(108a) High Temperature Biocatalyst Immobilization On Nanofibrous Supports By Reactive Electrospinning

Enzymes are highly efficient selective biological catalysts and hyperthermophilic enzymes are of particular interest due to their ability to function at elevated temperatures intrinsic to many industrial processes. In this work, we immobilize a model hyperthermophilic enzyme, α-galactosidase from Thermotoga maritima, on electrospun nanofibrous supports for potential use in a variety of applications, ranging from increasing nutritive value of animal feed to converting B-type blood to O-type blood, the universal blood type.  Immobilization offers inherent advantages: increased enzyme stability, ease of separation of the products so the enzyme can be reused, and lack of product contamination. However, immobilization can also impact the activity of the enzyme.  In all cases, the structure of the support material greatly affects the performance of the immobilized enzyme.  Fibers have high specific surface area, which increases with decreasing fiber diameter.  This makes nanofibers especially promising for enzyme immobilization.  Electrospinning is a common technique used to produce nanofibers in which high voltage is used to induce the formation of a liquid jet from a polymer solution. We have electrospun aqueous solutions of polyvinyl alcohol (PVA) and T. maritima α-galactosidase with glutaraldehyde, a chemical crosslinking agent, to generate water-insoluble, enzyme-loaded nanofibers of approximately 200 nm in diameter in a single reactive electrospinning process.  Using reactive crosslinking eliminates a post-electrospinning crosslinking step, thus accelerating the immobilization method by about 7-fold. After electrospinning, the fibrous structure remains intact when soaked in water and no enzyme leaching is observed. Most importantly, the enzyme retains catalytic activity and its hyperthermophilic characteristics (optimal activity between 90 and 95°C)  with  a two-fold increase in thermal stability following the reactive electrospinning process.  While the enzyme activity of the immobilized enzyme was about 5-fold lower than the free enzyme, the retained activity was significantly higher than following post-electrospinning treatment using a non-solvent or vapor phase crosslinking technique. We have determined that the presence of the enzyme can interfere with the PVA crosslinking, such that a maximum level of enzyme loading exists.  The effects of enzyme deactivation during crosslinking, as well as intra-fiber and inter-fiber diffusion limitations will also be discussed.