments, with a few of examples using porous supports or nanoparticles, which have limitations in some significant applications owing to high diffusional resistance, poor enzyme stability, and usually complicated preparation process. To breakthrough these limitations, the present work proposed a novel hollow nanofiber supported multi-enzyme system through co-axial electrospinning technology. By choosing bio-friendly solution with dissolved multienzymes as core solution and immiscible solvent with dissolved polymer as shell solution of co-axial electrospinning, an in-situ encapsulation of multi-enzyme inside the nano chamber of hollow nanofibers were realized. Compared to the existing multi-enzyme system in the form of particulates, novel hollow nanofiber supported multi-enzyme system is expected to offer several unique advantages: i.e., the woven-membrane formation of electrospun nanofibers enables an easily recycling and operation, facilitating molecular interactions between enzymes and possibly shared cofactor, as well as enhancing stability due to confining multi-enzyme system inside the nano-scale compartments.

To demonstrate the feasibility and advantages of the hollow nanofiber supported artificial cells, two multi-enzymatic systems were investigated. The first one is for bile acid assay, which includes 3α-hydroxysteroid dehydrogenase (3α-HSD), diaphorase (DP) and NAD(H), all of which were encapsulated in-situ inside chamber of hollow nanofiber via co-axial electrospinning. The activity recovery of immobilized 3-α-HSD and diaphorase reached 76% and 82%, respectively. When this nanofiber-supported multi-enzyme system was used for bile acid assay, a good linearity was obtained by varying bile acid concentrations within 0-200 μM, and this linear range was slightly broader than that obtained by using free multi-enzyme system (0-150μM). Investigation on storage stability of the multi-enzyme system showed that its half-life at 4^oC and 25^oC reached 160 days and 80 days, respectively. The second example is the fabrication of “ready-to-use” glucose test strip with glucose oxidase and horseradish peroxidase encapsulated inside the chamber and chromogenic agents embedded simultaneously in the shell of hollow nanofibers by co-axial electrospinning. The testing strip could be used either as colorimetric sensors in solution or as optical biosensor that can be operated in the “dip-and-read” mode. Both application modes yield a high sensitivity and broad testing range. The nano-confining effect of hollow nanofiber also provided the immobilized multi-enzyme system unique stability mechanism.