The carboxyl-functionalized polystyrene (poly(styrene-co-methacrylic acid), PSMAA) nanofibers with average diameters of 250 ± 20 nm was prepared by electrospinning. PSMAA nanofibrous membrane were employed for immobilization of horseradish peroxidase (HRP) enzyme on the fibrous surface by a chemical method. The parameters about immobilizing HRP on the PSMAA nanofibers were studied and the influence on the activity of the HRP is discussed. This study showed that soap-free emulsion method is an ideal technology to modify the polystyrene surface and ultimately achieve enzyme immobilization on electrospun PSMAA nanofibers surfaces. Compared with free HRP, the acid-base stability, thermal stability, and storage stability of HRP were increased after the immobilization. The immobilized HRP maintained about 60% of its initial activity during a 20-day storage period. However, the free HRP maintained only 40% of its initial activity. The removal percentages of o-methoxyphenol (OMP) reached 80.2% after 120 min for immobilized HRP. These results suggest that the proposed scheme for immobilization of HRP has potential in industrial applications for the treatment of phenolic wastewater.
Horseradish peroxidase (HRP) has been shown to be able to remove a variety of phenols and aromatic amines from aqueous solutions [
Electrospinning, being a simple and economical way to immobilize enzymes
In preparations for enzyme membranes, it is difficult to get bulk active groups on the membrane surface. The traditional plasma surface physics modification [
In this paper, poly(styrene-
Chemical route for synthesis of styrene-co-methacrylic acid copolymer and subsequent electrospinning nanofibers attachment of HRP.
The materials were used without further purification. N,N-Dimethylformamide (DMF) and o-methoxyphenol were purchased from the Shanghai Chemical Co. (China). The ingredients of phosphate buffer solution (PBS), such as orthophosphoric acid, dibasic sodium phosphate, and potassium phosphate monobasic, were of analytical grade and used as received. Horseradish peroxidase (HRP), Coomassie Brilliant Blue (G250), bovine serum albumin (BSA, molecular mass: 67,000 Da), phenol, 4-aminoantipyrine, and pyrocatechol (1,2-dihydroxybenzene) were purchased from Sigma-Aldrich co., China. The water used in all experiments was prepared in a three-stage Millipore Milli-Q Plus 185 purification system (Richmond Scientific Ltd. Great Britain) and had a resistivity higher than 18.2 MΩ/cm.
The styrene (Sty) and methacrylic acid (MAA) monomers were purchased from the Lingfeng Chemical Co. (China). These chemicals were distilled under vacuum at 75°C before use.
A typical synthesis was as follows: first, both Sty and MAA were distilled under vacuum at 75°C. 25 mL Sty and 1.0 mL MAA were then mixed, and the mixture and 100 mL water were added into a three-mouth flask with condenser and mechanical stirrer. The mixture was stirred at a speed of 300 rpm and heated under a heater cover. After 5 min of boiling, 0.1 g potassium persulfate powder (Sinopharm Chemical Reagent Co., Ltd, China) as initiator was added from the side mouth and the polymerization continued for 1.5 hours. The whole reaction was completed within 1.5 hours, with a conversion in excess of 90% (measured by the method of [
A 15 wt.% PSMAA solution was prepared by adding 1.5 g PSMAA powder to 8.5 mL DMF at room temperature with magnetic stirring until it finally became a viscous precursor solution. The solution was quickly loaded into a 5 mL syringe equipped with a steel needle with a tip diameter of 0.5 mm, whose tip was filed flat, which was connected to a high-voltage supply capable of generating voltage up to 30 kV. A copper wire-framed drum collection screen was placed at a horizontal distance of 15 cm from the tip of the needle. The copper wire drum was connected to a motor with two pulleys and rotated at a speed of 300 rpm. The feeding rate of the precursor solution was controlled at 1 mL/h using an automatic syringe pump so that a small drop was maintained at the capillary tip due to the surface tension of the solution. The solution on the tip of the needle was ejected under a strong electric field of the 20 KV, and the PSMAA fibers thus formed were dried initially for 5 h at 70°C under vacuum.
An appropriate amount of electrospun PSMAA nanofibrous membranes were immersed in 50 mL of BSA solution (1% wt) for about 1.5 h at 20°C in shakers while stirring continuously and then thoroughly washed with deionized water to remove the residue BSA. Subsequently, the pretreated membranes were submerged into 20 mL of the HRP solution (1 mg/mL in the PBS, pH 7.0) in a 25 mL beaker and shaken gently in an ice bath for the required time. Finally, the membranes were taken out and washed with the PBS until no protein was detected in the washings.
The concentration of HRP in the solutions was determined by the method of Bradford [
The PSMAA nanofibers morphology was examined using (S-3000N, Hitachi, Co., Ltd, Japan) scanning electron microscope (SEM). All samples were sputter coated with gold. Fourier transform infrared (FTIR) spectra (BRUKER IFS66/S Perkin-Elmer) were recorded using pressed KBr pellets over the wavenumber range of 4000–500 cm−1 at a resolution of 4 cm−1 to determine the PS and PSMAA.
The Worthington protocol was followed to calculate the activity of the HRP enzymes [
HRP-PSMAA (1 cm × 1 cm, total weight 20 ± 1 mg) membrane pieces were added one each to 10 mL solutions with the concentration of OMP at 8.06 × 10−4 mol/L; the molar ratio of the reaction between OMP and H2O2 was about 1 : 1. The reaction scheme was suggested as shown in Scheme
The mixture was incubated at
FTIR spectra of (a) the PS and (b) the PSMAA are shown in Figure
FT-IR spectrum of PS and PSMAA.
A SEM image of the original PSMAA nanofibers is displayed in Figure
Morphologies of PSMAA nanofiber. (a) Original PSMAA nanofibers. (b) PSMAA nanofibers with immobilization of HRP.
To determine the optimum adsorption time, the effect of adsorption time on the immobilized amount of protein was studied. As shown in Figure
Effect of immobilization time on enzyme loading capacity at pH 7.0 and room temperature.
The effect of temperature on the activity of free and immobilized HRP at pH 7.0 is shown in Figure
Effect of temperature on the activity of free and immobilized HRP.
The storage stability of immobilized and free enzyme is presented in Figure
Storage stability of free and immobilized HRP.
Figure
Effect of pH on the activity of HRP.
Figure
Degradation kinetics of OMP by PSMAA, free HRP, and HRP-PSMAA.
PSMAA nanofibrous membranes with uniform fiber diameters between 230 and 270 nm were prepared by an electrospinning method. HRP enzyme was successfully immobilized on the MAA carboxyl groups of the PSMAA nanofibers. The results of degradation experiments indicated that a removal efficiency of 80.2% was achieved. Immobilized HRP showed a better acid-base stability, thermal stability, and storage stability than free HRP, which is a very attractive aspect for real applications involving a sufficiently wide range of external conditions.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors acknowledge the financial support of the National Natural Science Foundation (no. 21102033), the Open Research Fund of MOIDAT (no. 201205), the Open Research Fund of State Key Laboratory of Fine Chemicals (no. KF1012), the Open Project Program of Key Laboratory of Ecotextiles, Ministry of Education, Jiangnan University (no. KLET1205), and the Scientific Research Fund of Liaoning Provincial Education Department (no. L2011122).