Preparation of Efficient, Stable, and Reusable LaccaseâCu3(PO4)2

Research Article pubs.acs.org/journal/ascecg

Preparation of Efficient, Stable, and Reusable Laccase−Cu3(PO4)2 Hybrid Microspheres Based on Copper Foil for Decoloration of Congo Red Jian Rong, Tao Zhang,* Fengxian Qiu,* and Yao Zhu School of Chemistry and Chemical Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, P. R. China S Supporting Information *

ABSTRACT: Issues with separation and recycling of suspension powder samples in wastewater hinder the practical application of hybrid enzyme materials for environmental pollution control. In this work, for the first time, laccase− Cu3(PO4)2 hybrid microspheres with hierarchical structure were successfully prepared and loaded on a treated copper foil surface. First, Cu 8 (PO 3 OH) 2 (PO 4 ) 4 ·7H 2 O nanoflowers (CPN) were synthesized on the copper foil surface by a solution-growth method. Then the laccase−Cu3(PO4)2 hybrid microspheres were loaded on the CPN surface via an immersion reaction method using a laccase-containing phosphate buffer solution (PBS) solution. The formation mechanisms of CPN and La−CPN (laccase−Cu3(PO4)2 hybrid microspheres on the CPN surface) are discussed in detail and mainly contain the following processes: crystal growth, coordination effect, in situ growth, and self-assembly. Compared with free laccase, the as-obtained La−CPN has a higher decoloration efficiency (more than 95%) and decoloration rate (nearly 3.6 times higher than that of free laccase) on Congo red dye (CR) solution in the short time of 3 h. Cyclic voltammetry results demonstrated that the oxidizability of immobilized laccase could be enhanced as a result of the presence of Cu2+. Meanwhile, the utilization of the CPN carrier and the unique nanostructure gave the laccase−Cu3(PO4)2 hybrid microspheres high and stable decoloration efficiency and improved the tolerance toward pH and temperature changes. La−CPN still maintained about 85% relative activity after storage for 10 days. The concept presented herein can be further expanded to the preparation of other hybrid enzyme materials for environmental control, medical treatment, and more applications. KEYWORDS: Laccase, Copper foil, Hybrid material, Decoloration efficiency, Congo red dye

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INTRODUCTION In just the past few decades, rapid economic development has also brought increasingly serious water pollution.1 An important cause of water pollution is the extensive use of various synthetic dyes, including Congo red (CR), Malachite green (MG), rhodamine B (RhB), methylene blue (MB), and so on.2 Different kinds of dyes have been widely used in the paper, leather, textiles, and wool industries.3 Every year, a large amount of dye wastewater is directly discharged into rivers and lakes without pretreatment, which has strong effects on the biota and animal groups in view of the potential carcinogenicity and genotoxicity.4 Moreover, wastewater with colors also could weaken the penetration of light in water, inhibiting the photosynthesis capacity of aquatic organisms. In recent years, because of increasing awareness of environmental protection and public health, various physical and chemical methods, such as physical adsorption and photocatalytic degradation, have been employed to treat colored wastewater.5,6 However, these traditional methods often contain some harmful heavy or noble metal (e.g., Bi, Ag, Au, Pt, etc.), which not only results in high © 2017 American Chemical Society

costs but also brings secondary pollution to the environment because of the difficulty of recycling.7,8 As an alternative, biological methods have been among the main studies on dye decoloration because of their ecological sociability, low cost, high efficiency, and absence of secondary pollution.9 Laccase is a multicopper blue oxidase enzyme that catalyzes the oxidation of several compounds coupled to molecular oxygen reduction.10,11 Free laccase is commercially available at low cost and is widely applied in wastewater treatment because of its relatively low substrate specificity and high catalytic activity. Although the application performance of free laccase is often inhibited by the complex wastewater environment, the advantages of laccase in wastewater treatment are still favored by many researchers. Previous studies have shown that a reasonable immobilization method for laccase could effectively increase the stability, reusability, durability, and operation Received: March 16, 2017 Revised: April 5, 2017 Published: April 9, 2017 4468

DOI: 10.1021/acssuschemeng.7b00820 ACS Sustainable Chem. Eng. 2017, 5, 4468−4477

Research Article

ACS Sustainable Chemistry & Engineering

inexpensive characteristics.19−21 Moreover, copper foil can be recycled just by simple washing and drying. With this in mind, in this work three-dimensional (3D) Cu8(PO3OH)2(PO4)4·7H2O nanoflowers (CPN) were fabricated on a copper foil surface as a monolithic carrier. The simple and low-energy-consumption solution-growth method shows high flexibility in terms of controlling the morphology and structure, allowing us to prepare porous nanoflowers on the surface of the copper foil. Then, for the first time, laccase− Cu3(PO4)2 hybrid microspheres composed of interactional nanosheets were prepared on the CPN surface via a simple onestep immersion reaction at ambient temperature. The formation mechanism of Cu8(PO3OH)2(PO4)4·7H2O nanoflowers and laccase−Cu3(PO4)2 hybrid microspheres were also studied in detail. Finally, the decoloration efficiency and stability of as-obtained samples were evaluated by the decoloration of CR aqueous solutions.

simplification in practical applications. To date, various strategies and carriers have been employed to improve laccase immobilization, thereby further increasing the stability to expand the range of applications.10,12 Typically, Champagne and Ramsay5 reported that the immobilization of laccase on silica beads effectively improved the laccase stability and increased the decoloration efficiency of Reactive Blue 19. Plagemann et al.13 investigated an inorganic ceramic support for the covalent immobilization of lassase, revealing excellent stability for 3 months. Recently, Yang et al.14 prepared magnetic cross-linked enzyme aggregates (M-CLEAs) for the immobilization of laccase, which not only showed a high tetracycline degradation efficiency but exhibited superior stability and easy recovery. These successful immobilization strategies have provided a significant impetus for the further development and utilization of laccase. However, it is well-known that pore diffusion limitation of the substrate and products is inevitable for enzyme immobilization because of the rate-controlling step in the catalytic reaction system. Nevertheless, there is steric hindrance between the carrier surface and the immobilized enzyme if the enzyme is directly immobilized on the surface.15 Therefore, it is still not enough to adequately ameliorate the performance (such as enzyme activity and reusability) by unilateral immobilization of laccase. It is desirable and challenging to find a facile immobilization strategy. Ge and co-workers16 described the first example of protein− inorganic hybrid nanoflowers using copper(II) ions as the inorganic component and various enzymes as the organic component. This has aroused wide attention in synthesizing protein molecule−metal phosphate hybrid materials. Simultaneously, two views on the enhancement of laccase performance were proposed: (i) the high surface area of the nanostructures (nanoflowers) and (ii) the presence of Cu2+ ions in the laccase nanoflowers. On the one hand, the high surface area of the hybrid nanoflowers could reduce the mass transfer limitation due to flexibility of the substrate and provide a large contact area for the target object. On the other hand, Zhang et al.12 also demonstrated the view that the presence of zinc ion could enhance the lipase activity in their work. Recently, Patel et al.17 reported the preparation of a metal−protein hybrid nanoflower system for efficient immobilization of the recombinant enzymes L-arabinitol 4-dehydrogenase from Hypocrea jecorina (HjLAD) and NADH oxidase from Streptococcus pyogenes (SpNox), and the immobilized enzymes exhibited significantly enhanced Lxylulose production under cofactor regeneration conditions compared with the free enzyme combination. Therefore, according to the above studies, the hybrid organic−inorganic materials with nanostructure bring an inspiration for the preparation of high-performance laccase materials for decolorizing dyes. Usually, recovery is a common disadvantage related to powder samples when used in wastewater treatment. Moreover, when as-obtained hybrid materials are nanosized, they often face the problem of serious agglomeration caused by high surface energy. In addition, a lot of effort has been put into the creation of monolithic carriers, which have merits such as ease of separation, rapid termination of reactions, mechanical stability, and adaptability to various practical applications for batch and continuous operations.18 Therefore, in order to overcome above two limitations of suspension powder samples, copper foil has been widely investigated as a carrier material because of its abundant, environmentally friendly, and

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EXPERIMENTAL SECTION

Chemicals and Materials. Several chemicals, including hydrochloric acid (HCl), ethanol, dibasic sodium phosphate (Na2HPO4), 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) (≥99.0%), Coomassie brilliant blue G250, bovine serum albumin (BSA), ammonium persulfate ((NH4)2S2O8), and phosphate buffer solution (PBS) (pH 7.4, 0.01 M) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All of the chemical reagents used were of analytical grade. CR (C32H22N6Na2O6S2, molecular weight = 696.67 g mol−1, λmax = 497 nm, molecular diameter ≈ 36 Å) used in the experiments was obtained from Aladdin Chemical Reagent Co. Ltd.; the molecular structure of CR is provided in Figure S1 in the Supporting Information. Laccase (BR, ≥120 units g−1) was purchased from Shanghai Yuanye BioTechnology Co., Ltd. Copper foil (0.1 mm × 200 mm, ≥99.9 purity) was purchased from the Shanghai Macklin Co. Ltd. All of the reagents were used as received without further purification. Deionized water was used throughout all of the experiments. Preparation of Cu8(PO3OH)2(PO4)4·7H2O Nanoflowers on the Copper Foil Surface. Preparation of Cu8(PO3OH)2(PO4)4·7H2O was performed according to the methods reported previously.18 In a typical experiment, copper foils with a size of 20 mm × 30 mm × 0.1 mm were washed with 3 M HCl for 15 min under ultrasound conditions and then washed with deionized water and ethanol for about 5 min, followed by immersion in 20 mL of mixed solution containing 3 mL of (NH4)2S2O8 (0.2 M) and Na2HPO4 (1.0 M) for 12 h. Then the copper foil was taken out of the solution, washed with deionized water several times, and dried at 40 °C for 2 h under nitrogen protection to obtain Cu8(PO3OH)2(PO4)4·7H2O nanoflowers (denoted as CPN) on the copper foil surface. Different reaction times were investigated to explore the formation mechanism of Cu8(PO3OH)2(PO4)4·7H2O nanoflowers. Preparation of Laccase−Cu3(PO4)2 Hybrid Microspheres on the CPN Surface. The laccase−Cu3(PO4)2 hybrid microspheres were synthesized by the immersion reaction method. Typically, 10 mg of laccase powder was dissolved in 20 mL of PBS (pH 7.4, 0.01 M) under magnetic stirring conditions for 5 min at room temperature. Then the CPN with a size of 20 mm × 30 mm was immersed in the laccase solution for 12 h without agitation, washed with deionized water several times to remove superfluous laccase that was not immobilized on the surface, and finally dried under vacuum at 30 °C to obtain the immobilized laccase−Cu3(PO4)2 hybrid microspheres on the CPN surface (denoted as La−CPN), which were stored at 4 °C for further use. Characterization. The morphology of the as-obtained products was examined with by scanning electron microscopy (SEM) (S-4800 microscope, Hitachi, Tokyo, Japan). High-resolution transmission electron microscopy (HRTEM) and selected-area electron diffraction (SAED) were performed on a Philips CM200 field-emission gun transmission electron microscope (FEI Tecnai G2 F30 S-Twin). The 4469


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Figure 1. SEM images of (A) copper foil and (B−D) Cu8(PO3OH)2(PO4)4·7H2O nanoflowers under different magnifications.

Figure 2. (A) XRD pattern of Cu8(PO3OH)2(PO4)4·7H2O nanoflowers on the copper film surface. (B) EDS spectrum and a standard atom counting result form. elemental contents of the as-prepared products were determined by energy-dispersive X-ray spectroscopy (EDS) (JEM-2100). The crystal structures of the products were characterized by X-ray diffraction (XRD) analysis (type HZG41 B-PC). Radial scans were recorded in the reflection scanning mode from 2θ = 5 to 70° at a scanning rate of 4° min−1. FTIR analysis was carried out in the range of 400−4000 cm−1 using an FTIR spectrophotometer (NEXUS, Thermo Nicolet) and KBr pellets. Assays of Free and Immobilized Laccase Activity. Determination of Laccase Loading Capacity on the CPN Surface. The amount of laccase immobilized on the CPN was determined by measuring the amount of protein lost in the supernatant after immobilization.22 The concentration of protein was determined using the Bradford protein assay method at 595 nm and BSA as the standard protein.23 The laccase loading capacity (Q, in mg (cm2 of carrier)−1) on the CPN surface was calculated according to eq 1:

Q=

(C0 − C)V S

measurement of the free laccase activity, 0.1 mL of the laccase solution was mixed with 1.9 mL of the ABTS solution (0.4 mM) in PBS (0.01 M, pH 7.4) at 25 °C. The absorbance change at 420 nm was followed for 5 min using a UV−vis spectrophotometer (Agilent Cary 8454). One unit of laccase activity was defined as the amount of enzyme required to oxidize 1 μmol of ABTS substrate per minute. To determine the immobilized laccase activity, a similar method as previously described for free laccase was used. Two pieces of La−CPN (2 cm × 3 cm) were added to 40 mL of the ABTS solution (0.4 mM) in PBS (0.01 M, pH 7.4) at 25 °C. All of the measurements were carried out in triplicate. Figure S2 shows the slope of the linear portion of the laccase activity assay. The laccase activity (0.00355 units mL−1) can be calculated by dividing the slope (0.00710) by the volume of the laccase solution (2 mL). The relative activity (in %) is defined by the ratio of the laccase activity at the end of each storage time to the initial laccase activity. The immobilization yield (IY) was calculated according to eq 2:

(1)

IY =

where C0 and C are the initial and final laccase concentrations in the solution (in mg mL−1), respectively, V is the volume of the solution (in mL), and S is the total area of the CPN carriers (in cm2). Determination of Laccase Activity. The activities of free laccase and La−CPN were assayed according to the method reported by Bourbonnais et al.24 A radical cation (ABTS+) is created during the oxidation of ABTS (ε = 36 000 M−1 cm−1) by laccase.14 Before

A−B × 100% A

(2)

where A is the initial laccase activity in the reaction solution and B is the residual laccase activity in the immobilization and washing solution after the immobilization process. Decoloration of Congo Red Dye. The decoloration of CR aqueous solution by La−CPN was performed at 30 °C in an incubator. The experiment details were as follows: two pieces of as-obtained La−CPN 4470


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Figure 3. (A−C) SEM images under different magnifications and (D) EDS spectrum of La−CPN hybrid nanoflowers. The inset in (B) makes the analogy to a pistil more evident, showing the growth process of the nanoflowers. (20 mm × 30 mm) were added to 40 mL of CR aqueous solution (10 mg L−1, pH 7.3), which was prepared by dissolving Congo red powder in deionized water. After the stipulated time period, 3 mL of decoloration solution was taken out and centrifuged at 10 000 rpm min−1. The percent color removal was analyzed by using a UV−vis spectrophotometer (Agilent Cary 8454) to record the characteristic absorption peak of CR at λ = 497 nm (ε = 2.70 × 105 M−1 cm−1), as shown in Figure S1. The decoloration efficiency (D) was calculated according to eq 3:

D=

C0 − Ct × 100% C0

the plentiful nanosheets could provide a large number of binding sites and release more Cu2+ for protein assembly. XRD analysis was performed to determine the crystal structure of the obtained CPN sample (Figure 2A). The XRD pattern displayed peaks at 2θ = 8.2°, 12.9°, 20.2°, 22.5°, 29.1°, 31.5°, 36.9°, and 40.5°, which could be indexed to the pattern of copper phosphate hydroxide hydrate 2 5 (Cu8(PO3OH)2(PO4)4·7H2O, JCPDS no. 35-0448), except for the copper foil substrate peaks (marked with triangles in Figure 2A).18,26 In addition, elemental analysis of the material surface layer was obtained by EDS. Figure 2B shows that the CPN contained O, Cu, and P. Moreover, the corresponding EDS results also determined that the standard Cu:P atom counting rate was approximately 4:3, which is consistent with the atom rate in Cu8(PO3OH)2(PO4)4·7H2O. According to all of above results, it could be concluded that the main component of the nanoflowers was Cu8(PO3OH)2(PO4)4· 7H2O. Preparation of La−CPN Hybrid Material. The asprepared CPN was immersed in laccase solution (PBS was selected as the solvent). Laccase−Cu3(PO4)2 hybrid microspheres were further prepared on the CPN surface via a onestep immersion reaction. From Figure 3A it can be seen that a large amount of laccase−Cu3(PO4)2 hybrid material was synthesized on the CPN surface. It was also found that the distribution of laccase−Cu3(PO4)2 had no distinct rule, which could be mainly determined by the initial deposition of the protein on the surface. As a comparison, Figure S3 shows SEM images of laccase deposited on the CPN surface without the growth process of the hybrid material. It was noticed that

(3)

where C0 and Ct are the concentrations of the CR solution initially and at time t (in h), respectively. Similarly, the decoloration efficiencies of CPN and free laccase (denoted as laccase) were also evaluated.

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RESULTS AND DISCUSSION Preparation of CPN. The morphologies of as-obtained CPN were investigated by SEM. From Figure 1A it can be seen that the surface of the untreated copper film showed a smooth surface, which served as the substrate for the preparation of CPN. Figure 1B shows an SEM image of CPN prepared at room temperature for 12 h. Interestingly, the CPN formed monodispersed 3D hierarchical superstructures that look like flowers. It was found that the CPN was constructed from plenty of interlaced nanosheets with diameters of 20−30 μm (Figure 1C). The high-magnification image (Figure 1D) revealed 2D nanosheets with a thickness of about 100 nm. The rough surface with macrospores of CPN facilitated deposition and held more laccase on the surface in the next experiment, and 4471


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cm−1 could be attributed to the ν4 bending mode of the O−P− O bond.32 La−CPN (Figure 4b) exhibited characteristic peaks at 3443 and 3355 cm−1, which could be assigned to −OH and −NH groups.22 The −CH group also was found in Figure 4b with the occurrence of the stretching peak at 2931 cm−1.33 Meanwhile, La−CPN showed a characteristic amide band (1539 cm−1, overlap of N−H bending and C−N stretching).34 Moreover, the weak absorption band at 1450 cm−1 could be attributed to the in-plane bending vibration of C−H and O−H bonds existing in laccase protein. The above comparison results suggested the presence of proteins in the hybrid materials. In addition, the changes in the relative intensities of several peaks indicated the coordination between Cu 2+ and laccase protein.12,26 Tentative Formation Mechanisms of CPN and La− CPN Hybrid Material. In order to explore the formation mechanism of the Cu8(PO3OH)2(PO4)4·7H2O nanoflowers, the morphology evolution with reaction time was checked by SEM. The SEM images shown in Figure 5 exhibit the morphological evolution of the copper foil surface. As shown in Figure 5A, the copper foil notably changed from a smooth surface to one with developed crystals after 1 h of immersion. With increasing reaction time, the Cu8(PO3OH)2(PO4)4·7H2O nanoflowers were gradually formed on the copper foil surface according to the SEM images in Figure 5B−D. On the one hand, the density of Cu8(PO3OH)2(PO4)4·7H2O nanosheets increased, and it was apparently observed that the nanosheets changed from a scattered distribution to full coverage of the whole surface. On the other hand, it is interesting to see that this growth process is very similar to the growth procedure of a flower, which is from the opening flower bud to blooming flowers. On the basis of the above experimental results and previous studies,35,36 the formation mechanisms of CPN and the La− CPN hybrid material were speculated, and schematic diagrams of these mechanisms are shown in Figure 6. It is well-known that copper is readily oxidized in the presence of oxidant. Hence, the copper foil can be oxidized by S2O82− ions (Cu + S2O82− → Cu2+ + 2SO42−). During a prolonged reaction time, an obvious phenomenon was noted, namely, a layer of blue material was formed on the copper foil surface (as shown in the photograph in Figure 6A). Next, metallic phosphate is ready to form nanosheets when the nucleation and growth processes are controlled by the Cu2+ concentration with the increase in oxidation reaction time. Finally, the generated Cu2+ had a strong tendency to combine with PO43− ions to generate Cu8(PO3OH)2(PO4)4·7H2O nanosheets (8Cu2+ + 2HPO42− + 4PO43− + 7H2O → Cu8(PO3OH)2(PO4)4·7H2O).25 Laccase is a copper-containing enzyme. By the use of a laccase-containing PBS solution, La−CPN hybrid microspheres were further prepared, as illustrated in Figure 6B. First, the Cu2+ released by CPN forms complexes with laccase molecules by means of the coordination effect between copper ions and amide groups in the laccase backbone (step 1 in Figure 6B).37 Meanwhile, the Cu2+ also reacts with PO43− (present in the PBS buffer) to form the primary crystal structure (3Cu2+ + 2PO43− → Cu3(PO4)2) (step 2 in Figure 6B).16 Second, the laccase/Cu2+ agglomerates could serve as the nucleation sites for the subsequent in situ phosphate growth. This crystal growth process is dominated by the binding sites of Cu2+, thus leading to self-assembly of nanosheets with different orientations. Finally, as-obtained hybrid microspheres with uniform size (5−8 μm) were prepared after 12 h.

plentiful protein molecules without uniform structure were deposited on the CPN surface in disorder. Next, Figure 3B demonstrates that the laccase−Cu3(PO4)2 hybrid materials were assembled into microspheres with interactional nanosheets, resembling a ripe flower in appearance. Meanwhile, it was noticed that a porous block material (marked with a red circle) could be attributed to the formation of primary crystals of Cu3(PO4)2 enclosed by laccase, which was similar in appearance to a pistil in the center of a flower (inset of Figure 3B). The high-magnification images in Figure 3C show that the diameter of the laccase−Cu3(PO4)2 microsphere was 5−8 μm. As is well-known, these microspheres are conducive for decoloring dyes or removing pollutants because the high specific surface area can provide a high contact area and a large number of active sites.27,28 As shown in Figure 3D, the presence of peaks for C, N, and O in the EDS spectrum of the microsphere (and the absence of the corresponding peaks in CPN) proved that laccase− Cu3(PO4)2 hybrid materials were synthesized successfully. It should be emphasized that the Cu:P atom counting rate changed from 4:3 to 3:2. In addition, the corresponding HRTEM image (Figure S4) shows clear lattice fringes of one of the nanosheets existing in a hybrid microsphere, indicating that the nanosheets were well-crystallized and had a high degree of crystallinity. The SAED pattern (Figure S4 inset) exhibited several diffraction ring patterns, indicating a polycrystalline structure. From these results, combined with those of previous studies,16,29 it could be inferred that phosphate was present in the microspheres in the form of Cu3(PO4)2. FTIR Analysis. FTIR analysis was carried out to further confirm that the La−CPN hybrid material was prepared successfully. For FTIR measurements, a small amount of sample was collected from the surface of the treated copper foil. As shown in Figure 4, the FTIR spectra of CPN and laccase−

Figure 4. FTIR spectra of (a) CPN and (b) the La−CPN hybrid material.

Cu3(PO4)2 showed discriminatory absorption bands. In Figure 4a, the broad absorption bands at around 3405 and 1652 cm−1 resulted from the structural water (O−H/H2O vibration) of copper phosphate hydroxide hydrate (Cu8(PO3OH)2(PO4)4· 7H2O).30 The peaks at 1145, 1049, and 992 cm−1 were assigned to the bending vibration of Cu−OH (ν3 bending mode) and the asymmetric and symmetric stretching vibrations of PO43−, respectively.31 Meanwhile, the peaks at 625 and 561 4472


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Figure 5. SEM images of Cu8(PO3OH)2(PO4)4·7H2O nanoflowers at different reaction times: (A) 1, (B) 3, (C) 6, and (D) 12 h.

Figure 6. Schematic diagram of the formation mechanisms of (A) CPN and (B) the La−CPN hybrid material.

Decoloration of CR Solution. According to previous studies, many fungi, free enzymes, and immobilized enzymes have been used to decolorize and degrade azo dyes.9 A comparative study of CR solution decoloration was carried out over CPN, free laccase (laccase), and La−CPN. From Figure 7A it can be seen that the decoloration of CR with CPN and laccase reached 13.5% and 58.4%, respectively, which were still dissatisfactory with respect to decoloration efficiency. Figure S5 shows that the decoloration efficiency of CR over the free laccase had no significant increase upon incubation for a long period of time (5.5 h). Nevertheless, it is noteworthy that the decoloration efficiency of as-obtained La−CPN hybrid materials reached 95.8% after the same 3.5 h with the identical dye concentration. By comparison, it could be concluded that the laccase plays a leading role in decoloration of CR solution according to the results in Figure 7A, and the creation of such laccase−Cu3(PO4)2 hybrid materials significantly enhances the decoloration performance. From Figure 7B it can be clearly

seen that the intensity of the absorption peak at 497 nm decreased evidently with extension of the reaction time. Figure 7C shows a visual comparison that the CR solution underwent a significant color change (from red to colorless). It was also found that the decoloration efficiency of La−CPN had no significant increase when decoloration time was extended from 3 to 3.5 h (see Figure 7A,B). Thus, the optimum time for decoloration was set to 3 h in the next batch of experiments. From the above results, the preparation of La−CPN hybrid materials is simple, and the enhancement of the decoloration efficiency is evident. In order to further study the reasons for the enhanced decoloration efficiency, an SEM image of free laccase powders is provided in Supporting Information for comparison (see Figure S6). It can be seen that the free laccase is an amorphous solid particle with a size of ca. 10 μm. Therefore, one of the possible reasons could be that the asprepared hybrid nanoflowers provide high surface area and developed pore structure, which is an effective measure to 4473


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Figure 7. (A) Decoloration curves for CR decoloration by CPN, laccase, and La−CPN. (B) Sequential UV−vis absorption spectra of CR solution (10 mg L−1) after addition of La−CPN. (C) Noticeable color change (from red to colorless) of the CR solution after 3 h. (D) Kinetics of CR decoloration by CPN, laccase, and La−CPN.

decrease mass transfer limitations. In addition, a positive correlation between decoloration efficiency and laccase activity exists according to previous studies.22,38 Laccase is rich in histidine residues, which can form special covalent bonds: coordinate bonds with transition metal ions such as Cu2+. For the enhancement of laccase activity, the reason could be that the cooperative binding of the substrate to the active site of laccase is caused by the entrapment of laccase molecules through the interactions with Cu2+ in the phosphate crystals.15,16 Furthermore, cyclic voltammetry was employed to study the effect on the redox potential of laccase by the immobilization methods, as shown in Figure S7. The results demonstrated that the oxidizability of immobilized laccase could be enhanced by the presence of Cu2+ compared with free laccase. Hence, the enhancement of the decoloration efficiency may be attributed to the high contact area of laccase− Cu3(PO4)2 hybrid microspheres and increased enzyme activity caused by the synergistic effects between laccase and cupric ion (Cu2+). The decoloration rate is another way to directly explore the decoloration efficiency of the as-obtained samples. Here, a firstorder kinetics model was applied to describe the experimental data (eq 4): ln

C0 = kt Ct

Table 1. Decoloration Efficiency and Kinetics Parameters for CR with As-Prepared Samples first-order kinetics sample

decoloration efficiency of CR dye (%)

R2

k (h−1)

La−CPN Laccase CPN

95.8 58.4 13.5

0.979 0.993 0.988

0.947 0.266 0.0473

values of the correlation coefficient (R2 ≥ 0.979) demonstrated that the decoloration process fitted the first-order kinetics perfectly. In Table 1, La−CPN showed the highest decoloration rate constant (k = 0.947 h−1) among the three samples; this value is nearly 3.6 times higher than that of free laccase (k = 0.266 h−1). It is well-known that a higher rate constant (k) indicates better decoloration efficiency. Stability of the La−CPN Hybrid Material. The immobilization yield (IY) was 42.6%, and the loading capacity (Q) of CPN was 0.358 mg (cm2 of carrier)−1. Then the stability of storage is an important factor for its potential large-scale industrial production and application.39,40 Therefore, the storage stability of the La−CPN was also investigated in detail. As-prepared La−CPN and free laccase samples were stored at 4 °C for 7 days, and the decoloration efficiencies were measured with a time interval of 24 h. As can be seen from Figure 8A, the decoloration efficiency of La−CPN was slightly reduced (as high as 89.0%) over a 7 day storage period, while the free laccase had a relatively obvious decrease from 55.1 to 32.7%. Furthermore, the storage stabilities of free laccase and La−CPN also were investigated by measuring their relative activities at 25 °C (at about room temperature). From Figure S8 it can be seen

(4)

where C0 and Ct are the concentrations of the CR solution and initially and at time t (in h), respectively, and k is the rate constant (in h−1). Plots of ln(C0/Ct) versus t are shown in Figure 7D. Combined with Table 1, both the good linearity between ln(C0/Ct) and t (shown in Figure 7D) and the high 4474


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Figure 8. (A) Storage stability and (B) reusability of La−CPN hybrid material and free laccase for the decoloration of CR solution (conditions: temperature, 30 °C; time, 3 h; CR concentration, 10 mg L−1). (C, D) Effects of (C) pH and (D) temperature on the decoloration efficiency of La− CPN and free laccase.

that free laccase lost above 60% of its initial activity after storage for 10 days when incubated in PBS (pH 7.4). However, it was interesting to find that La−CPN still maintained about 85% relative activity under the same conditions. As is well-known, Cu2+ (a soft Lewis acid) has a preference for nonbonding lonepair electrons from the laccase amino acids.10 Hence, this may be the reason that the immobilized laccase chelated with Cu2+ led to a high decoloration efficiency and smaller reduction in laccase activity under long-time storage. In addition, the La−CPN and free laccase were collected from the reaction solution and washed with PBS several times after each test. As shown in Figure 8B, the decoloration efficiency of La−CPN did not show a significant decrease after the fifth cycle (from 95.3% to 85.9%). The decoloration efficiency retention of La−CPN (90.1%) was apparently higher than that of free laccase (63.7%). Figure S9 shows an SEM image of La−CPN for the CR decoloration after the fifth cycle. Compared with Figure 3B, although the morphology of the laccase−Cu3(PO4)2 hybrid microspheres was destroyed to some extent, a large number of hybrid nanosheets still existed on the CPN surface. This may be the reason that the decoloration efficiency still remained above 85% after the fifth cycle. In consideration of the complexity of environmental wastewater, the effects of pH and temperature on the decoloration efficiencies of free and immobilized laccase were investigated in detail (Figure 8C,D). From Figure 8C it can be seen that the La−CPN still had relatively high decoloration efficiency over a broad pH range. Additionally, it is worth noting that the decoloration efficiency of La−CPN was significantly higher than that of free laccase. This increased

pH resistance is probably due to the reduction of molecular mobility caused by multipoint binding between laccase and Cu2+, which could reduce drastic conformational changes with changing pH.15 The effect of temperature on the decoloration efficiency of La−CPN and free laccase was studied over the temperature range 15−50 °C, as shown in Figure 8D. It can be seen that the relatively high decoloration efficiencies of laccase were achieved from 20 to 30 °C. Interestingly, it should be pointed out that the as-prepared La−CPN was effective from 20 to 50 °C, which is a wider temperature range than for free laccase. The increased temperature resistance could also be attributed to a similar reason as for the pH resistance, namely, the strengthening of the protein molecule’s structural rigidity when it binds to the phosphate. The increased structural rigidity of the protein molecule decreases the extent of conformational change when the enzyme is exposed at higher or lower temperatures.41 In addition, it was reported that polycationic amino groups also provide support to resist harsh chemicals and high temperatures.15 All of the results of the above experiments demonstrate that the as-prepared La−CPN has excellent stability.

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CONCLUSIONS An organic−inorganic hybrid material (La−CPN) was successfully fabricated by a simple combination of solutiongrowth and immersion reaction methods. Laccase−Cu3(PO4)2 hybrid microspheres were attached onto a CPN surface through the coordination effect between copper ions and amide groups. Thus, the CPN surface served as both a source of copper ions and a carrier for laccase immobilization. The formation mechanism of CPN and the La−CPN hybrid material have 4475


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been described as a crystal growth, coordination effect, in situ growth, and self-assembly process. The La−CPN demonstrated high decoloration efficiency (more than 95%) and decoloration rate (nearly 3.6 times higher than that of free laccase) in the short time of 3 h. Besides, the as-prepared La−CPN exhibited excellent storage stability (7 days) and reusability (five cycles) and also appeared to have improved stability against pH and temperature. This work provides a new strategy for assembling organic−inorganic hybrid materials on the copper foil surface by a facile method. This not only improves the application performance of the enzyme but also effectively overcomes difficulties of separation and recycling. The concept presented herein can be further expanded to the preparation of other functional hybrid film materials for environmental control, medical treatment, and more applications.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00820. UV−vis spectrum and chemical structure of CR; slope of the linear portion of the laccase activity assay; SEM images of laccase on CPN surface and free laccase powders; HRTEM crystal lattice structure and SAED pattern of the laccase−Cu3(PO4)2 nanosheet; detailed explanation of the decoloration behavior on the laccase; detailed explanation of the cyclic voltammograms of laccase and La−CPN; relative activities free laccase and La−CPN; and an SEM image of La−CPN for CR decoloration after the fifth cycle (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel./fax: +86 511 88791800. E-mail: [email protected] (T. Zhang) *E-mail: [email protected] (F. Qiu). ORCID

Tao Zhang: 0000-0001-9255-9802 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (U1507115 and 21576120), the Natural Science Foundation of Jiangsu Province (BK20160500, BK20161362, and BK20161264), the Scientific Research Foundation for Advanced Talents, Jiangsu University (15JDG142), the China Postdoctoral Science Foundation (2016M600373), the China Postdoctoral Science Foundation of Jiangsu Province (1601016A), and the 333 High-Level Personnel Training Project of Jiangsu Province (BRA2016142).

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Preparation of Efficient, Stable, and Reusable LaccaseâCu3(PO4)2

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