Protease Immobilization in Solution-Blown Poly(ethylene oxide) Nanofibrous Nonwoven Webs

Abstract

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.

Developing Enzyme Immobilization with Fibrous Membranes: Longevity and Characterization Considerations

“…Tyler et al demonstrated the use of triatomic Bi 3 + as the primary ion and a maximum autocorrelation factors (MAF) image-processing method for generating a clear contrast (boundaries confirmed by fluorescence microscopy) between two similar proteins (human serum albumin versus bovine serum albumin) and two very different proteins (human serum albumin versus hemoglobin) immobilized on non-flat polystyrene micro-bead surfaces [ 269 ]. This demonstration of the technique on complex-shaped surfaces instead of regular flat substrates, such as silicon wafers, indicates that TOF-SIMS can also be applied to study the distribution of enzymes immobilized on fibrous supports, as was carried out with protease incorporated in poly(ethyleneoxide) solution, blown, nonwoven webs [ 270 ]. The technical parameters of different surface characterization techniques are compared in Table 7 .…”

Controllable Water-Triggered Degradation of PCL Solution-Blown Nanofibrous Webs Made Possible by Lipase Enzyme Entrapment

“…Solution-blown nonwoven webs were produced using the same instrument and method described in our previous work [27]. PCL-based spinning solutions were prepared by dissolving PCL pellets (1.5 g) in AOT surfactant-loaded (1 mg AOT/mL solvent) chloroform (10 mL) with continuous (2 h) stirring (250 rpm).…”

“…The protein (enzyme) content in EFSBN-PCL was determined by modifying Lowry assay method described in our previous work [27]. A Thermo Scientific Pierc Rapid Gold Bicinchoninic Acid (BCA) Protein Assay Kit was used for this analy where two molecules of BCA combine with one cuprous ion to form an orange-gold lution with an absorbance intensity at 480 nm that correlates to protein concentrat EFSBN-PCL webs were first incubated at 40 °C in tris buffer (pH 8.0, 0.1 M) for 120 m to degrade the webs entirely.…”

“…The pressurized air stream causes the central polymer jet to stretch and bend while solvent evaporates which solidifies the polymer into nanofibrous nonwoven webs that can be collected on any form of collector [25,26]. An illustration and mechanism of a single needle solution blow spinning unit is shown in our previous work [27]. SBS produces fiber morphology that is similar to electrospinning [28,29], but at a much larger production rate and without high voltage requirements [30,31].…”