Abstract

Laccase was immobilized in polyvinyl alcohol beads containing halloysite nanotubes (PVA/HNTs) to improve the stability and reusability of enzyme. The porous structure of PVA/HNTs beads facilitates the entrapment of enzyme and prevents the leaching of immobilized laccase as well. Halloysite nanotubes act as bridge to connect the adjacent pores, facilitating the electron transfer and enhancing the mechanical properties. PVA/HNTs beads have high laccase immobilization capacity (237.02 mg/g) and activity recovery yield (79.15%), indicating it can be used as potential support for laccase immobilization. Compared with free laccase, the immobilized laccase on hybrid beads exhibits enhanced pH tolerance (even at pH 8.0), good thermal stability (57.5% of the initial activity can be maintained at 75 °C), and excellent storage stability (81.17% of enzyme activity could be retained after storage at 4 °C for 5 weeks compared with that for free enzyme of 60%). Also, the removal efficiency for reactive blue can reach as high as 93.41% in the presence of redox mediator 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate), in which adsorption and degradation exist simultaneously. The remarkable pH tolerance, thermal and storage stability, and reuse ability imply potential application of porous PVA/HNTs immobilized enzyme in environmental fields.

INTRODUCTION

In the past decades, the release of dye effluents from industries such as textile, paper and pulp, food, and cosmetics has become one of the major environmental concerns owing to their carcinogenic, genotoxic and/or mutagenic nature (Bilal et al. 2017). Employment of enzymes as biocatalysts offers one option for the current demands of sustainable green methodologies in industrial processes, due to their benefits of mild reaction conditions, biodegradability and catalytic efficiency (Köse et al. 2016). However, the utilization of free enzymes in industries to dispose of dye wastewater is usually hampered by their low stability in a broad working pH and temperature range, poor long-term stability, difficulty in recovery and recycling, and propensity to be inhibited by high concentrations of reaction components (Hernandez & Fernandez-Lafuente 2011). Immobilization is almost compulsory for most industrial uses to facilitate the recovery of the enzyme, which can expand the practical application potential of enzymes (Peirce et al. 2016; Piacentini et al. 2017). Immobilization may also be utilized to improve enzyme stability, for example, if the reaction is performed at alkaline pH value and the enzyme stability/activity is higher at acidic pH value (Rodrigues et al. 2013). For now, there are lots of solid activated support for enzyme immobilization, such as natural polymers, synthetic polymers, and inorganic materials (Datta et al. 2013). However, the catalytic behavior of immobilized enzymes strongly depends on the properties of their carriers, such as material types, structures, and compositions. At present, there is no universal support that can immobilize all types of enzymes for various applications (Mohamad et al. 2015). An ideal support should possess some adequate properties, relating to, for example, internal geometry, specific surface area, superficial activation degree, mechanical resistance, and pore diameter (Santos et al. 2015). Also, the multipoint or multisubunit attachment (ionic bonding, covalent bonding, entrapment/encapsulation, cross-linking. etc.) between the enzyme and support should be strong enough to avoid the leaching of absorbed enzyme from its support upon change in the reaction pH, ionic strength or temperature (Betancor & Luckarift 2008; Bergamasco et al. 2013; Guzik et al. 2014; Gyles et al. 2017).

Polyvinyl alcohol (PVA), which is a non-toxic and biologically friendly synthetic polymer with easy availability, low cost, and good chemical and thermal stability, has been widely used in biomedical applications (Park et al. 2001; Zajkoska et al. 2013; Piacentini et al. 2017). According to the previous reports, the porous PVA matrix in the form of beads or microspheres, fibers and films can be used as support for enzyme immobilization owing to their ordered uniform pore structure, large pore size, huge surface area, and good biocompatibility (Morelli et al. 2016; Zheng et al. 2016). However, PVA has poor water-soluble resistance and mechanical properties, leading to the leakage of cells and enzymes, thereby decreasing the enzyme activity and immobilization efficiency. Researchers find that the adding of inorganic additives such as carbon nanotubes, montmorillonite, or kaolin can enhance the mechanical properties and facilitate electron transfer (El-Mohdy & Ghanem 2009; Gong et al. 2010; Cheng et al. 2012; Shameli & Ameri 2017). Halloysite nanotubes (HNTs), which are a low-cost aluminosilicate clay mineral with tubular structure, are chemically similar to kaolin and formed by rolling of kaolin sheets during natural hydrothermal processes (Lvov et al. 2016). HNTs with ca. 50 nm diameter, 10 nm lumen and 1 μm length have great potential in removing cationic dyes from wastewater without modification or loading because they have chemical and mechanical stability, high adsorption capacity and biocompatibility (Zhao et al. 2013). HNTs can also be dispersed into a polymer matrix to make polymer/clay nanocomposites, which exhibit unexpected properties such as improved thermomechanical and barrier properties (Lvov et al. 2016). Furthermore, HNTs have been investigated as support for enzyme immobilization owing to the oppositely charged inner and outer surfaces (Kumar-Krishnan et al. 2016; Tully et al. 2016). Integration of HNTs into porous PVA beads can facilitate the transport of the dyes toward both the surface and the interior. It could also provide certain advantages for immobilizing enzymes in terms of reusability, biocompatibility, mechanical strength and open spaces within the matrix (Rawtani et al. 2012; Tully et al. 2016; Gaaz et al. 2017).

However, it has been reported that the diffusion resistance between non-porous PVA/clay and enzyme is very high, leading to low enzyme immobilization capacity (Cheng et al. 2012). Meanwhile, the weak binding between the enzyme and non-porous PVA/clay has a disadvantage in that the absorbed enzyme might leach from its support upon a change in the reaction environment. To overcome these limitations, the cross-linking of PVA/clay with a crosslinker containing two or more functional groups to act as a bridge between enzyme and support to enhance the electron transfer is necessary (El-Mohdy & Ghanem 2009; Gong et al. 2010; Cheng et al. 2012). Glutaraldehyde is a widely used cross-linker containing two aldehyde groups, which can react with amino groups on an enzyme to form a Schiff base (RC¼N). After cross-linking, the connection between the enzyme and substrate is firm, giving good stability and reusability (Shameli & Ameri 2017).

Therefore, we prepared porous PVA/HNTs beads by phase separation method with PVP/acetyldimethylamine as pore formation agent, followed by cross-linking with glutaraldehyde. Their morphology analysis and thermal decomposition behavior were characterized using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy, which was followed by immobilization of laccase onto PVA/HNTs, investigation of enzymology properties of the immobilized laccase, and biodegradation of reactive blue from aqueous solution. The schematic illustration of PVA/HNTs formation is shown in Figure 1. The immobilized laccase was developed as an alternative for the biodegradation of reactive blue since the free laccase has low efficiency and poor operational stability. The cross-linking of PVA/HNTs beads with glutaraldehyde can increase the loading capacity and improve the binding between laccase and PVA/HNTs beads. The addition of HNTs to beads can not only increase the adsorption of reactive blue to the beads, but also enhance the physiological stability and mechanical strength of the beads. The mechanism in the degradation of reactive blue by laccase immobilized on PVA/HNTs beads may be divided into two phases: the adsorption of reactive blue on the PVA/HNTs beads followed by the degradation of reactive blue by the immobilized laccase on the beads.

Figure 1

Schematic illustration of PVA/HNTs beads formation.

Figure 1

Schematic illustration of PVA/HNTs beads formation.

MATERIAL AND METHODS

Materials

The reactive blue used in this study was obtained from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). Halloysite was milled and sieved to obtain fine powder (Henan, China). Laccase (EC 1.10.3.2, 0.1 U/mg) from Aspergillus species and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were purchased from Sigma. Coomassie brilliant blue G-250 and bovine serum albumin were purchased from Solarbio Company (Beijing, China). PVA and acetone were purchased from Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd. Polyvinylpyrrolidone (PVP) and acetyldimethylamine (DMAC) were purchased from Sinopharm Chemical Reagent Co., Ltd. All of the chemicals were analytical grade and used without purification, which means the effect of the impurities in the chemicals can be ignored.

Characterization

The microstructures of the samples were observed using SEM (JSM-7500F). The FTIR spectra of the samples were measured by an FTIR spectrometer (IR300, Nocolet) to determine the surface functional groups. A Shimadzu UV–visible spectrophotometer (UV-2450, Shimadzu) was used to analyze the activity of enzyme and the concentrations of reactive blue at λmax of 420 nm (Bradford 1976) and 592 nm (Peralta-Zamora et al. 2003), respectively.

Preparation of porous PVA/HNTs beads

The procedure for porous PVA/HNTs beads was as follows (Khanna et al. 2005; Lin et al. 2013). (1) HNTs were dispersed in distilled water under ultrasound to form a homogeneous solution (2 wt%) and PVA was dissolved in deionized water (10 wt%) and rested for 30 minutes to eliminate the bubbles. (2) The same amounts (15 ml) of the HNTs solution and PVA solution were mixed uniformly under vigorous stirring to ensure the successful coating of PVA on HNTs. (3) PVP (40 mg) was dissolved in 15 ml of DMAC solution and magnetically stirred for 30 min. (4) The PVP/DMAC solution was poured into PVA/HNTs solution and treated by ultrasound to form a homogeneous mixture. (5) This mixture was dropped into acetone drop by drop to form PVA/HNTs beads. (6) After soaking for 8 h, the beads were filtered and cross-linked by glutaraldehyde solution in 10 mM citric acid/Na2HPO4 buffer at pH 5 and 25 °C for 1 h, under mild stirring. The suspension was then filtered and washed with 10 mM citric acid/Na2HPO4 buffer at pH 5 to remove the excess of glutaraldehyde. Finally, the separated product was washed to neutral with distilled water to remove the nonspecific-adsorbed glutaraldehyde and dried at 25 °C in vacuum for 24 h for further enzyme immobilization.

Enzyme immobilization

In a typical experiment, the immobilization of laccase was carried out by incubating 1,000 mg of porous PVA/HNTs beads in the citric acid/Na2HPO4 buffer (100 ml, 0.1 M, pH 5) containing 2 mg/ml laccase at 4 °C for 4.5 h, followed by washing three times with citric acid/Na2HPO4 buffer to remove free laccase. Then the immobilized enzyme was collected and stored at 4 °C for subsequent enzyme assays. The supernatant was also collected and used to measure the concentration of the residual enzyme. According to our previous work (Chao et al. 2014), the enzyme loading was calculated as the difference between the total enzyme used and the residual enzyme in the supernatant after immobilization. As one of the most widely used reagents in the design of biocatalysts, glutaraldehyde could activate an enzyme support and stabilize the immobilized enzyme in a polymeric form resulting from aldol condensation (Monsan 1978). It is a powerful cross-linker, which is able to react with itself and can improve stability or inhibit distortion of the enzyme conformation (Barbosa et al. 2014; Rueda et al. 2016). We chose glutaraldehyde as a cross-linker in this study.

Determination of enzyme activity

The activity of free and immobilized laccase was determined at 30 °C using ABTS as a color-generating substrate according to a previous work (Bourbonnais & Paice 1990), where the rate of color formation was proportional to enzyme activity. The reaction mixture contained 1 mM ABTS, 100 mM citric acid/Na2HPO4 buffer (optimum pH for free and immobilized laccase) and a suitable amount of free and immobilized laccase, as reported in our previous work (Chao et al. 2013). The enzyme activity assay was performed in triplicate, and the standard deviations in measurements were consistently below 3%.

Removal of reactive blue

All degradation tests were performed by adding immobilized laccase to 50 ml reactive blue solution with known initial concentration. Removal efficiency (R) was measured by recording the remaining concentration of reactive blue using a spectrophotometer (UV-2450, Shimadzu) since reactive blue has a maximum characteristic absorption peak at 592 nm, and calculated with the following equation:
formula
(1)
where C0 and C are the initial and final reactive blue concentrations (mg/ml) in the solution, respectively. All the measurements were repeated for three times with errors less than 1%.

RESULTS AND DISCUSSION

Characterization

Figure 2 shows the SEM and transmission electron microscopy (TEM) images of the HNTs. It can be seen from Figure 2(a) that HNTs have regular tubular shape with diameter of 50–70 nm and length of ca. 800 nm. Figure 2(b) further shows the inner diameter of HNTs is 15–20 nm. Figure 3 depicts a photograph and the inner morphology of the PVA/HNTs beads. It can be clearly seen that the beads have regular spherical shape with diameter of 0.1–0.2 mm (Figure 3(a)). There are plenty of large open pores with diameter of 15–20 μm internally (Figure 3(b)). Meanwhile, a more porous structure with diameter 0.5–1 μm can be observed after further magnifying the walls of the large pores (Figure 3(c) and 3(d)). These macro- and micro-pores have the ability to entrap the enzyme, thus enhancing the enzyme loading capacity (Gutiérrez et al. 2007). In addition, it is observed from Figure 3(d) that HNTs are dispersed uniformly in the PVA matrix, which acts as bridge to connect the adjacent pores, which greatly facilitates the electron transfer and enhances the mechanical properties.

Figure 2

SEM and TEM images of HNTs.

Figure 2

SEM and TEM images of HNTs.

Figure 3

(a) Photograph of PVA/HNTs beads and (b)–(d) SEM images of the PVA/HNTs beads.

Figure 3

(a) Photograph of PVA/HNTs beads and (b)–(d) SEM images of the PVA/HNTs beads.

The FTIR spectra of HNTs, PVA, laccase, PVA/HNTs, and laccase/PVA/HNTs are presented in Figure 4. In the FTIR spectra of HNTs, absorption bands at 3,696, 3,621, and 3,484 cm−1 are ascribed to the stretching vibrations of hydroxyl groups. The band at 1,629 cm−1 is attributed to the deformation vibration of interlayer water. The other peaks observed at 1,100–500 cm−1 are caused by the vibrations of Al-O-Si, Si-O, and Al-O. For PVA, the peaks at 3,280, 2,909, and 1,413 cm−1 are ascribed to the stretching vibrations of -OH groups, asymmetric stretching vibration of -CH2 groups, and bending vibration of -CH2 groups. In the spectra of porous PVA/HNTs beads, the peaks appearing at 2,938 and 1,430 cm−1 are caused by stretching vibration and bending vibration of -CH2 groups, which means PVA and HNTs combined completely. The absorption band at 2,867 cm−1 is associated with the symmetric vibration of the -CHO group on glutaraldehyde, which indicates that the surface of hybrid beads has been successfully modified by glutaraldehyde. However, after immobilization of laccase on the PVA/HNTs beads, the peaks of the -CHO group (2,867 cm−1) disappear, and the adsorption of the -NH stretching vibration (3,300–3,000 cm−1), amide I stretching vibration (near 1,650 cm−1) and amide II stretching vibration (near 1,550 cm−1) of proteins (Tripathi et al. 2009; Jin et al. 2001) are increased to different extents. These results suggest that the interaction between glutaraldehyde and laccase changes the stretching vibration of both the -NH2 group of enzyme and -CHO group of glutaraldehyde. The enzyme may be loaded on hybrid beads through adsorption and covalent attachment.

Figure 4

FTIR spectra of HNTs, PVA, PVA/HNTs, laccase and immobilized laccase on PVA/HNTs.

Figure 4

FTIR spectra of HNTs, PVA, PVA/HNTs, laccase and immobilized laccase on PVA/HNTs.

Enzymatic activity of the immobilized laccase

The immobilization capacity, specific activities and recovery yield of the immobilized laccase on PVA/HNTs hybrid beads (at initial concentration of 4.0 mg/ml) are summarized and compared with free enzyme in Table 1. The laccase specific activity was measured as the oxidation of ABTS according to the method in the literature. (Bourbonnais & Paice 1990). The activities of free and immobilized laccase were obtained spectrophotometrically by generation of the ABTS radical. The relative enzymatic activity was related to a percentage of this highest activity (100% represents the highest enzymatic activity). The activity recovery was calculated from the value of the activity of the initial laccase solution divided by the activity value of immobilized laccase obtained immediately after the immobilization procedure. It can be seen that the hybrid beads have very high laccase immobilization capacity of 237.02 mg/g compared with HNTs/laccase of 11.30 mg/g (Chao et al. 2013). The high enzyme loading capacity of PVA/HNTs hybrid beads is because of the porous structure inside the support using DMAC as pore formation agent, which makes the enzyme molecules fully dispersed and results in less contact of immobilized enzyme with the external hydrophobic interface, such as gas bubbles (Bolivar et al. 2006). Compared with free enzyme, the laccase supported on PVA/HNTs hybrid beads has only a modest decrease of specific activity (Mateo et al. 2007) and recovery yield, indicating it can be used as potential support for enzyme immobilization.

Table 1

Comparison of free and PVA/HNTs hybrid beads immobilized laccase

LaccaseImmobilization capacity (mg/g)Specific activity (U/g)Recovery yield (%)
Free – 11.51 100 
Immobilized 237.02 9.11 79.15 
LaccaseImmobilization capacity (mg/g)Specific activity (U/g)Recovery yield (%)
Free – 11.51 100 
Immobilized 237.02 9.11 79.15 

Enzymatic properties of PVA/HNTs-immobilized laccase

The activity of laccase is known to be strongly dependent on pH, since the solution pH determines the level of electrostatic or molecular interaction between the loading surface and enzyme. As illustrated in Figure 5(a), the immobilized laccase shows higher activity compared with free laccase. The reason may be that the support acts as a ‘solid’ buffer, which generates a pH microenvironment inside the biocatalyst beads that could dramatically differ from that in the reaction medium (Jin et al. 2001). Note that the solution pH of both free and immobilized laccase was adjusted identically for each comparison test. The nest-like inner channels provide a favorable microenvironment for maintaining the enzyme activity at a higher level. Also, both the free and immobilized laccase show optimum activity at pH 3.2, and the free enzyme becomes inactivated above pH 6.0. However, immobilized laccase still has activity even at pH 8.0. The reason for this extended activity at pH 8 is still unclear and will be investigated in further study.

Figure 5

Activity of free and immobilized laccase at (a) different pH values, (b) different temperatures, (c) different times at 75 °C, (d) different storage times at 4 °C.

Figure 5

Activity of free and immobilized laccase at (a) different pH values, (b) different temperatures, (c) different times at 75 °C, (d) different storage times at 4 °C.

To investigate the thermal stability of the immobilized laccase, the activities of the free and immobilized laccase were studied at different temperatures of 15–85 °C (Figure 5(b)) and constant temperature of 75 °C in a buffer solution of pH 5 for varied incubation periods (Figure 5(c)). It can be observed that free laccase and immobilized laccase exhibit maximum activity at 35 °C and 55 °C, respectively. The immobilized laccase exhibits a significantly higher temperature tolerance and enhanced activity within the temperature range of 45–85 °C compared with those of free laccase, which means that the thermal tolerance of the enzyme could increase after immobilization. This tendency might be attributable to the reduction of enzyme mobility after multipoint covalent attachment on hybrid beads and the conformational changes caused by immobilization, which could prevent the dissociation of the subunits when the pH curves (Barth 2007; Hwang & Gu 2013; Rodrigues et al. 2013).

In addition, it can be seen from Figure 5(c) that the activity of the free laccase decreased much faster than that of the immobilized laccase when keeping at 75 °C. The free laccase lost almost all the activity in the first 45 min, whereas the immobilized laccase could remain at 57.5% of the initial activity after an incubation period of 2 h. This result proves that the support can effectively protect laccase and afford a stable microenvironment, which could resist the drastic changes of the external environment. The increased resistance to thermal deactivation offers a potential advantage in wastewater treatment applications. This is because a large number of pretreatment systems (such as heat treatment, adding dilute acid) are employed to remove impurities from wastewater prior to the main treatment process itself. The increased resistance of immobilized enzyme to thermal deactivation offers a potential advantage especially in the heat pretreatment process. On the other hand, foam is a dispersion of gas in a liquid, which is usually generated by stirring the solution. In order to reduce the foam, hot water treatment or using porous materials seems to be economical and efficient.

The storage stability was studied over a period of 5 weeks at 4 °C as shown in Figure 5(d), and it showed that the immobilized enzyme retained more than 81.17% of its initial activity after storage for 5 weeks, whereas an activity loss of more than 60% was observed in free enzyme. The high storage stabilities of immobilized enzymes show that immobilization of laccase on insoluble supports is a useful strategy to optimize their operational performance in large-scale industrial applications (Rodrigues et al. 2013; Zheng et al. 2016).

Dye removal by immobilized laccase

To evaluate the potential application of PVA/HNTs-immobilized laccase, reactive blue was chosen as a model dye to investigate the removal properties. In a typical experiment, the removal of reactive blue (200 mg/l, 50 ml) was conducted in citric acid/Na2HPO4 buffer (0.1 M, pH 5) at room temperature for a predetermined period of time (8 h). As shown in Figure 6(a), PVA/HNTs bead has removal efficiency of 16.3%, meaning that reactive blue can be adsorbed on PVA/HNTs. However, after laccase was immobilized onto PVA/HNTs, the removal efficiency for reactive blue can be improved up to 40.97%. The increase in removal efficiency suggests that both adsorption and degradation exist simultaneously in the process of reactive blue removal. When 0.3 mM redox mediator ABTS was added into the dye solution, the removal efficiency dramatically increased to 93.41%. The reason may that ABTS can facilitate the transfer of electrons from the substrates to the active site of laccase, thus increasing the removal efficiency of reactive blue. Laccase belongs to copper-containing oxidases that can catalyze the oxidation of electron-rich substrates such as phenols. Each monomer has four copper atoms with one distributed in each T1/T2 redox site and two in the T3 redox site (Metin 2013). It is supposed that catalysis by laccase firstly involves T1 Cu reduction by the substrate, followed by internal electron transfer from T1 Cu to T2 and T3 Cu, and finally, dioxygen reduction at T2 and T3 sites (Ba et al. 2013). The redox potential of laccases is in the range of 500–800 mV, which is lower than that of non-phenolic dyes (about 1,000 mV) (Fernández-Sánchez et al. 2002). So it is difficult to degrade non-phenolic compounds by laccase. When mediator is added into laccase system, the low redox potential of a small molecule mediator (such as ABTS, 680 mV) makes it possible to functionalize as a bridge for the transfer of electrons from laccase to substrates. With the inclusion of mediator, the substrate specificities of laccase become broader (Gianfreda et al. 1999; Camarero et al. 2005). This is because in the presence of ABTS as a redox mediator, laccase reacts with non-phenolic substrates, which are more difficult to oxidize, such as benzyl alcohols. ABTS was first adsorbed physically around the copper cluster of immobilized laccase, and then changed into ABTS2+ after electron transfer reaction happened. So the oxidation-reduction could easily take place between obtained active ABTS2+ and non-phenolic substrates (Peralta-Zamora et al. 2003; Riva 2006).

Figure 6

(a) Time-course of reactive blue degradation by PVA/HNTs beads, immobilized laccase on PVA/HNTs beads, and immobilized laccase on PVA/HNTs beads with 0.3 mM ABTS. (b) Effect of different amounts of ABTS on PVA/HNTs-immobilized laccase.

Figure 6

(a) Time-course of reactive blue degradation by PVA/HNTs beads, immobilized laccase on PVA/HNTs beads, and immobilized laccase on PVA/HNTs beads with 0.3 mM ABTS. (b) Effect of different amounts of ABTS on PVA/HNTs-immobilized laccase.

The amount of ABTS also plays an important role in reactive blue removal as shown in Figure 6(b). It can be seen that only 40.97% reactive blue can be removed when adding 0.1 mM ABTS, whereas 93.41% reactive blue can be removed with 0.3 mM ABTS. However, when further increasing the mediator amount to 0.5 mM, the removal efficiency of reactive blue decreases to 80%. The reason may be that the excessive amounts of free ABTS2+ make the color of reaction mixture deeper (Lu et al. 2007; Peralta-Zamora et al. 2003). Thus, the appropriate amount of ABTS was chosen as 0.3 mM in the following experiments.

We also studied the removal rate of reactive blue by PVA/HNTs-immobilized laccase at different pH values in the presence of 0.3 Mm ABTS. The experiment was performed with 50 ml of 200 mg/l reactive blue in citric acid/Na2HPO4 buffer (0.1 M, pH 2–8) at room temperature for 8 h. After each reaction, the PVA/HNTs beads were separated from the reaction mixture by centrifugation. As illustrated in Figure 7(a), PVA/HNTs-immobilized laccase has the highest removal efficiency for reactive blue when pH was ranging from 4 to 6. This might be attributed to the suitable micro environment at pH 4–6. Since the oxidation of ABTS was not affected by pH, the variation tendency of removal efficiency may be due to the diffusion limitations of hybrid beads.

Figure 7

(a) Effect of pH on the removal of reactive blue for PVA/HNTs-immobilized laccase. (b) Reusability of immobilized laccase for reactive blue removal.

Figure 7

(a) Effect of pH on the removal of reactive blue for PVA/HNTs-immobilized laccase. (b) Reusability of immobilized laccase for reactive blue removal.

In order to study the reusability of the immobilized laccase in dye decolorization, we determined the degradation of reactive blue (200 mg/l, 50 ml) in citric acid/Na2HPO4 buffer (0.1 M, pH 5) at room temperature for 8 h. After each cycle, the reaction mixture was centrifuged to remove the supernatant. The beads were washed with deionized water and reused to remove reactive blue (Figure 7(b)). PVA/HNTs beads immobilized laccase can keep over 80% of its initial removal rate in the third cycle, and more than 60% of initial removing rate was retained in the sixth cycle, which means that PVA/HNTs-immobilized laccase possesses very good reusability. The decrease of the removal rate is probably because some laccase may be detached from the support during cycling (Rodrigues et al. 2013). The porous structure of PVA/HNTs beads provides space for enzyme immobilization and prevents enzyme leaching from the pore channels. Since the immobilized enzyme has enhanced pH tolerance and good thermal stability and reusability, the PVA/HNTs-immobilized laccase can be used in removing dye pollutants and has potential for use in other environmental applications.

CONCLUSION

Porous PVA/HNTs hybrid beads were successfully prepared for enzyme immobilization. The microstructure of the composite showed the internal network of porous structure, which provided a buffer ‘solid’ for enzyme biocatalysis. It is expected that PVA/HNTs hybrid beads could be of great potential as promising supports for biomacromolecule immobilization. Compared with free enzyme, the immobilized laccase exhibited enhanced pH and temperature tolerance, thermal stability, and storage stability. Furthermore, the immobilized laccase exhibited a rapid removal rate, high removal efficiency, and good reusability for reactive blue, suggesting that the PVA/HNTs-immobilized laccase can be used for removing dyes in wastewater and has potential application in other environmental fields.

ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (Grant No. 21576247 and 21706242), Key Scientific Research Project of University in Henan (Grant No. 17A530005).

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