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

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Preparation of efficient, stable and reusable laccase-Cu3(PO4)2 hybrid microspheres based on copper foil for decolouration of Congo red Jian Rong, Tao Zhang, Fengxian Qiu, and Yao Zhu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b00820 • Publication Date (Web): 09 Apr 2017 Downloaded from http://pubs.acs.org on April 9, 2017

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ACS Sustainable Chemistry & Engineering

Preparation of efficient, stable and reusable laccase-Cu3(PO4)2 hybrid microspheres based on copper foil for decolouration of Congo red Jian Rong, Tao Zhang*, Fengxian Qiu* , Yao Zhu School of Chemistry and Chemical Engineering, Jiangsu University, 301Xuefu Road, Zhenjiang 212013, P. R. China

*Corresponding authors: Tel./fax: +86 511 88791800. E-mail: [email protected] (T, Zhang); [email protected] (F. Qiu) 1

ACS Paragon Plus Environment


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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 firstly time, laccase-Cu3(PO4)2 hybrid microspheres with hierarchical structure were successfully prepared and loaded on the treated copper foil surface. Firstly, the Cu8(PO3OH)2(PO4)4·7H2O nanoflowers (CPN) was synthesized on copper foil surface by solution-growth method. Then the laccase-Cu3(PO4)2 hybrid microspheres were loaded on the CPN surface via immersion reaction method by using a laccase-containing PBS solution. The formation mechanisms of CPN and La-CPN (laccase-Cu3(PO4)2 hybrid microspheres on CPN surface) have been discussed in detail and mainly contain the following processes: crystal growth, coordination effect, in-situ growth and self-assembly. Compared with the free laccase, the as-obtained La-CPN has higher decolouration efficiency (more than 95%) and decolouration rate (nearly 3.6 times higher than that of free laccase) on Congo red dye (CR) solution in the short 3 hours. The results of cyclic voltammograms demonstrated that oxidizability of immobilized laccase could be enhanced due to the presence of Cu2+. Meanwhile, the utilization of CPN carrier and the unique nanostructure made laccase-Cu3(PO4)2 hybrid microspheres high and stable decolouration efficiency and improved the tolerance towards 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 enzymes materials for environmental control, medical treatment and more applications. 2


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Keywords: Laccase; Copper foil; Hybrid material; Decolouration efficiency; Congo red dye Introduction In just the past few decades, the rapid economic development has also brought to increasingly serious water pollution.1 An important cause of water pollution is the extensive use of various synthetic dyes, including Congo red, Malachite green (MG), Rhodamine B (RhB), Methylene blue (MB) and so on.2 Different kinds of dye have been widely used in 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 have strong effects on the biota and animal groups in consideration of the potential carcinogenicity and genotoxicity.4 Moreover, wastewater with colors also could weaken the penetration of light in water, inhibiting photosynthesis capacity of aquatic organisms. In recent years, owing to increasing awareness of environmental protection and public health, various physical, chemical methods, such as photocatalytic degradation and physical adsorption, have been employed to treat coloured wastewater.5,6 However, these traditional methods often contained some harmful heavy or noble metal (such as Bi, Ag, Au, Pt and so on), which not only required high costs but also brought secondary pollution to environment due to the difficulty of recycling.7,8 As an alternative, biological method has been one of the main studies on dye decolouration due to their ecological sociability, low cost, high efficiency and no secondary pollution.9 Laccase is a multi-copper blue oxidase enzyme that catalyzes the oxidation of 3



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several compounds coupled to molecular oxygen reduction.10,11 Meanwhile, free laccase is commercially available at low cost and is widely applied in wastewater treatment due to its relatively low substrate specificity and high catalytic activity. Although the application performances of free laccase were often inhibited by 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 simplification in practical applications. Up to now, various strategies and carriers have been carried out to improve laccase immobilization, thereby further increasing the stability for expanding the range of applications.10,12 Typically, Champagne et al.5 reported that the immobilization of laccase on silica beads effectively improved the laccase stability and increased decolouration efficiency of Reactive blue 19. Plagemann et al.13 investigated an inorganic ceramic support for the covalent immobilization of lassase, revealing an excellent stability within 3 months. Recently, Yang et al.14 prepared a 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 provided a significant impetus for 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 4


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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 ions (II) as the inorganic component and various enzymes as the organic component, which has arouse wide attention in synthesizing protein molecules-metal phosphate. Simultaneously, views on the enhancement of laccase performance were proposed as follows: (i) the high surface area of nanostructure (nanoflower); (ii) the presence of Cu2+ ions in laccase nanoflowers. On the one hand, high surface area of hybrid nanoflowes could reduce the mass transfer limitation due to flexibility of substrate and provide large contact area for 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 L-xylulose production under co-factor regeneration conditions than the free enzyme combination. Therefore, according to the above studies, the hybrid organic-inorganic materials with nanostructure bring an inspiration to the preparation of high performance laccase material for decolorizing dyes. 5



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Usually, recovery is a common disadvantage related to powder samples when used in wastewater treatment. Moreover, when as-obtained hybrid materials are nano-sized, they often face the problem of serious agglomeration caused by high surface energy. In addition, a lot of efforts have been put into 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 limitation of suspension powder samples, copper foil has been widely investigated as a carrier material because of its abundant, environmentally friendly, and inexpensive characteristics.19-21 Moreover, copper foil can be recycled just by simple washing and drying. With this in mind, herein, 3D Cu8(PO3OH)2(PO4)4·7H2O nanoflowers (CPN) were fabricated on the copper foil surface as a monolithic carrier. The simple and low energy consumption solution-growth method shows high flexibility in term of controlling the morphology and structure, which allows us to prepare porous nanoflowers on the surface of 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 one-step 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 illustrated in detail. Finally, the decolouration efficiency and stability of as-obtained samples were evaluated by the decolouration of CR aqueous solution. 6


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Experimental section Chemicals and materials. Several chemicals of hydrochloric acid (HCl), ethanol, dibasic

sodium

phosphate

(Na2HPO4),

2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS, ≥ 99.0%), coomassie brilliant blue G250, bovine serum albumin (BSA), ammonium persulphate (NH4)2S2O8 and phosphate buffer solution (PBS, pH 7.4, 0.01 M) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All chemicals reagents used were of analytical grade. CR (formula: C32H22N6Na2O6S2, molecular weight: 696.67 g mol−1, λmax: 497 nm, molecular diameter: ~36 angstrom) used in the experiments was obtained from Aladdin Chemical Reagent Co. Ltd., and the molecular structure of CR was provided in supplementary information (Fig. S1). Laccase (BR, ≥120 U g-1) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Copper foil (0.1 mm × 200 mm, ≥99.9 purity) was purchased from the Co. Ltd. of Shanghai Macklin. All reagents were used as received without further purification. Deionized water was used throughout all experiments. Preparation of Cu8(PO3OH)2(PO4)4·7H2O nanoflowers on the copper foil surface. Preparation of Cu8(PO3OH)2(PO4)4·7H2O is 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 immersing in a 20 mL of mixed solution containing 3 mL of (NH4)2S2O8 (0.2 M) and Na2HPO4 (1.0 M), respectively, for 12 h. Then the copper foil was taken out of the 7



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solution washed with deionized water for 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 time 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 powders were dissolved in 20 mL of PBS solution (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 above laccase solution for 12 h without agitation, followed by washing with deionized water for several times to remove superfluous laccase which was not immobilized on the surface, and finally dried under vacuum at 30 °C to obtain the immobilized laccase-Cu3(PO4)2 hybrid microsphere on the CPN surface (expressed as La-CPN), storing at 4 °C for further use. Characterization. The morphology of the as-obtained products was examined with a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan). High resolution transmission electron microscopy (HRTEM) and selected area electron diffraction (SAED) were performed on a Philips CM200 filed-emission gun transmission electron microscope (FEI Tecnai G2 F30 S-Twin). The element 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 8


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reflection scanning mode from 2θ = 5–70° with a scanning rate of 4° min-1. FTIR analysis was carried out in the range of 400-4,000 cm−1 using a FTIR spectrophotometer (Thermo Nicolet, NEXUS, TM) in 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) on the CPN surface was calculated as the following Equation (1): Q ⁄ carrier =

(1)

where C0 and C are the initial and final laccase concentrations in the solution (mg mL-1), respectively. V is the volume of the solution (mL); S is the total area of the CPN carriers (cm2). Determination of laccase activity. The activity of free laccase and La-CPN was assayed according to the method reported by Bourbonnais et al.24 A radical cation (ABTS+) was created during the oxidation of ABTS (ɛ = 36,000 M-1 cm-1) by laccase.14 Before measuring the free laccase activity assay, 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. Absorbance change at 420 nm was followed for 5 min by UV-vis spectrophotometer (Agilent Cary 8454). One unit of laccase activity was defined as

9



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the amount of enzyme required to oxidize 1 µmol of substrate ABTS 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 in 40 mL of the ABTS solution (0.4 mM) in PBS (0.01 M, pH 7.4) at 25 °C. All measurements were carried out in triplicate. Fig. S2 shows the slope of the linear portion of the laccase activity assay. The laccase activity (0.00355 U mL-1) can be calculated by dividing the slope (0.00710) by the volume of the laccase solution (2 mL). Relative activity (%) is defined by the ratio of the laccase activity at the end of each storage time to the initial laccase activity. Immobilization yield (IY) was calculated as the following Equation (2): IY% =

× 100

(2)

where A is the initial laccase activity in the reaction solution, and B is the residual laccase activity in immobilization and washing solution after immobilization process. Decolouration of Congo red dye experiment. The prepared La-CPN for the decolouration of CR aqueous solution was performed at 30 °C in an incubator. The experiment details were as follows: two pieces of as-obtained La-CPN (20 mm × 30 mm) were added to 40 mL CR aqueous solution (10 mg L-1, pH 7.3), which was prepared by dissolving the Congo red powder in deionized water. After stipulated time period, 3 mL of decolouration solution was taken out and centrifuged at 10000 rpm min-1. The percentage color removal was analyzed by UV-vis spectrophotometer (Agilent Cary 8454) recording the characteristic absorption peak of CR at λ = 497 nm (ɛ = 2.70×105 M-1 cm-1), as shown in Fig. S1. The decolouration efficiency (D%) was 10


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calculated as the following Equation (3): % =

× 100

(3)

where C0 and Ct are the initial concentration and concentration at time t (h) of CR solution, respectively. Similarly, the decolouration efficiency of CPN and free laccase (denoted as laccase) were also evaluated. Results and discussion Preparation of CPN. The morphologies of as-obtained CPN were investigated by SEM. From the Fig. 1A, it could be seen that the surface of untreated copper film showed a smooth surface, which will serve as the substrate for the preparation of CPN. Fig. 1B showed the SEM images of CPN prepared at room temperature for 12 h. Interestingly, the CPN performed the mono-dispersed 3D hierarchical superstructures which like flowers. It could be found that the CPN was constructed by the plenty of interlaced nanosheets with the diameter of 20 ~ 30 µm. (Fig. 1C). Then high magnification image (Fig. 1D) revealed that the 2D nanosheets with a thickness of about one hundred nanometer. The rough surface with macrospores of CPN will be facilitate to deposit and hold more laccase on the surface in next experiment, and the plentiful nanosheets could provide a large number of binding sites and release more Cu2+ for protein assembly.

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Fig. 1. SEM images of Copper foil (A) and Cu8(PO3OH)2(PO4)4•7H2O nanoflowers (B, C and D) under different magnifications XRD analysis was performed to determine the crystal structure of the obtained CPN sample (Fig. 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°, could be indexed to the pattern of copper phosphate hydroxide hydrate25 (Cu8(PO3OH)2(PO4)4·7H2O, JCPDS No. 35-0448) except for those copper foil substrate peaks marked with a triangle.18,26 In addition, elemental analysis of material surface layer was obtained by electron dispersive X-ray spectroscopy (EDS). Fig. 2B showed that the CPN contained O, Cu and P. Moreover, the corresponding EDS results also determined that the standard atom counting rate of Cu : P was approximately 4 : 3, which was consistent with the atom rate in Cu8(PO3OH)2(PO4)4·7H2O. According to all above results, it could be concluded that the main component of nanoflower was Cu8(PO3OH)2(PO4)4·7H2O.

12


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Fig. 2. XRD pattern of Cu8(PO3OH)2(PO4)4·7H2O nanoflowers on the copper film surface (A), EDS spectrum and a standard atom counting result form (B) Preparation of La-CPN hybrid materials. The as-prepared CPN was immersed in laccase solution (the PBS was selected as the solvent). Laccase-Cu3(PO4)2 hybrid microspheres were further prepared on the CPN surface via a one-step immersion reaction. From the Fig. 3A, it could be seen that a large amount of laccase-Cu3(PO4)2 hybrid materials was synthesized on the CPN surface. It also could be 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, Fig. S3 showed the SEM images of laccase were deposited on the CPN surface without the growth process of hybrid materials. It was noticed that plentiful protein molecules without uniform structure deposited on the CPN surface in disorder. Next, Fig. 3B demonstrated that the laccase-Cu3(PO4)2 hybrid materials were assembled into microsphere with interactional nanosheets, which was like a ripe flower. 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 to a pistil (inset of Fig. 3B) in the center of a flower. As 13



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shown in Fig. 3C, high magnification images showed that the diameter of laccase-Cu3(PO4)2 microsphere was 5 ~ 8 µm. As is well known, these microspheres are conducive to decolor dye or remove pollutant because high specific surface area could provide a high contact area and a large number of active sites.27,28 From the EDS result (Fig. 3D), the presence of peaks of C, N, O in EDS of microsphere (absence of corresponding peaks in CPN) proved that Laccase-Cu3(PO4)2 hybrid materials were synthesized successfully. It should be emphasized that the atom counting rate of Cu : P changed from 4 : 3 to 3 : 2. In addition, the corresponding high-resolution TEM (HRTEM) image (Fig. S4) showed clear lattice fringes of one of the nanosheet existing in hybrid microsphere, indicating that the nanosheets were well-crystallized and had a high degree of crystallinity. The SAED pattern (the inset Fig. S4) exhibited several diffraction ring patterns, indicating a polycrystalline structure. Herein, combined with previous studies,16,29 it could be inferred that phosphate could be presented in microsphere in the form of Cu3(PO4)2.

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Fig. 3. SEM images under different magnification (A, B and C) and EDS spectrum of La-CPN hybrid nanoflower (D). To make the analogy to pistil (inset of (B)) more evident showing the growth process of flowers FTIR analysis. FTIR analysis was carried to further confirm that La-CPN hybrid material was prepared successfully. For FTIR measurement, a small amount sample was collected from the surface of the treated copper foil. As shown in Fig. 4, the FTIR spectra of CPN and laccase-Cu3(PO4)2 showed discriminatory absorption bands. From the Fig. 4(a), the broad absorption bands at around 3405 and 1652 cm-1 resulted from the structure 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, asymmetric, and symmetric stretching vibration of PO3-4 (ν3 bending mode), respectively.31 Meanwhile, the peaks at 625 and 561 cm-1 could be attributed to the ν4 bending mode of O-P-O bond.32 Compared with the CPN, the La-CPN (Fig. 4(b)) exhibited characteristic peaks at 3443 and 3355 cm-1, which 15



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could be assigned to –OH and –NH groups.22 The –CH group also was found in Fig. 4(b) with the occurrence of the stretching peaks at 2931 cm-1.33 Meanwhile, the La-CPN showed characteristic amide (1539 cm-1, overlap of N-H bending and C-N stretching) bands.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 existed in laccase protein. The above comparison results suggested the presence of proteins in the hybrid materials. In addition, the change in relative intensities of several peaks indicated the coordination effect between Cu2+ and laccase protein.12,26

Fig. 4. FTIR spectra of CPN (a) and La-CPN hybrid material (b) A tentative formation mechanism of CPN and La-CPN hybrid materials. In order to explore the formation mechanism of the Cu8(PO3OH)2(PO4)4·7H2O nanoflowers, the morphology evolution with the reaction times was checked by SEM. 16


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The SEM images shown in Fig. 5 exhibited the morphological evolution of the copper foil surface. As shown in Fig. 5A, the copper foil notably changed from smooth surface to developed crystals after 1 h immersion. With the increasing of reaction times, the Cu8(PO3OH)2(PO4)4·7H2O nanoflowers were gradually formed on the copper foil surface according to the SEM images of Fig. 5(B, C and D). On the one hand, the density of Cu8(PO3OH)2(PO4)4·7H2O nanosheets was increasing, which apparently observed that the nanosheets changed from scattered distribution to full of whole surface. On the other hand, it is interesting to see that this growth process is very similar to a growth procedure of a flower, which is from opening flower bud to blooming flowers.

Fig. 5. SEM images of Cu8(PO3OH)2(PO4)4·7H2O nanoflowers with different reaction time: 1 (A), 3 (B), 6 (C) and 12 h (D) Based on the above experimental results and previous studies,35,36 the formation mechanism of CPN and La-CPN hybrid materials were speculated and the schematic diagram was shown in Fig. 6(A and B). It is well known that copper is readily 17



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oxidized in the presence of oxidant. Hence, the copper foil can be oxidized by S2O2-8 ions (Cu + S2O2-8 → Cu2+ + 2SO2-4 ). During a prolonged reaction time, an obvious phenomenon was noted that a layer of blue material was formed on copper foil surface (Photograph of experimental Process was provided in Fig. 6A). Next, metallic phosphate is ready to form nanosheets when the nucleation and growth process are controlled by the Cu2+ concentration with the increase of oxidation reaction time. Finally, the generated Cu2+ had a strong tendency to combine with PO 3-4 ions to generate Cu8(PO3OH)2(PO4)4·7H2O nanosheets (8Cu2+ + 2HPO2-4 + 4PO3-4 + 7H2O → Cu8(PO3OH)2(PO4)4·7H2O).25 Laccase is a copper-containing enzyme. By using a laccase-containing PBS solution, La-CPN hybrid microspheres were further prepared, as illustrated in Fig. 6B. Firstly, the Cu2+ released by CPN will form the complexes with laccase molecules by means of the coordination effect between copper ions and amide groups in the laccase backbone (Step 1, Fig. 6B).37 Meanwhile, the Cu2+ also will react with the PO3-4 (existed in PBS buffer) to form the primary crystal structure (3Cu2+ + 2PO3-4 → Cu3(PO4)2) (Step 2, Fig. 6B).16 Secondly, the agglomerates of laccase/Cu2+ could serve as the nucleation site for the subsequent in-situ phosphate growth. This crystal growth process was dominated by the binding sites of Cu2+, thus leading to self-assembly nanosheets with different orientations. Finally, as-obtained hybrid microspheres with uniform size (5 ~ 8 µm) were prepared after 12 h.

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Fig. 6. Schematic diagram of the formation mechanism of CPN (A) and La-CPN hybrid materials (B) Decolouration of CR solution. According to previous studies, many funguses, free enzymes and immobilized enzymes were carried out to decolourize and degrade azo dyes.9 A comparative study for CR solution decolouration was carried out over the CPN, free laccase (laccase) and La-CPN. From the Fig. 7A, it could be found that the decolouration of CR with CPN and laccase reached 13.5% and 58.4%, respectively, which were still dissatisfactory in decolouration efficiency. From the Fig. S5, it could be found that the decolouration efficiency of CR over the free laccase had no significant increase when incubated for a long period of time (5.5 h). Nevertheless, it is noteworthy that the decolouration 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 played a leading role in decolouration of CR solution according to the result of Fig. 7A, and the creation of

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such laccase-Cu3(PO4)2 hybrid materials significantly enhanced the decolouration performance. From the Fig. 7B, it can be clearly seen that the intensity absorption peak (497 nm) decreased evidently with the extension of reaction time. Fig. 7C illustrated a visual comparison that the color of CR had undergone significant change (from red to colorless). It was also found that decolouration efficiency of La-CPN had no significant increase when decolouration time was extended from 3 h to 3.5 h (see Fig. 7(A and B)). Herein, the optimum time for decolouration will be set to 3 h in the next batch experiments. From the above results, the preparation of La-CPN hybrid materials is simple and the enhancement decolouration efficiency comparable. In order to further study the reasons for enhanced decolouration efficiency, the SEM image of free laccase powders was provided in supplementary information for comparison (see Fig. S6). It can be seen that the free laccase is an amorphous solid particle with size of ca. 10 µm, as is shown in Fig. S6. Therefore, one of the possible reasons could be that the as-prepared hybrid nanoflowers provide high surface area and developed pore structure, which is an effective measure to decrease mass-transfer limitation. In addition, a positive correlation existed between decolouration efficiency and laccase activity according to previous studies.22,38 Laccase is rich of 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 was caused by their entrapment of laccase molecules through the interactions with Cu2+ in the phosphate 20


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crystals.15,16 Furthermore, the cyclic voltammograms (CVs) were employed to study the effect on the redox potential of laccase by the immobilization methods, as shown in Fig. S7. The result demonstrated that oxidizability of immobilized laccase could be enhanced due to the presence of Cu2+ compared with free laccase. Hence, the enhancement decolouration efficiency may attribute 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 decolouration rate is another way to directly explore the decolouration efficiency of the as-obtained samples. Here, a first-order kinetics model was applied to describe the experimental data as following Equation (4):

(4)

= !"

where C0 and Ct are the initial concentration and concentration at time t (h) of CR solution, respectively; k is the rate constant (h-1). A plot ln(C0/Ct) versus the t was shown in Fig. 7D. Combined with Table 1, both a good linearity between ln(C0/Ct) and t (shown in Fig. 7D) and high values of correlation coefficient R2 (≥0.979) demonstrated that the decolouration process fitted the first-order kinetics perfectly. In Table 1, the La-CPN showed the highest decolouration rate constant (k, 0.947) among three samples that is nearly 3.6 times higher than that of free laccase (k, 0.266). It is well known that higher rate constant (k) indicates the more excellent decolouration efficiency.

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Fig. 7. The decolouration curves (A) of CR decolouration on the CPN, laccase and La-CPN; the sequential UV-vis adsorption spectra of CR solution (10 mg L-1) after adding La-CPN (B) and noticeable colour change illustration (from red to colourless) of CR after 3 hours (C) and the kinetics (D) of CR decolouration on the CPN, laccase and La-CPN Table 1 Decolouration efficiency and kinetics parameters for CR with as-preparation samples Samples

The decolouration

First-order kinetics

efficiency of CR dye(%)

Correlation

Kinetic constant (k,

coefficient (R2)

h-1)

La-CPN

95.8

0.979

0.947

Laccase

58.4

0.993

0.266

CPN

13.5

0.988

0.0473

22


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Stability of the La-CPN hybrid material. The immobilization yield (IY) was 42.6%, and loading capacity (Q) of CPN was 0.358 mg cm-2 carrier. 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 in 4 °C for 7 days and the decolouration efficiencies were measured with a time interval of 24 h. As can be seen from Fig. 8A, the decolouration 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 stability of free laccase and La-CPN also was investigated by measuring relative activity at 25 °C (at about room temperature). From the Fig. S8, it could be found 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 we all know, Cu2+ (soft Lewis acids) has a preference for non-bonding lone pair electrons from the laccase amino acids.10 Hence, it may be the reason that the immobilized laccase chelated with Cu2+ leaded a high decolouration efficiency and smaller reduction in laccase activity under the long time storage. In addition, the La-CPN and free laccase were collected from the reaction solution and washed with the PBS buffer for several times after each test. As shown in Fig. 8B, the decolouration efficiency of La-CPN did not show a significant decrease (from 95.3% to 85.9%) after the fifth cycle. The decolouration efficiency retention of 23



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La-CPN (90.1%) was apparently higher than free laccase (63.7%). Fig. S9 showed the SEM image of La-CPN for the CR decolouration after the fifth cycle. Compared with the Fig. 3B, although the morphology of laccase-Cu3(PO4)2 hybrid microsphere was destroyed to some extent, a large number of hybrid nanosheets still existed on the CPN surface. It may be the reason that the decolouration efficiency still reached more than eighty-five percent after the fifth cycle. In consideration of the complexity of environmental wastewater, the effects of pH and temperature on the decolouration efficiency of the free and immobilized laccase were investigated in detail (Fig. 8C and D). From the Fig. 8C, it was found that the La-CPN still had relatively high decolouration efficiency in a broad pH range. Additionally, it is worth noting that the decolouration efficiency of La-CPN was significantly higher than that of free laccase. This increased pH resistance was probably due to the reduction of molecular mobility caused by multi-point binding between laccase and Cu2+, which could reduce drastic conformational changes during pH changing.15 The effect of temperature on decolouration efficiency of La-CPN and free laccase was studied in the temperature range 15 – 50 °C, as shown in Fig. 8D. It can be seen that the relatively high decolouration efficiencies of laccase were achieved range from 20 to 30 °C. Interestingly, it should be pointed out the as-prepared La-CPN with adaptable temperature from 20 to 50 °C was wider than the range of free laccase. The increased temperature resistance could also be attributed a similar reason for the pH resistance that the strengthening of the protein molecule’s and structural rigidity when it binds to the phosphate. The structural rigidity of the 24


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protein molecule decreased the extent of conformational change when the enzyme was exposed at higher or lower temperatures.41 In addition, it was reported that polycationic amino groups also provided support to resist to harsh chemicals and high temperatures.15 All the results of the above experiments demonstrate that the as-prepared La-CPN has excellent stability.

Fig. 8 The storage stability (A) and reusability (B) of La-CPN and free laccase hybrid material for the decolouration efficiency of CR solution (Condition: temperature, 30 °C; time, 3 h; CR concentration, 10 mg L-1); Effects of pH (C) and temperature (D) on decolouration efficiency of La-CPN and free laccase Conclusions In summary, organic–inorganic hybrid material (La-CPN) was successfully fabricated by a simple combination of solution-growth and immersion reaction 25



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method. Laccase-Cu3(PO4)2 hybrid microspheres were attached onto CPN surface through the coordination effect between copper ions and amide groups. Thus the CPN surface served both as source of copper ions and carrier for laccase immobilization. The formation mechanisms of CPN and La-CPN hybrid material has been described as crystal growth, coordination effect, in-situ growth and self-assembly process. The La-CPN demonstrated high decolouration efficiency (more than 95%) and decolouration rate (nearly 3.6 times higher than that of free laccase) in the short 3 hours. Besides, the as-prepared La-CPN exhibited excellent storage stability (in 7 days) and reusability (5 cycles), and also appeared an improved stability against pH and temperature. This work provided a new strategy for assembling organic–inorganic hybrid materials on the copper foil surface by a facile method, which not only improves the application performance of the enzyme but effectively overcomes difficulty 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. Associated content Supporting Information UV-vis spectrum and chemical structure of CR; slope of the linear portion of the laccase activity assay; laccase on CPN surface and free laccase powders, SEM; HRTEM crystal lattice structure and the SAED pattern of the laccase-Cu3(PO4)2 nanosheet; decolouration behavior on the laccase, detailed explanation; CVs of laccase and La-CPN, detailed explanation; relative activity free laccase and La-CPN; 26


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La-CPN for the CR decolouration after cycle, SEM Author Information Corresponding Authors Tel./fax: +86 511 88791800. E-mail: [email protected] (T, Zhang); [email protected] (F. Qiu) Notes The authors declare no competing financial interest. Acknowledgements This work was financially supported by National Nature Science Foundation of China (U1507115 and 21576120), Natural Science Foundation of Jiangsu Province (BK20160500, BK20161362 and BK20161264), Scientific Research Foundation for Advanced Talents, Jiangsu University (15JDG142), the China Postdoctoral Science Foundation (2016M600373) and China Postdoctoral Science Foundation of Jiangsu Province (1601016A), 333 High-Level Personnel Training Project of Jiangsu Province (BRA2016142). References (1) Shu, Z.; Wu, H.; Lin, H.; Li, T.; Liu, Y.; Ye, F.; Mu, X.; Li, X.; Jiang, X.; Huang, J. Decolorization of remazol brilliant blue R using a novel acyltransferase-ISCO (in situ chemical oxidation) coupled system. Biochem. Eng. J. 2016, 115, 56-63. (2) Jo, W.-K.; Tayade, R. J. Facile photocatalytic reactor development using nano-TiO2 immobilized mosquito net and energy efficient UVLED for industrial dyes effluent treatment. J. Environ. Chem. Eng. 2016, 4, 319-327. 27



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Preparation of efficient, stable and reusable laccase-Cu3(PO4)2 hybrid microspheres based on copper foil for decolouration of Congo red Jian Rong, Tao Zhang*, Fengxian Qiu*, Yao Zhu School of Chemistry and Chemical Engineering, Jiangsu University, 301Xuefu Road, Zhenjiang 212013, P. R. China

Table of contents graphic

Synopsis: Laccase-Cu3(PO4)2 hybrid microspheres were synthesized under ambient conditions, which are efficient, stable and reusable for dye decolorizing.

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

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