Preparation of electrospun polyurethane nanofibers for immobilization of peroxidases

HAURE P.; CARACCIOLO P; MORALES URREA, D. A.; CONTRERAS, E. M.

Mar del Plata

Simposio; XVI Simposio Latinoamericano de Polímeros SLAP 2018 ? Argentina; 2018

The enzyme peroxidase is well known for its capacity to decolorize textile effluents in the presence of hydrogen peroxide. The immobilization of enzymes shows great perspective due to the possibility of recycle and improved stability. However, the activity of the enzyme is affected by the structure of the supporting material. Electrospinning produces sub-micron polymer fibers that form membranes with extremely high surface areas on which enzymes can be immobilized covalently, thus increasing the catalytic activity of the biocatalyst. In this study, polyurethane (PUR) nanofibers produced via electrospinning were used for the immobilization of a commercial horseradish peroxidase (HRP) to catalyse the decolorization of orange II (OII) with hydrogen peroxide (P). Electrospun scaffolds were produced from commercial polyurethane Avalon 65 DB (PUR). The preparation of PUR nanofibrous membrane (PURM) was performed with solutions containing 12.5% w/v N,N-dimethylformamide (DMF)/ tetrahydrofuran (THF) 50/50, using a flow rate of 0.9 mL/h, a nozzle to collector distance of 15 cm and a voltage of 15 kV. The morphology of PURM was observed with a field emission scanning electron microscope.HRP was immobilized on PURM (HRP-PURM) according to the technique of Caracciolo et al. The presence of enzyme in the membrane was verified immersing the HRP-PURM in a toluidine blue solution (pH 10). The effects of washing with NaCl 2 M and temperature during HRP immobilization were analyzed. The decolorization activity of HRP-PURM was determined by using OII as the reducing substrate. Decolorization assays were performed in 3 mL quartz spectrophotometric cells at room temperature (20 ± 2 oC). During the experiments, one or two pieces of HRP-PURM (1 cm2) were immersed into 2.2 mL assay mixture consisting of 2 mL phosphate buffer (100 mM, pH 8), 0.1 mL OII (1 mM) and 0.1 mL P (2 mM). OII decolorization was monitored by the change of absorbance at 485 nm. Obtained PURM fibers were uniform. Mean fiber diameter resulted lower than 1 μm. Toluidine assay suggested that HRP had been successfully immobilized onto the nanofibers. In preliminary assays no change in absorbance at 485 nm was obtained. This result suggested the inactivation of HRP during its immobilization and that the developed membrane did not adsorb OII under the tested conditions. Studies performed at 40 °C during 60 h showed that the temperature affect the activity of HRP. Also, the washing of the membrane with NaCl 2 M strongly inhibited HRP. Therefore, a new HRP-PURM was made avoiding the washing step with NaCl (HPR-PURMnew). Additionally, the effect of the immobilization time was optimized. Under the optimized conditions, immobilization of HRP of 30% at 60 h was obtained (Fig. 6). HPR-PURMnew showed an initial specific decolorization rate (q0) between 2.1x10-5 and 3.3x10-5 u.a/min/mm3. Moreover, when the HPR-PURMnew was withdrawn from the solution, the decolorization stopped. This result demonstrated that the enzyme did not leach to the solution.