In order to get the best values of the binding parameters from the experimental results, Equation 1 can be rearranged as below:

According to experimental results, plots of q vs. Ce and 1/q vs. 1/Ce are shown in Figures 1 and 2.

It is clearly observed that the adsorption of Cu(II) increased significantly with the rising concentration from 1.88 mmol/L to 327.79 mmol/L and gradually leveled off. The initial increase of Cu(II) adsorption might be due to many available chelating hydroxyl groups and carbonyl groups in PVA/PA6 composite nanofibers, then reached saturation gradually with the increase in the concentration of Cu(II).

The adsorption equilibrium data could be interpreted by the Langmuir absorption equation. The basic assumption of the Langmuir theory is once a metal ion occupies a reaction site, then no further adsorption occurs at the same location [15]. The reaction kinetics parameters could be calculated through straight line slope and ordinate intercept of plots. The reaction rate constant (K_l) was 0.0593 L/mmol, and the maximum coordinate capacity (q_m) of PVA/PA6 composite nanofibers to Cu(II) was 3.731 mmol/g (dry fiber). Comparing with our previous work, PVA/PA6 composite nanofibers showed obviously higher adsorption capability for Cu²⁺ ions than that of PVA nanofibers [2].

The FTIR spectra of the PVA/PA6 composite nanofibers and the Cu(II)-PVA/PA6 metal chelated nanofibers are presented in Figure 3. The FTIR spectrum of the PVA/PA6 (Figure 3a) showed the following characteristic peaks: the bands of 3500 cm⁻¹–2800 cm⁻¹ correspond to the N–H, O–H and C–H stretching vibrations. The band at 1173 cm⁻¹ was assigned to the C–O stretching vibration in PVA, and the band at 1642 cm⁻¹ indicated the C=O stretching vibration in PA6.

The C-O band at 1173 cm⁻¹ (Figure 3a) moved to 1165 cm⁻¹ (Figure 3b), the C=O band at 1642 cm⁻¹ (Figure 3a) moved to 1630 cm⁻¹ (Figure 3b) due to the decrease of bond force constant coupled with decrease of the electron density, which indicated that the corresponding groups of PVA/PA6 coordinated with Cu(II). Moreover, the wide band of 3,287 cm⁻¹ (Figure 3a) corresponding to the O–H stretching vibration moved to 3,341 cm⁻¹ (Figure 3b) due to the increase of the relative mass of hydroxyl group, which was caused by the coordination of metal ions.

The SEM images of the original PVA/PA6 composite nanofibrous membranes are displayed in Figure 4a. The electrospun PVA/PA6 composite nanofibrous membranes formed a fibrous membrane with random orientations. The nanofibrous membranes looked very even and the average diameter of the electrospun nanofibers ranged between 90 nm and 110 nm. The diameter of composite nanofibrous membranes did not change substantially after the reaction with aqueous solution of Cu(II) ions for 24 h, and the fibrous structures was not obviously distorted, as revealed in Figure 4b. However, the constructions of PVA nanofibrous membranes showed obvious damages because of swelling after the reaction with aqueous solution of Cu(II) ions for 24 h. The stability of nanofibrous membranes was obviously improved when PA6 was added to the composite, and the nanofibrous structure of PVA/PA6 composite nanofibers was still maintained in the metal ions aqueous solution.

Maximum reaction rate V_max and Michaelis-Menten constants K_m are shown in Table 1. K_m of the immobilized catalases was higher than that of free catalases, and V_max of the immobilized catalases was smaller than that of free catalases.

These data indicated that affinity between immobilized catalases and substrate decreased comparing with free catalases. This might be affected by the structural changes of immobilized catalases, with less accessibility between substrate and active points of immobilized catalases caused by the space barriers presented by the supports [16,17].

The effect of pH on the activity of free and immobilized catalase is shown in Figure 5.

According to the experimental results, no significant shift of the optimal pH was observed. The optimal pH value was about 7.0 for free and immobilized enzyme, but the residual relative activity of the immobilized catalase was higher than that of the free one in the pH range between 5.0 and 8.5. It was found that the immobilized enzyme showed less sensitivity to pH than that of the free enzyme, probably because of the production of oxygen, forming foams, and causing slight diffusion limitation on the surface of nanofibrous membrane [18]. The effect of temperature on the activity of free and immobilized enzyme is depicted in Figure 6.

It is obvious that the initial relative activity was increased with the increase of temperature and decreased while the temperature was further increased for immobilized and free catalases. The optimum temperatures for the free and immobilized catalases were observed to lay at approximately 35 °C and 40 °C, respectively. At higher temperature range, the immobilized catalase exhibited higher stability than the free one, and it showed the anti-thermal properties of the immobilized enzyme. The multipoint interactions between enzyme and Cu(II)-PVA/PA6 support might reduce the degree of freedom of the spatial structure of enzyme, protecting it from deactivation at high temperatures.

The storage stability of immobilized and free enzyme is presented in Figure 7. The residual activity of immobilized and free catalase was 54% and 19% after 20 days, and the half-lives of free and immobilized catalase were 8 days and 24 days, respectively. The results indicated that the storage stability of immobilized catalases was better than that of free catalases, which might be attributed to the immobilization of enzyme to a matrix. The immobilization of enzyme could limit the freedom of conformational changes, resulting in increasing stability towards denaturalization [19].

According to the effect of repeated use on activity of immobilized catalase (Figure 8), after five repeated uses, immobilized catalases retained about 74% of their initial activity, and the residual activity of the immobilized enzyme was about 51% of the initial activity after 10 repeated uses, which showed its potential value in actual applications.

PA6 (weight-average molecular was 1800, characteristic viscosity was 2.8) and PVA (weight-average molecular was 84,000–89,000) obtained from Shanghai Kanghu Chemical (Shanghai, China). Bovine liver catalase (hydrogen peroxide oxidoreduction; EC. 1.11.1.6) were purchased from Sigma (Shanghai, China). Copper dichloride, hydrogen peroxide, formic acid and Coomassie Brilliant Blue (G250) for the Bradford protein assay were purchased from Shenyang Sinopharm Chemical Reagent (city, China). Hydrogen peroxide (30%) and the ingredients of phosphate buffer solution (PBS) such as NaCl, KCl, KH₂PO₄, K₂HPO₄ were analytical grade and used as received. Water used in all experiments was de-ionized.

Electrospinning was carried out to fabricate PVA/PA6 composite nanofibers. PVA and PA6 were dissolved in formic acid (of 88% purity) at room temperature with stirring. The mass ratios of PVA:PA6 were 4%:12% in blended solutions. PVA/PA6 blended solutions were placed in a syringe (content of 20 mL) equipped with a 0.7 mm diameter spinner jet, and the solution flow rate was controlled by a microinfusion pump (WZ-50C2, Zhejiang, China). The high-voltage supplier (DW-P503-4AC, Tianjin, China) was used to connect the metal needles and the grounded collector to form the electrostatic fields. The blended polymer solutions were electrospun at the positive voltage of 15 kV with a collecting distance of 14 cm (the distance between the syringe needle tip and the collection roller covered with the aluminum foil). Nanofibers were collected by the roller for 24 h. On the other hand, PVA nanofibers were also prepared with the same parameters (weight ratio of PVA in solution was 8%) for comparison.

PVA/PA6 composite nanofibers (0.05 g) were put in flasks, each of them containing various concentrations of copper dichloride solution (50 mL). The flasks were stirred at 20 °C for 24 h. The nanofibrous membranes with adsorbed Cu(II) were washed with de-ionized water. The amount of adsorbed Cu(II) was calculated by using the concentrations of the Cu(II) in the initial solution and in the resulting solution. The Cu(II) concentration was determined by atomic absorption spectrophotometry (AAS). The Cu(II)-PVA/PA6 nanofibrous membranes were dried in a vacuum drying oven, at a temperature of 40 °C for 24 h. The reaction kinetics parameters were studied by the Langmuir isothermal adsorption model:

where q_m is the maximum amount of the coordinated Cu(II), K_l is the binding constant, C_e is the equilibrium concentration. Fourier transform infrared spectroscopy (FTIR) spectra of the PVA/PA6 composite nanofibers and Cu(II)-PVA/PA6 nanofibers were recorded using a FTIR spectrometer (NICOLET NEXUS 470). Surface morphologies of Cu(II)-PVA/PA6 and Cu(II)-PVA nanofibers were examined using FE-SEM (S-4800). All samples were coated with the platinum by sputtering before scanning electron microscope observation.

Cu(II)-PVA/PA6 chelated nanofibrous membranes (coordinated Cu²⁺ ions = 3.03 mmol/g) were immersed in 50 mL of catalase solution (0.3 mg/mL) for 4 h at 20 °C in shakers while stirring continuously. Catalases were dissolved in 50 mM PBS (value of pH was 7.0). Then, the nanofibrous membranes were removed from the solution and rinsed with the same PBS until no soluble protein was detectable. The method of Bradford was applied to determine the concentration of enzyme, the amount of the catalase was determined by spectrophotometry according to the absorbance of Coomassie Brilliant Blue and catalases at 595 nm, and the amount of the bound enzyme was calculated as:

where Q is the amount of catalases bound onto unit mass of nanofibrous membranes (mg/g), C₀ and C are the initial and equilibrium enzyme concentrations in the solution (mg/mL), V is the volume of the catalases solution, and m is the mass of the nanofibrous membranes.

The activity of the free and immobilized catalases was determined spectrophotometrically by the measurement of decrease in the absorbance change of hydrogen peroxide at 240 nm. Immobilized catalases were mixed with hydrogen peroxide solution (100 mL, 50 mmol/L). The reaction was kept at 35 °C for 3 min, and the specific activity of enzyme was calculated by the following formula:

where v is specific activity of enzyme and immobilized enzyme (μmol/mg·min), ΔA is the absorbance decrease of the solution at 240 nm, V is the volume of the hydrogen peroxide solution (mL), T is the time of reaction (min), K is the molar extinction coefficient of hydrogen peroxide at 240 nm (K = 0.033 L/mmol cm), and E_w is the amount of enzyme (mg).

The catalytic mechanism of catalases is presented as follows [20]:

The effect of hydrogen peroxide concentration on the activity was tested. V_max and K_m values of free and immobilized catalases were calculated by the Lineweaver-Burk plots:

where K_m is the Michaelis constant, v and V_max represent the initial and maximal rate of the reaction, [S] is the concentration of the hydrogen peroxide. Kinetics parameters for immobilized and free enzyme were investigated at 35 °C and the concentrations of hydrogen peroxide ranged from 20 to 200 mmol/L (pH 7.0).

The immobilized and free enzyme were mixed with hydrogen peroxide solution (100 mL, 50 mmol/L) respectively, at temperatures ranging from 15 °C to 65 °C. The optimum pH values of free and immobilized enzyme were investigated at 35 °C for 3 min. PBS (pH 5.0–8.5) was used for pH dependence study.

The stability of free and immobilized catalases at storage was measured by calculating their activity retention during 20 days at 4 °C in 50 mM PBS (pH 7.0), using 2 days intervals, then a sample was removed and determined for the enzyme activity as described above. The half-lives of free and immobilized catalases were calculated by the following formulas.

where K_d is the decay constant, E₀ is the initial activity of enzyme, E_t is the residual activity of enzyme, t_1/2 is the half-life of enzyme. In addition, the reusability of immobilized catalases was studied. Immobilized catalases were reused 10 times within 10 days (once a day). After each reaction run, the enzyme was washed with PBS to remove any residual substrate, and the retention of activity of the immobilized enzyme was conducted under the optimum conditions.