Nanofibrous membranes for single-step immobilization of hyperthermophilic enzymes

We report a single-step method to immobilize hyperthermophilic enzymes within chemically crosslinked polyvinyl alcohol (PVA) nanofibrous membranes. The polymer crosslinking that entraps the enzyme within the fiber is not affected by the particular enzyme and can thus be applied to any enzyme. Using a reactive electrospinning process, the chemical crosslinking that occurs during processing effectively entraps the enzyme within the fiber preventing enzyme leaching at elevated temperature establishing that the system is sufficiently robust for immobilization of hyperthermophilic enzymes. Upon immobilization, the enzyme retains 20% of its catalytic activity as well as its hyperthermophilicity, as the maximum activity occurs at ~90°C, and that activity at 90°C is an order of magnitude higher than at 37°C. Furthermore, thermostability of the enzyme is enhanced upon immobilization as indicated by the 2-fold increase in half-life at 90°C and pH 5.5 which extends the use of these biocatalysts at high temperatures. Compared to alternative methods, the apparent activity using the single-step method is significantly higher than alternative two-step methods (4 orders of magnitude higher than non-solvent based crosslinking and 3-fold higher than vapor-phase crosslinking). Analysis of this immobilization method indicates that the apparent decrease in specific activity could be attributed to enzyme deactivation arising from the crosslinking reaction, whereas mass transfer limits the apparent activity using alternative two-step immobilization methods. Based on this understanding, enzyme activity upon immobilization may be improved by using enzymes with higher intrinsic stability. Since significant enzyme activity is observed upon immobilization and the stability under high temperatures is enhanced, this versatile approach leverages the unique properties of hyperthermophilc enzymes and electrospun nanofibers providing a platform to produce catalytically active nanofibrous membranes appropriate for high temperature processes. [Display omitted] •Entrapment of enzymes within electrospun nanofibers using in situ crosslinking.•Single-step method at least 6× faster than alternative 2-step methods.•Enzyme activity up to 4 orders of magnitude higher than alternative 2-step methods.•No observed intra-fiber diffusion limitations.•Nanofibrous membranes are sufficiently robust for high temperature biocatalysis.