Immobilization improves enzyme stability, allows easy enzyme separation from reaction mixtures, and enables repeatable use over prolonged periods, especially in systems requiring continuous chemical reactions.
Enzyme-functionalized solution-blown nonwoven (EFSBN) fibers were produced by a single-step solution blow spinning method that utilizes high-velocity gas to simultaneously extrude the codissolved polymer−solvent−enzyme spinning solution and evaporate solvent at mild conditions to form a nanofibrous web with preserved enzyme activity. A broad concentration range of 0.6−7.4 wt % protein of subtilisin A protease from Bacillus licheniformis was successfully entrapped by poly(ethylene oxide) (PEO) during solution blow spinning nonwoven web production. The presence of enzyme protein in the solid nanofibers was detected by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Time of flight-secondary ion mass spectroscopy and laser confocal microscopy revealed the immobilized enzymes were mainly positioned inside the fibers and homogeneously distributed throughout the webs. Scanning electron microscopy showed that the fiber shape and diameter of PEO nanofibers containing enzymes were irregular compared to PEO-only nanofibers. Residual enzyme activity in the webs was measured by redissolving fibers in buffer and comparing the released enzyme activity to nonimmobilized free enzyme using a casein substrate-based assay. Immobilized protease (1.3% (w/w) protein in solid dry nanofiber webs) retained more than 90% of the free enzyme activity. Protease immobilized in solid nanofiber webs exhibited long storage stability at ambient (∼22 °C) and 4 °C temperature storage conditions, with more than 60% remaining catalytic activity after 300 days compared to the initial activity. Immobilized protease had equally good thermal stability as a stabilized liquid commercial protease, both retaining above 95% of their initial activity after treatment for 12 h at 65 °C. In contrast, the same liquid protease diluted in buffer lost activity within 2 h at that temperature. The nondusting, readily aqueoussoluble EFSBN solid materials are easy to handle and have good storage stability compared to liquid products.
Polymers in nanofibrous forms offer new opportunities for achieving triggered polymer degradation, which is important for functional and environmental reasons. The polycaprolactone (PCL) nanofibrous nonwoven polymer webs developed in this work by solution blow spinning with entrapped enzymes were completely, rapidly and controllably degraded when triggered by exposure to water. Lipase (CALB) from Candida antarctica was successfully entrapped in the PCL webs via an enzyme-compatible water-in-oil emulsion in the PCL–chloroform spinning solution with added surfactant. Protein (enzyme) in the nanofibrous webs was detected by Fourier Transform Infrared Spectroscopy (FTIR), while time of flight-secondary ion mass spectroscopy (ToF-SIMS) and laser confocal microscopy indicated that enzymes were immobilized within solid fibers as well as within microbead structures distributed throughout the webs. Degradation studies of CALB-enzyme functionalized solution-blown nonwoven (EFSBN)-PCL webs at 40 °C or ambient temperature showed that EFSBN-PCL webs degraded rapidly when exposed to aqueous pH 8 buffer. Scanning electron microscopy (SEM) images of partially degraded webs showed that thinner fibers disappeared first, thus, controlling fiber dimensions could control degradation rates. Rapid degradation was attributed to the combination of nanofibrous web structure and the distribution of enzymes throughout the webs. CALB immobilized in the solid dry webs exhibited long storage stability at room temperature or when refrigerated, with around 60% catalytic activity being retained after 120 days compared to the initial activity. Dry storage stability at ambient conditions and rapid degradation upon exposure to water demonstrated that EFSBN-PCL could be used as fibers or binders in degradable textile or paper products, as components in packaging, for tissue engineering and for controlled-release drug or controlled-release industrial and consumer product applications.
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