Graphene oxide-enzyme hybrid nanoflowers for efficient water soluble dye removal

Accepted Manuscript Title: Graphene oxide-enzyme hybrid nanoflowers for efficient water soluble dye removal Authors: Hui Li, Jingwei Hou, Linlin Duan, Chao Ji, Yatao Zhang, Vicki Chen PII: DOI: Reference:

S0304-3894(17)30357-6 http://dx.doi.org/doi:10.1016/j.jhazmat.2017.05.014 HAZMAT 18573

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

21-2-2017 7-5-2017 10-5-2017

Please cite this article as: Hui Li, Jingwei Hou, Linlin Duan, Chao Ji, Yatao Zhang, Vicki Chen, Graphene oxide-enzyme hybrid nanoflowers for efficient water soluble dye removal, Journal of Hazardous Materialshttp://dx.doi.org/10.1016/j.jhazmat.2017.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphene

oxide-enzyme

hybrid

nanoflowers

for

efficient water soluble dye removal Hui Li, a Jingwei Hou, b* Linlin Duan, a Chao Ji, b Yatao Zhang, a** and Vicki Chen b

a

School of Chemical Engineering and Energy, Zhengzhou University, Science Road 100,

Zhengzhou 450001, China b

UNESCO Centre for Membrane Science and Technology, School of Chemical Engineering,

University of New South Wales, Sydney, Australia

E-mail address for corresponding authors: [email protected]; [email protected]

Graphical abstract

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Research highlights 1. A facile method is applied to build a 3D structure for enzyme immobilization. 2. Biocatalytic nanoflower has high laccase loading and improved activity. 3. Nanoflower on electrodes shows improved direct electron transfer efficiency 4.

Efficient organic dye and micropollutant removal is achieved.

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ABSTRACT High efficient enzyme immobilization on carbon based conductive supports could provide wide applications in energy and environmental science. Here, we synthesized a 3D flower-like structured self-assembly hybrid nanocomposite with copper phosphate, laccase, graphite oxide (GO) and carbon nanotubes (CNTs) via a facile one-pot strategy under mild conditions. The prepared nanocomposite exhibited very high enzyme loading and improved laccase activity. During the formation of the nanocomposite, the copper phosphate-laccase petals were interwined by CNTs, and GO nanosheets were further coated on the petal surface. Such a configuration ensured high enzyme loading between the GO sheets and good mass transfer efficiency between immobilized enzyme and substrate, which was confirmed by the kinetics test. We further deposited the immobilized enzyme onto electrodes and observed significantly improved direct electron transfer efficiency. Furthermore, higher dye removal efficiency was observed with the immobilized enzyme. The highly efficient enzyme immobilization strategy provides significant opportunity for its application in bioelectronics and wastewater treatment.

KEYWORDS: enzyme immobilization; self-assembly; graphene oxide; improved enzymatic activity; dye adsorption

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1. Introduction Enzyme technology meets the “green chemistry” trend as it provides high efficient substrate conversion, less by-product, lower energy requirement and lower environmental toxicity when compared with traditional catalysts. As a result, the enzyme catalyzed biocatalytic reaction has been applied in various areas like pollutant removal, biosensor, drug synthesis and food processing.1-4 As the stability and reusability of the bioentities are crucial to their practical applications, the recent progress of enzyme immobilization technology stimulates the advance in the biocatalytic reactor developments and applications. The immobilization supports provide microenvironments which can protect the enzymes from sudden change of ambient conditions like temperature, pH, solvent, mechanical force and inhibitors, allowing better stability and easier biocatalyst reuse.5 So far, the main factors affecting the performance of immobilized enzyme are immobilization methods, immobilization carriers, and enzyme loading.6 Particularly, the structure and property of immobilization carriers and enzyme loading have strong influence on the enzyme performance via the following aspects: 1) mass transfer between substrate and immobilized enzyme; 2) interaction between the immobilized enzyme molecules; 3) surface area and further enzyme loading on the support; and 4) electron transfer from the active site of the enzyme. For example, a hydrophilic support is generally preferable for the immobilization of most enzymes as the interaction between the hydrophobic cores and hydrophobic support surface can change the conformation of the enzyme and subsequently lead to the loss of enzyme activity. Therefore, advanced material synthesis processes have been applied to prepare nanomaterials as immobilization supports such as nanoparticles (0D), nanotubes (1D), nanosheets (2D), and nanocomposites (3D).5 Recently, carbon based nanomaterials, including one-dimensional (1D) 4

carbon nanotubes (CNTs) and two-dimensional (2D) graphene oxide (GO), have been demonstrated to have excellent capabilities for enzyme immobilization and further development of biocatalytic reactor, biofuel cell and biosensor.7-9 These materials have remarkable mechanic and structural characteristics, which ensure high enzyme adsorptive capability. 10-11 In addition, it has been demonstrated that the carbon based material can facilitate the direct electron transfer (DET) from the enzyme’s active site to the support, leading to flexible bio-electrode design for biosensor and biofuel cell without extra electron transfer mediators. 12 However, when compared with the traditional metal based catalysts like Pt, Pd, Ru and Ir, enzyme molecules are large, leading to low catalytic density per unit area.13 For example, a monolayer of a typical enzyme on an electrode only has a theoretical current density of 80 µA cm-2, which is much lower than the Pt based electrode.14 As a result, a multilayer 3D structure is desirable to improve immobilized enzyme’s catalytic density. CNTs and GO can be modified with carboxyl, hydroxyl, epoxide groups, allowing further surface functionalization and metal ion binding to construct a 3D structure for enzyme immobilization.15-19 However, to the best knowledge of the authors, such CNTs and GO based 3D structure for enzyme immobilization has not been reported to date. The improvement of enzyme stability after immobilization is expected as a certainty. However, this is normally accompanied with the loss of activity due to the loss of affinity to substrates, distortion of enzyme’s natural conformation and the random orientation of the immobilized enzyme. Recently, some novel immobilization approaches have been demonstrated to improve the immobilized enzyme activity. Lyu and co-workers applied a relatively facile co-precipitation approach for Cyt c in-situ encapsulation within a 3D metal-organic framework (MOF), with up to 10-fold increase of Cyt c activity.20 The favorable interaction between Zn2+ and the immobilized enzyme, together with the well accessible MOF structure, led the improvement of

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the peroxidase activity. However, the co-precipitation approach was carried out in a methanol solution, which would lead to denaturation for most proteins. Another problem associated with the MOF material is its sub-nanoscale pore aperture which significantly increases the mass transfer resistance for large-sized substrates. For the application of most biocatalytic system, mesoporous structures are considered preferable, allowing easier diffusion of mediators and substrates to the redox sites of the enzymes.13,

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In another line of work, 3D inorganic

nanocrystals with mesopores were applied to anchor enzymes. For example, Ge et al. 22 prepared a copper phosphate-enzyme hybrid flower-like crystal nanostructure, and improved enzymatic activity was observed with epinephrine, norepinephrine, dopamine and phenolic substrates. However, due to the poor conductivity of the copper phosphate crystals, it is difficult to adopt such a hybrid nanostructure for biofuel cell and biosensor development. Another challenge is to achieve high enzyme loading. High loading is preferable as it could effectively reduce the reactor size by providing higher catalytic capability density. However, the enzyme loadings in the MOF and inorganic crystal nanostructures were usually lower than 10 wt %.22-23 Herein we demonstrate a facile one-step 3D self-assembly of GO, CNTs and copper phosphate structure for enzyme immobilization. And our groups have prepared GO/lysozyme nanocomposites with ten-fold higher of antimicrobial activity24 and GO/CNTs/Carbonic anhydrase with better stability than that of the free enzyme (Figure S1-S3, Table S1) via the above technology. Based on the above, Laccase was selected as the model enzyme due to its broad substrates and wide applications in micropollutant removal, biosensor and biofuel cells. 25 Previously we have successfully immobilized laccase onto TiO2 nanoparticles and TiO2 functionalized membranes, but the loss of activity was inevitable.25-27 In this study, we reported a high efficient laccase immobilization on the copper phosphate/CNTs/GO hybrid nanostructure

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(referred as GCCL), which improved both enzyme activity and stability while achieving a very high laccase loading on the support. In addition, the nanostructure without CNTs (referred as GCL) was synthesized as a benchmark to demonstrate the function of CNTs in maintaining hybrid nanostructure and enzyme immobilization. Finally, the bio-electrode performance and the dye removal with the immobilized laccase were investigated to demonstrate the potential application of the 3D nanocomposite. Cumulatively, this work highlighted a facile approach of high efficient enzyme immobilization and its application in energy and environmental technologies.

2. METHODS 2.1 Materials. Graphite powders (spectral pure) were purchased from Sinopharm Chemical Reagent Co., Ltd., and were used as received. Multi-wall carbon nanotubes (CNTs) were purchased from Chengdu Organic Chemicals Co., Ltd., Chinese Academy of Sciences. Laccase (high pure grade) from trametes versicolor was obtained from Hefei Bomei Biotechinology Co., Ltd., China. Dialysis bag with molecular weight cut-off 8000-14000 Da was purchased from Solarbio, China. Disodium hydrogen phosphate dodecahydrate (AR) was purchased from Guangdong Chemical Reagent Engineering-technological Research and Development Center, China. Sodium phosphate monobasic dehydrate (AR) was purchased from Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd., China. Copper (II) sulfate pentahydrate (AR) and other chemicals were purchased from Tianjin Kermel Chemical Reagent Co., Ltd., China, and all chemical reagents were used as received. The water used in the chemical solution preparation was deionized water.

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2.2 Preparation of GO and CNTs-COOH. The preparation of GO was based on the method reported earlier with slight modification.28 1 g CNTs was added into the mixture of 30 ml oil of vitriol (98%) and 10 ml phosphoric acid (60%), followed by ultrasonication with frequency of 60 kHz and power outputs of 300 W under room temperature for 8 h. The mixture was diluted by deionized water and vacuum filtered by sand cored funnel, then washed several times with deionized water, and eventually dried in vacuum freeze dryer for 10 h. 2.3 Fabrication of the GCCL, GCL and CuP-CNTs. 100 mM phosphate buffered saline (PBS, pH=5.7) was preparation by the disodium hydrogen phosphate dodecahydrate solution and sodium phosphate monobasic dehydrate solution. 10 mg GO and 6 mg CNTs-COOH were added into 100 ml PBS, and dispersed by ultrasonication frequency of 60 kHz and power outputs of 300 W for 20 min. 15 mg laccase and 0.667 ml CuSO4 (120 mM) were sequentially added into the solution. The mixture was left in a shake culture box (HZQ-F100 shake culture box, China) under 25 oC, 140 rpm shaking speed for 2 days. Then the solids were separated by centrifugation at 5000 rpm for 10 min. The solids were cleaned several times by deionized water and freezedried. In this work, the nanocomposite prepared without CNTs-COOH was also conducted following the same procedure. The resulted nanocomposite was referred as GCL in this work. 2.4 Determination of Enzyme Loading. The concentration of the enzyme was measured as a protein concentration using the Bradford assay with a UV-vis spectrophotometer. The ratio of enzyme loading was defined by Eq. (1): Ratio loading (%) =

MI 100 MT

(1)

Where MI and MT are immobilized and initially added enzyme amounts.

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2.5 Activity Assay of Free and Immobilized Enzymes. Laccase activity was determined by measuring the oxidation of 2 mM ABTS buffered with 50 mM citrate phosphate buffer (pH 5.4). Formation of the cation radical was monitored at 420 nm (Ɛ420=36.0 mM-1 cm-1) at room temperature. In terms of free laccase, the reaction mixture (3 ml) contained 0.045 mg of free laccase. For the immobilized laccase, GCCL, GCL and CuP-CNTs containing 0.045 mg immobilized laccase were incubated in 3 ml ABTS solution. Prior to the UV test, the suspension liquid was retracted from the mixture and quickly filtered through a 0.1 µm syringe filter. In this work, one unit of activity was defined as the amount of enzyme needed to oxidize 1 µmol of ABTS per minute. 2.6 Calculation of the kinetic parameters. The Michaelis-Menten kinetic constant values for both free and immobilized laccase were determined by measuring the enzyme activity with ABTS as substrates under various concentrations ranging from 0.08 to 2 mM. The values of the kinetic values were obtained by non-linear curve fitting of the plot of the reaction rate versus substrate concentration. More detailed process could be found in our earlier publication. 26 2.7 Electrochemical Characterization. The cyclic voltammetry pattern was measured using the CHI 650D Electrochemical Workstation, and the cyclic voltammetry curve was obtained with CHI 650D software. In this work, the glass carbon (GC) electrode was the working electrode, the platinum electrode (2 mm in diameter) was the counter electrode, and the Ag/AgCl electrode was the reference electrode. The measurement was carried out in a potential range of -0.8 V to +1.0 V (vs Ag/AgCl) with a scan rate of 100 mV/s. In terms of the sample electrode preparation, the GC electrode was polished with alumina slurry and rinsed thoroughly with Milli-Q water and absolute ethanol. 10 ml of 0.5 mg/ml GCCL or GCL suspension was filtered through a 20 cm2 0.45 µm PVDF membrane, then the GCCL or

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GCL layer was carefully peeled off from the membrane and attached onto the GC electrode surface. 20 µl of Nafion was dropped on the electrode surface, wetted the GCCL or GCL layer and allowed to dry for 10 min. The final sample electrode contained around 7 µg of immobilized laccase. The sample electrode only with Nafion, GO, CNTs and free laccase were prepared with similar process. The cyclic voltammetry test was carried out under O2 saturated 0.1 M KCl electrolyte. 2.8 Removal of Crystal Violet (CV) and Neutral Red (NR). In this work, two dye solutions were selected to explore the dye removal by both free and immobilized laccase. For the biocatalytic dye removal, 3.66 mg free or immobilized laccase was suspended into 100 ml dye solution (initial concentration of CV 2.5 mg/L and NR 7.5 mg/L). The suspension was incubated under room temperature under constant stirring, and the dye removal was determined with certain time interval with UV-vis spectrophotometer. In order to examine the effect of the GO adsorption, thermal inactivated GCCL was applied in this work as a benchmark. In brief, the prepared GCCL were treated by calcination for an hour at 100 ˚C using a tube furnace under the protection of nitrogen.

3. RESULTS AND DISCUSSIONS 3.1 Characterization of nanocomposite. Herein, we constructed a 3D structure with laccase, GO, CNTs and Cu3(PO4)2·3H2O. The morphology of the GCL and GCCL were analyzed with the scanning electron microscope (FEI Nova NanoSEM 230 FESEM), and the results were shown in Figure 1. In this work, both GCL and GCCL formed microscale particles with nanoscale flowerlike 3D structures. Such an observation was in accordance with the previously reported proteincopper phosphate hybrid structure.22 The separated individual flower-like particles had very minor aggregations. In terms of the nanoscale hierarchical petals the flower petal for GCCL was

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well exposed (Figure 1 b&c). In comparison, the flower petals of GCL were mostly covered by GO sheet layers (Figure 1 d). Furthermore, the size of the nanocomposite particles was analyzed by DLS (Malvern Zetsizer Nano), and the mean diameter of GCL and GCCL were 3.6 ± 0.4 and 3.3 ± 0.2 µm. This value was in good agreement with the SEM images. Such an observation confirmed the addition of GO and CNTs did not lead to the aggregation of the hybrid particles, which ensured high surface-to-volume ratios, benefiting the enzyme immobilization and the mass transfer. The nanoscale structure of the flower petals was further investigated with the transmission electron microscope (TEM, FEI, TECNAI G2) methods. As shown in Figure 1 e-f, the nanoscale copper phosphate crystals were covered by GO sheet for both GCL and GCCL. In terms of GCCL, the addition of CNTs introduced more wrinkles for the GO sheets. Such an observation suggested the formation of the inter-layer channels by CNTs, which acted as a spacer between neighboring GO sheets. As shown in Figure 2, the XRD pattern of pure GO exhibited a sharp diffraction line at 9.5º, which was in good agreement with other works. 29 The XRD diffraction line for GO sheets shifted to 7.7º (for GCL) and 7.4º (for GCCL). Such an observation suggested the increase of the GO layer spacing. The crystallization of copper phosphate acted as a spacer between GO layers for GCL, and further the intercalation of CNTs increased the spacing between the neighboring GO sheets. In this work, the XRD results for GCL and GCCL did not show obvious peaks of CNTs (XRD peak at 20º and 40º) and copper phosphate crystals (XRD peak at 25º).30-31 This observation suggested that the surface of GCCL and GCL was mainly covered by GO sheets. Furthermore, Raman spectroscopy (Nanofinder 30) (Figure 3) was employed to study the change of surface chemical properties of the hybrid nanocomposite. The characteristic D band (1352 cm-1) and G bands (1591 cm-1) were attributed to the local

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defects/disorders and the sp2 graphitized structure. Thus, smaller ID/IG peak intensity ratios were assigned to lower defects/disorders. As depicted in Figure 3, the addition of laccase and copper phosphate did not change the ID/IG peak intensity ratio for GCL, while the addition of CNTs slightly reduced the ID/IG peak intensity. This observation confirmed the presence of CNTs in the GCCL nanocomposite. Similarly, the peaks of laccase and copper phosphate were not clearly observed in the Raman spectrum of GCL and GCCL, indicating the laccase and copper phosphate were mainly located inside the nanocomposite matrix.32 As a result, the GO layer structure would impose significant effect on the immobilized laccase performance. The schematic diagram of the proposed nanocomposite formation process is displayed in Figure 4, and the SEM images during the GCCL and GCL formation process are presented in Figure S12. Initially, primary copper phosphate crystals formed immediately when mixing copper ions with the phosphate ions, and laccase was encapsulated within the nanoscale crystals.22 The amine backbone of laccase molecules could further form coordination with the copper crystals, which facilitated the formation of laccase-copper phosphate lamella.22,

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Such a lamella acted as

nucleation center for further crystal growth, and the protein on the crystal served as a joint to link the copper phosphate lamella in an edge-to-edge way, forming the backbone of the flower-like structure (Figure 4 c, Figure S2 c and Figure S3 c). In terms of the GO and CNTs, due to the abundance of oxygen-containing groups on their surface, the electrostatic interaction and coordination between Cu2+ and carboxyl groups of GO/CNTs could occur.34-35 As presented in Figure S2 c, after 4 hours’ growth of the GCCL, the lamella was interwined with CNTs. Such a lamella complex stabilized the 3D structure of the GCCL.28 Further attachment of GO onto the lamellas complex forms a cage-like structure which encapsulated laccase within the neighboring GO sheets and between the GO and the lamella complex. In terms of the GCCL, the accessibility

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of the encapsulated laccase could be better preserved due to the CNTs acting as the interlayer spacer between GO sheets. In comparison, in the absence of CNTs (GCL), the GO sheets would form a more compact layer-by-layer structure on the lamella complex, which might be beneficial for the enzyme encapsulation but it also limited the accessibility of the enzyme by substrate. 3.2 Activity Assay. One major challenge of enzyme immobilization is to achieve high loading of enzyme and to preserve high apparent activity. In this work, the 3D nanocomposite structure was applied for laccase encapsulation and the immobilized enzyme performance is presented in Table 1. In terms of laccase loading, GCCL, GCL and CuP-CNT could all achieve over 30 wt % of laccase loading, and slightly higher loading was obtained with GCL. Compared with copper phosphate-laccase nanocomposite without GO and CNTs (less than 10 wt % of enzyme loading),22 the high laccase loading achieved in this work could be attributed to the 3D structure formed by GO sheets and CNTs. Therefore, the structural property of the GO layers would impose significant effect on the immobilized laccase performance. In terms of the activity recovery, an increase of 15 % laccase activity was observed with GCCL. In comparison, laccase immobilized on GCL only preserved 67.3 % of its original activity, this could be due to the intermolecular steric hindrance.36-38 The increase of activity could be attributed to the following aspects: the strongly negatively charged CNTs and GO sheets absorbed the substrate to the nanocomposite surface and increased the contacting of the substrate and immobilized enzyme; the CNTs acted as the nanospacer between the GO sheets and increased the mass transfer efficiency; the highly conductive GO and CNTs facilitated the electron transfer from the active site; and the crystallization of copper phosphate with laccase formed preferable orientation or conformation with exposed laccase activity site. 22 In order to better understand the effect of immobilization, the kinetic parameters of both free and immobilized laccase were studied in this

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work. Km for free laccase was 13.7 µM. The increase of Km after immobilization is expected due to the increased mass transfer resistance and limited accessibility of active sites. In this work, the Km for GCL increased to 15.3 µM, indicating the encapsulated laccase was covered by GO sheets and less accessible to the substrate. However, after the addition of CNTs, the nanotube performed as a nanospacer between different GO layers which made the immobilized laccase more accessible. These aspects led to the reduction of Km for GCCL when compared with free enzyme and GCL. In terms of the laccase catalytic efficiency, the decrease of kcat/Km for GCL indicated the loss of laccase efficiency to convert substrate into product, which could be attributed to the distortion of natural laccase conformation, resulted from the over-crowded enzyme within the firmly packed GO sheets. However, the GCCL had an improved kcat/Km compared with free laccase. The reason could be the improved electron transfer efficiency between different active clusters on laccase molecule due to the addition of CNTs.39-40 On the other hand, the presence of Cu2+ ions could also improve the enzyme activity.22 The improved activity of laccase for GCCL, together with the kinetic parameters, confirmed the synergistic effect of the GO, CNTs and copper phosphate crystals. The addition of CNTs for GCCL could facilitate the direct electron transfer from the active site of laccase, and such property would be beneficial for further development of biosensors and biofuel cells. This aspect will be discussed in the next section. The immobilization of laccase on various supports has been well documented in literature, and the results of some recent literatures were summarized in Table 3. Due to different enzyme source, support material and immobilization process applied, it is difficult to directly compare the laccase immobilization performance. Generally, the immobilization support with 3D structure could provide higher surface area and more enzyme anchoring position, thus its laccase

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loading was relatively higher. The highest laccase loading reported was on porous microspheres.41 Most immobilization was accompanied with the loss the activity: up to 90 % of activity could be preserved with nanoparticles and nanotubes.42-43 The laccase immobilization performance reported in this work exhibited significant improvement in terms of loading and activity recovery when compared with the previous reported by our group.41 A much higher activity recovery (650 %) was reported when immobilize laccase onto copper phosphate crystal.22 This could be partially attributed to different substrate applied. Syringaldazine had smaller molecular size than ABTS, which ensured better substrate mass transfer efficiency for the immobilized laccase. However, the laccase loading on the copper crystals was relatively low, and the poor conductivity of the copper phosphate crystals limited its further application in biosensors and biofuel cells. To further investigate the time-dependent storage stability of the immobilized laccase, the free and immobilized laccase (GCCL) were stored at room temperature in a phosphate buffer solution of pH 5.4 (Figure 5). The free laccase lost over 50 % of its activity during the initial 4 days. In comparison, the GCCL still preserved over 80 % of its original activity after 6 days’ storage. Another important concern is the gradual release of Cu2+ ions due to its toxicity towards aqueous environment. At the end of the storage test, the suspension solution (total volume of 100 ml) was filtered and the permeation liquid was tested by ICP-MS. The result indicated the Cu2+ ion content was lower than 100 ppm, indicating great stability of the copper phosphate crystal and attached laccase. This observation also indicated the gradual loss of the GCCL activity was due to the change of the laccase conformation rather than the enzyme detachment from the support. Furthermore, thermal stability of free laccase and immobilized laccase were also observed under different temperature ranging from 30 oC to 70 oC for 1 h (Figure 6). The activity of free laccase

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and immobilized laccase decreased as temperature increased from 30 oC to 70 oC. For free laccase, only 57 % of the maximum activity was preserved at 70 oC, while immobilized laccase were more stable at high temperature (70 oC) and retained 71%, 69% and 68% of their initial biological activities ( GCCL, GCL, CuP-CNT), separately. And the results revealed that it could react in harsh environments. It is consistent with those of the previous study that the increased thermal property of the immobilized enzymes is contributed to the conformation of laccase after immobilization. 44-45 3.3 Electrochemistry of the immobilized laccase. The cyclic voltammetry of the immobilized laccase was investigated in this work with the scanning range of -0.8 V to + 1.0 V with a scanning rate of 100 mV/s. The results are shown in Figure 7. Background tests carried out with the GC electrode, Nafion, CNTs and GO had peaks around -500 mV (vs. Ag/AgCl) (Figure a), which was attributed to the reduction of the dissolved oxygen in the electrolyte. 46 For the CNTs sample, the peak around 350 mV (vs. Ag/AgCl) indicated the oxidization of CNTs occurred under this potential. In terms of laccase, its main electrocatalytic property is to couple the reduction reaction of oxygen to water by a four-electron transfer process. In this work, both GCCL and GCL had a well-defined redox wave with a mid-point of around 180 mV (vs. Ag/AgCl) (Figure 7b), which was consistent with the T1 copper reduction potential determined by potentiometric titration and electrochemical characterization.47 Compared with free laccase, the reduction peaks for dissolved oxygen and T1 copper became much more distinct, indicating the significant improvement of the catalytic capability and the electron transfer efficiency for the immobilized laccase, especially for GCCL. The oxidizing wave of CNTs was also observed on GCCL (350 mV, vs. Ag/AgCl). The presence of CNTs in

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GCCL ensured the intimate contact between the enzyme’s active site and conductive support, facilitating the direct electron transfer process. 3.4 Removal of CV and NR. Batch experiments were conducted to investigate the aqueous dye removal performance by both free and immobilized enzyme. The background adsorption of GO and CNTs were also investigated with the thermal inactivated GCCL. Figure 8 shows the removal of dyes after 8 days’ incubation. While all test results showed certain level of dye removal, the complete dye removal was only achieved with GCCL. The laccase catalyzed dye removal has been well documented.48-49 However, the removal efficiency of dyes by laccase would be negatively affected due to the rapid loss of free enzyme activity. In this work, improved dye removal was observed with the immobilized laccase. This observation could be attributed to the enhanced enzyme stability. In addition, the GO and CNTs were absorbent for dye owing to the electrostatic interaction and the π-π stacking interaction.50-52 The background adsorption test with the thermal inactivated GCCL also indicated around 70 % removal for CV and around 45 % removal for NR after 8 days’ treatment. The adsorbed dye would also improve the affinity between immobilized laccase and substrates, which eventually benefited the biocatalytic dye removal. It should be noted that the structure of nanoflower did not experience significant change after the degradation test, indicating the structure was relatively stable.

4. CONCLUSIONS In this work, we have demonstrated a facile synthesis of a novel 3D structure under mild conditions for enzyme immobilization. The nanocomposite exhibited excellent laccase loading capacity, and the laccase activity increased by 15 % when compared with equal amount of free laccase. After storage under room temperature for 6 days, the nanocomposite still preserved over

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80 % original activity for laccase, compared with around 30 % for free laccase. The improved laccase activity could be attributed to the GO and CNTs composite structure, which ensured good affinity between laccase and substrates and enhanced electron transfer efficiency. An additional advantage observed in this work was the improved electrical properties of the immobilized enzyme. The immobilized laccase could more efficiently catalyze the reduction reaction of the dissolved oxygen and the released electrons could be better transferred due to the good conductivity of GO and CNTs. Based on the dye removal test with CV and NR, the immobilized laccase exhibited significantly improved removal performance compared with free laccase. As such, this hybrid nanocomposite 3D material offers an opportunity to broaden its application in pollutant removal, biosensor and biofuel cell preparation.

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Acknowledgment This work was financially sponsored by the National Natural Science Foundation of China (No. 21376225) and Excellent Youth Development Foundation of Zhengzhou University (No. 1421324066). We sincerely acknowledge the financial assistance of visiting research program in University of New South Wales by the China Scholarship Council (No. 201208410135). References [1] D. Wesenberg, I. Kyriakides, S.N. Agathos, White-rot fungi and their enzymes for the treatment of industrial dye effluents, Biotechnology Advances, 22 (2003) 161-187. [2] N.C. Foulds, C.R. Lowe, Enzyme entrapment in electrically conducting polymers. Immobilisation of glucose oxidase in polypyrrole and its application in amperometric glucose sensors, Journal of the Chemical Society Faraday Transactions, 82 (1986) 12591264. [3] K.P. Rao, Recent developments of collagen-based materials for medical applications and drug delivery systems, Journal of Biomaterials Science Polymer Edition, 7 (1996) 623-645. [4] A. Amine, H. Mohammadi, I. Bourais, G. Palleschi, Enzyme inhibition-based biosensors for food safety and environmental monitoring, Biosensors & Bioelectronics, 21 (2006) 14051423. [5] D.H. Zhang, L.X. Yuwen, L.J. Peng, Parameters Affecting the Performance of Immobilized Enzyme, Journal of Chemistry, 7 (2013) 946248. [6] J.N. Talbert, J.M. Goddard, Enzymes on material surfaces, Colloids & Surfaces B Biointerfaces, 93 (2012) 8-19. [7] H. Chang, H. Wu, Graphene-based nanocomposites: preparation, functionalization, and energy and environmental applications, Energy & Environmental Science, 6 (2013) 34833507. [8] E.T. Thostenson, Z. Ren, T.W. Chou, Advances in the science and technology of carbon nanotubes and their composites: a review, Composites Science & Technology, 61 (2001) 1899-1912. [9] X. Wen, D. Zhang, T. Yan, J. Zhang, L. Shi, Three-dimensional graphene-based hierarchically porous carbon composites prepared by a dual-template strategy for capacitive deionization, Journal of Materials Chemistry A, 1 (2013) 12334-12344. [10] D.T. Pham, T.H. Lee, D.H. Luong, F. Yao, A. Ghosh, V.T. Le, T.H. Kim, B. Li, J. Chang, Y.H. Lee, Carbon Nanotube-Bridged Graphene 3D Building Blocks for Ultrafast Compact Supercapacitors, Acs Nano, 2015, 9(2): 2018-2027. [11] X.L. Pang, H.L. Peng, H.S. Yang, K.W. Gao, X.L. Wu, A.A. Volinsky, Comparative study of methylene blue dye adsorption onto activated carbon, graphene oxide, and carbon nanotubes, Chemical Engineering Research & Design, 91 (2013) 361–368. [12] D. Leech, P. Kavanagh, W. Schuhmann, Enzymatic fuel cells: Recent progress, Electrochimica Acta, 84 (2012) 223–234. [13] X.Y. Yang, T. Ge, J. Nan, B.L. Su, Immobilization technology: a sustainable solution for biofuel cell design, Energy & Environmental Science, 5 (2012) 5540-5563. 19

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Figure 1 SEM and TEM images of the biocatalytic nanocomposite material: (a) (b) (c) SEM image of GCCL under different magnifications; (d) SEM image of GCL; (e) TEM image of the GCCL petal structure and (f) TEM image of GCL surface structure.

23

Figure 2 XRD patterns of GCCL (Red line), GCL (green line) and GO (black line).

24

Figure 3 Raman spectra of GCCL (Red line), GCL (green line) and GO (black line).

25

Figure 4 Preparation procedure and proposed crystal growth process: (a) primary crystallization of the copper phosphate and laccase; (b) growth of the copper phosphate lamella; (c) interconnect of separated lamellas and (d) crystal growth and the formation of final flower-like structure

26

Figure 5 Storage stability of free and immobilized laccase as a function of time at room temperature.

27

Figure 6 Thermal stability of free laccase and immobilized laccase stored at different temperature ranging from 30 oC to 70 oC for 1 h.

28

(a)

2.0x10

-5

1.0x10

-5

Current (A)

0.0 -1.0x10

-5

-2.0x10

-5

-3.0x10

-5

-4.0x10

-5

-5.0x10

-5

GC Eletrode Nafion CNTs GO

-0.8

-0.4

0.0

0.4

0.8

1.2

Potential (V)

Current (A)

( b) -5 6.0x10

4.0x10

-5

2.0x10

-5

Pure Laccase GCL GCCL

0.0

-2.0x10

-5

-4.0x10

-5

-0.8

-0.4

0.0

0.4

0.8

1.2

Potential (V) Figure 7 Representative cyclic voltammetry of the free and immobilized laccase.

29

Figure 8 Removal of Crystal Violet (CV) (I) and Neutral Red (NR) (II) at pH 5.4, 25oC: (a) benchmark of 2.5 mg/L CV; (b-e) the final effluent of CV after treated by free laccase, GCL, thermal inactivated GCCL, GCCL; (f) benchmark of 7.5 mg/L NR; (g-j) the final effluent of NR after treated by free laccase, GCL, thermal inactivated GCCL, GCCL.

30

Table 1 Performance of the immobilized laccase Apparent activity

(U/mg support)

Laccase activity (U/mg laccase)

Laccase loading (µg/mg support)

Activity recovery (%)

Free laccase

-

4.1

-

-

GCL

1.1

2.8

384.7

67.3

GCCL

1.6

4.7

335.7

115

CuP-CNTs

0.57

1.8

317.8

43.9

31

Table 2 Kinetic parameters of the laccase Free laccase

GCL

GCCL

Km

13.7 µM

15.3 µM

9.9 µM

kcat

69.4 µmol s-1 g-1

44.4 µmol s-1 g-1

98.0 µmol s-1 g-1

kcat/Km

5.1 L s-1 g-1

2.9 L s-1 g-1

9.89 L s-1 g-1

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Table 3 Summary of the laccase immobilization on various supports Sources of laccase Trametes versicolour Trametes versicolor Aspergillus oryzae Trametes versicolor Trametes versicolor Trametes versicolor Trametes versicolor Pleurotus ostreatus Myceliophthora thermophila

Laccase loading (mg/g) 384.7 335.7

Activity recovery (%) 67.3 115

98.1

92.5 (catechol)

34

Mesoporous silica (0D)

187

-

43

TiO2 nanoparticles (0D)

Up to 25

90 (ABTS)

20

168.8

90 (ABTS)

35

150

15 (ABTS)

Support type GCL (3D) GCCL (3D) Magnetic mesoporous silica nanoparticles (0D)

Halloysite nanotubes (1D) CNTs (1D)

References This study

44

GO (2D)

270

35 (ABTS)

311.2

80 (ABTS)

33

nanofibrous membrane (3D)

220

72 (ABTS)

45

Mesoporous (3D)

187

-

46

~ 44

38(ABTS)

22

90

650 (syringaldazine)

17

Porous microspheres (3D)

organosilicas

Trametes versicolor

TiO2 coated membranes (3D)

PVDF

Trametes versicolor

Copper nanoflower (3D)

phosphate

33