Reduced Graphene Oxide Particles for Efficient Enzyme Immobilization

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Eco-friendly Composite of Fe3O4-Reduced Grapene Oxide Particles for Efficient Enzyme Immobilization Sanjay Kumar Singh Patel, Seung Ho Choi, Yun Chan Kang, and Jung-Kul Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05165 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 24, 2016

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ACS Applied Materials & Interfaces

Eco-friendly Composite of Fe3O4-Reduced Grapene Oxide Particles for Efficient Enzyme Immobilization

Sanjay K. S. Patela, ‡, Seung Ho Choib,‡, Yun Chan Kangb,*, Jung-Kul Leea,*

Addresses: aDepartment of Chemical Engineering, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul, 143-701, Republic of Korea; bDepartment of Materials Science and Engineering, Korea University, Anam-Dong, Seongbuk-Gu, Seoul, 136-713, Republic of Korea ‡These authors contributed equally to this work.

*

Correspondence authors. E-mail: [email protected], (Jung-Kul Lee, Fax: (+82) 2-458-3504),

[email protected] (Yun Chan Kang, Fax: (+82) 2-928-3584)

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ABSTRACT A novel type of spherical and porous composites were synthesized to dually benefit from reduced graphene oxide (rGO) and magnetic materials as supports for enzyme immobilization. Three magnetic composite particles of Fe3O4 and rGO containing 71% (rGO-Fe3O4-M1), 36% (rGO-Fe3O4-M2), and 18% (rGO-Fe3O4-M3) Fe were prepared using a one-pot spray pyrolysis method, and were used for the immobilization of the model enzymes, laccase and horseradish peroxidase (HRP). The rGO-Fe3O4 composite particles prepared by spray pyrolysis process had a regular shape, fine size, and uniform composition. The immobilization of laccase and HRP on rGO-Fe3O4-M1 resulted in 112% and 89.8% higher immobilization efficiency than that of synthesized pure Fe3O4 and rGO particles, respectively. The stability of laccase was improved by approximately 15-fold at 25 °C. Furthermore, rGO-Fe3O4-M1-immobilized laccase exhibited 92.6% of residual activity after 10 cycles of reuse, and was 192% more efficient in oxidizing different phenolic compounds than the free enzyme. Therefore, these unique composite particles containing rGO and Fe3O4 may be promising supports for the efficient immobilization of industrially important enzymes with enhanced electrochemical properties and lower acute toxicity towards Vibrio fischeri than commercial pure Fe3O4 particles. Keywords: Acute toxicity; Immobilization; Laccase; Magnetic composite particle; One pot synthesis; Stability

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INTRODUCTION Graphene oxide (GO) based materials have diverse biotechnological and industrial applications.15

Due to their large surface area and structural properties, they have become suitable support

materials for the immobilization of various enzymes.4-7 Enzyme immobilization is considered a suitable method to provide stability and facilitate reuse. Several enzyme immobilization methods have been designed based on physical, covalent, or affinity interactions.8 However, a major challenge that remains is the significant loss of enzyme activity after immobilization.4,9,10 Among the different types of immobilization supports, nanostructure composite and hybrid materials are gaining significant attention for their potential to improve immobilization efficiency.11 Thus, the interaction between enzyme and support is critical to achieve high enzyme activity after immobilization, which is significantly influenced by the properties of both the enzyme and the support. GO-based materials interact with enzymes through covalent bonding via a cross-linker for the groups present on the enzyme surface, or through non-covalent bonding such as electrostatic, van der Waals, π-π stacking, and/or hydrophobic interactions. These materials have the ability to immobilize different types of enzymes irrespective of their properties by modification or reduction of functional groups on the surface.4,5 Recently, various efforts have been made to achieve high stability and reusability after enzyme immobilization, as well as to make improvements in enzyme loading through different immobilization methods.4,12-15 To date, various support and composite materials have been tested for the immobilization of enzymes.4,13,15-17 Compared with pure support or non-magnetic materials, magnetic composite particles are widely used for enzyme immobilization due to the ease of separation by magnetic fields.18-20 For efficient enzyme immobilization, there is still

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ongoing debate regarding different support materials due to significant variations in their properties and enzyme-loading capacity. Horseradish peroxidase (HRP, EC 1.11.17) and laccase (EC 1.10.3.2) are the most industrially important enzymes used as model systems for immobilization studies.4,6,21-26 Laccase has gained significant attention due to its broad substrate specificity for the oxidation of various phenolic and non-phenolic compounds in the presence of mediators.27-31 Industrial effluents contain various toxic compounds, which can have severe effects on the environment and living organisms. Thus, methods to instantly remove, minimize concentrations below the toxic levels, or degrade the toxins are urgently required. While GO particles are well-known for their enzyme immobilization abilities, few reports are available regarding the immobilization of enzymes on reduced GO (rGO) and its composite particles with magnetic materials.16,17,32,33 Thus, the synthesis of different composite structures with different properties may provide great opportunities to improve enzyme immobilization loading and efficiency. In the current study, spherical-shaped magnetic composite particles of Fe3O4 and rGO (rGO-Fe3O4-M) with a fine size and varying Fe3O4 content were synthesized and successfully used as support for the immobilization of laccase and HRP as model enzymes. The results of this study demonstrate that rGO-Fe3O4 composite particles prepared by one-pot spray process are suitable supports to achieve high loading and efficiency of immobilized enzymes. Furthermore, the stability and electrochemical properties of immobilized laccase were significantly improved as compared to free form of enzyme. Additionally, the low acute toxicity of synthesized rGO-Fe3O4-M1 particles towards Vibrio fischeri suggests that they are more environment friendly than commerciallyavailable Fe3O4 particles.

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RESULTS AND DISCUSSION Synthesis and characterization of composite particles

Scheme 1. Schematic diagram for the synthesis and immobilization of enzyme on rGO-Fe3O4 composite particles.

In this study, the low-cost spray pyrolysis process (Figure S1) for large-scale production of rGO-Fe3O4 composite particles with regular shapes, fine size, and uniform composition was applied. In the spray pyrolysis process, the characteristics of the rGO-Fe3O4 composite particles, such as the ratio of rGO and magnetic Fe3O4 nanoparticles and mean size, could be easily controlled by changing the preparation conditions.34 Therefore, the spray pyrolysis process was efficient for the preparation of magnetic rGO-Fe3O4 composite particles for enzyme immobilization. The synthesis scheme of the rGO-Fe3O4 composite particles with different magnetic material content using a spray pyrolysis process is shown in Scheme 1. Briefly,

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spherical composite particle with uniform distribution of iron chloride and GO nanosheets was formed as an intermediate product by drying of droplet, and then combustion of the powder at the rear part of the reactor under an argon (Ar) atmosphere produced the rGO-Fe3O4 composite particles. Decomposition of iron chloride under the Ar atmosphere led to the formation of oxygen-deficient Fe3O4, and thermal reduction of the GO nanosheets formed the rGO nanosheets. In the spray pyrolysis process, one composite particle was formed from one droplet containing iron chloride and GO nanosheets without losing any of the components except for the water solvent. Therefore, the Fe3O4 content in the rGO-Fe3O4 composite particles was easily controlled by changing the concentration of iron chloride dissolved in the colloidal solution of the GO nanosheets. In the current study, three rGO-Fe3O4 composite particles with different magnetic material content were prepared by the simple one-step process shown in Scheme 1. These particles contained 71, 36, and 18% Fe and were designated as rGO-Fe3O4-M1, rGO-Fe3O4-M2, and rGO-Fe3O4-M3, respectively.

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Figure 1. TEM (a,c,e) and HR-TEM (b,d,f) images of the rGO-Fe3O4 composite particles prepared using a one-pot spray pyrolysis method: a,b) rGO-Fe3O4-M1, c,d) rGO-Fe3O4-M2, and e,f) rGO-Fe3O4-M3.

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The morphologies of the rGO-Fe3O4-M1, rGO-Fe3O4-M2, and rGO-Fe3O4-M3 particles obtained by the spray pyrolysis process are shown in Figure 1. The particles had crumpled structures irrespective of their compositions due to the high aspect ratio of the GO nanosheets. The high-resolution transmission electron microscopy (HR-TEM) images shown in Figures 1b, 1d, and 1f revealed that there was an increase in the mean size of the Fe3O4 nanocrystals corresponding to increased Fe3O4 content in the rGO-Fe3O4 composite particles. The Martin diameters of the Fe3O4 nanocrystals as determined on the basis of the HR-TEM images of rGOFe3O4-M1, rGO-Fe3O4-M2, and rGO-Fe3O4-M3 composite particles were 15 ± 2.7, 9 ± 1.3, and 7 ± 1.1 nm, respectively. The rGO-Fe3O4-M1, rGO-Fe3O4-M2, and rGO-Fe3O4-M3 composite particles showed similar mean sizes when measured from the scanning electron microscope (SEM) images (Figure S2). The morphology of the composite particles changed from sphericallike shape to crumpled shape with increasing the rGO content in the composite particles. The selected area electron diffraction (SAED) patterns shown in Figures S3 confirmed the formation of Fe3O4 nanocrystals within the rGO matrix. The elemental mapping images shown in Figure S4 revealed the uniform distribution of the ultrafine Fe3O4 nanocrystals over the spherical rGO matrix. The X-ray diffraction (XRD) pattern shown in were 16, 9, and 8, respectively. The thermogravimetric (TG) data of the GO, rGO, and rGO-Fe3O4-M particles are shown in Figures S5 and 2b, respectively. In the TG curve, the GO powders showed a two-step weight decrease by adsorbed water evaporation and thermal reduction of GO into rGO at temperatures below 300°C and combustion of rGO at temperatures between 400°C and 500°C. However, the pure rGO powders showed a one-step weight decrease by combustion of rGO at temperatures between 400°C and 500°C. The TG curves of the rGO-Fe3O4-M particles also showed a two-step weight decrease by thermal

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Figure 2. Characteristics of the rGO-Fe3O4 composite particles prepared by a one-pot spray pyrolysis method: a) XRD patterns, b) TGA analysis, and c) N2 adsorption and desorption isotherms.

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reduction of GO into rGO below 300°C and combustion of rGO under atmospheric conditions at temperatures between 400°C and 500°C. The weight loss due to thermal reduction of GO into rGO was minimized by the increase in the weight caused by the oxidation of Fe3O4 into Fe2O3. The rGO content (equivalent to % weight) of the rGO-Fe3O4-M1, rGO-Fe3O4-M2, and rGOFe3O4-M3 composite particles, as measured by thermogravimetric analysis (TGA), was 32%, 58%, and 61%, respectively (Figure 2b). The isotherms of the composite particles shown in Figure 2c had a clear hysteresis loop that resembles type-H2 IUPAC (International Union of Pure and Applied Chemistry) isotherm classification, demonstrating the existence of mesopores. The Brunauer-Emmett-Teller (BET) surface areas of the rGO-Fe3O4-M1, rGO-Fe3O4-M2, and rGO-Fe3O4-M3 composite particles were 30.0, 136, and 177 m2/g, respectively. The size distribution of the rGO-Fe3O4-M1 composite particles measured by dynamic light scattering analysis is shown in Figure S6. The mean particle size and relative standard deviation of the rGO-Fe3O4-M1 composite particles, as measured by dynamic light scattering analysis, were 900 nm and 21%, respectively. The magnetic properties of the rGO-Fe3O4-M1 composite particles were evaluated using a vibrating sample magnetometer (VSM) at room temperature. A hysteresis loop for the rGO-Fe3O4-M1 composite particles is shown in Figure S7. The saturation magnetization of the rGO-Fe3O4-M1 particles was 38 emu g-1. The chemical state of the Fe component in the rGO-Fe3O4-M1 composite particles was characterized by X-ray photoelectron spectroscopy (XPS) analysis (Figure S8). The main peaks of the rGO-Fe3O4-M1 composite particles (Fe 2p spectrum) occurred at binding energies of 710.4 eV for Fe 2p1/2 and 724.4 eV for Fe 2p/2; these are characteristic of the Fe3O4 phase.35 The morphologies and crystal structures of the pure Fe3O4 and rGO powders formed as comparison samples using spray pyrolysis process are shown in Figures S9 and S10. The pure Fe3O4 powders without phase impurity showed

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completely spherical shape and filled structures (Figure S9). However, the pure rGO powders exhibited crumpled structures (Figure S10). The BET surface areas of the pure Fe3O4 and rGO powders were 2 and 145 m2/g, respectively (Figure S11).

Immobilization of enzymes A diagram of the enzyme immobilization process is presented in Scheme 1. GO is a well-known support material for the immobilization of enzymes.1,4,6,7,24 Primarily, the immobilization of enzymes on pure GO particles has resulted in a significant loss of their activity.4,5 To improve immobilization efficiency and enzyme activity, various modifications such as chemical reduction of GO and composite with magnetic material have previously been adopted.4-6 Here, the combined beneficial effects of rGO and the magnetic nature of synthesized composite particles (rGO-Fe3O4-M1) were employed for the immobilization of laccase and HRP as model enzymes. To compare the variables of the immobilization process, two additional synthesized pure magnetic (Fe3O4) and rGO particles were used as immobilization support controls. Enzyme immobilizations were carried out through adsorption on synthesized rGO-Fe3O4 (-M1, -M2, andM3) and control particles. Immobilization yields (IYs) of laccase and HRP on these supports were in the ranges of 49.0-91.1% and 41.8-86.4%, respectively (Table S1). Among the different particles, maximum IYs of 91.1% and 86.4% for laccase and HRP were observed on rGO-Fe3O4M1, respectively. The immobilization profiles of laccase and HRP at different pH values are presented in Figure S12. The optimum pH values for laccase adsorption were 5, 5, and 4, whereas for HRP they were 6, 7, and 7 on rGO-Fe3O4-M1, rGO, and Fe3O4 particles, respectively. Laccase and HRP immobilized on different synthesized particles resulted in immobilization efficiencies (IEs) in the ranges of 45.3-112% and 39.3-89.8%, respectively

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Figure 3. Activities of immobilized laccase and HRP on different particles. The activity of free or immobilized enzyme was measured under standard assay conditions at room temperature. Free enzyme activity was considered as 100%.

(Table S1). RGO-Fe3O4-M1 particles showed significantly higher IEs of 89.8% and 112% for HRP and laccase than those of the pure rGO and Fe3O4 particles as supports, which resulted in IEs in the ranges of 39.3-43.5% and 45.3-70.4%, respectively (Figure 3). Here, synthesized composite particles containing different amounts of Fe resulted in significant variation in the IEs of both immobilized laccase and HRP (Table S1). The reductions in IEs may be due to high hydrophobic interactions between supports and enzymes as rGO content increased in the composite particles.4 RGO-Fe3O4-M1 and rGO particles had similar IYs. The IEs observed herein on rGO-Fe3O4-M1 particles were much higher than those reported previously for the immobilization of Trametes versicolor laccase, where IEs were reported in the ranges of 20.091.0%,13,18,36-39 and HRP, where IEs were reported to be 55.5%32 and 77.5%21, on magnetic composite particles. HRP immobilized on rGO-Fe3O4-M1 particles retained up to a 7-fold higher

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IE than that of the chemically different rGO particles.4 These results suggest that rGO-Fe3O4-M1 is a suitable support material for retaining high enzyme activity of laccase and HRP after immobilization.

Figure 4. Immobilization yields of enzymes on the particles: a) laccase and b) HRP. Loading of enzymes on the particles: c) laccase and d) HRP. All experiments evaluating enzyme immobilization were performed at 4 ºC with shaking of 60 rpm using 10 mg of particles at optimum pH values in buffer (50 mM). To determine optimum incubation time, 1 mg of enzyme was added to the reaction mixture and immobilization was monitored over a period of 12 h. Further, enzyme loading onto the particle was measured by increasing the enzyme concentration from 50 to 600 mg/g of support in the reaction mixture under optimum incubation conditions.

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Enzyme loading The amount of laccase and HRP immobilized on the rGO-Fe3O4-M1 and rGO particles was found to increase through the first 4 h of incubation, and was nearly stable thereafter for up to 12 h, whereas immobilization increased on Fe3O4 particles over the entire 12 h incubation period (Figure 4a and 4b). The optimum incubation periods for efficient immobilization of both laccase and HRP on rGO-Fe3O4-M1, rGO, and Fe3O4 were 4, 4, and 8 h, respectively (Figure S13). To examine the maximum loading capacities of laccase and HRP on rGO-Fe3O4-M1, rGO, and Fe3O4 particles, immobilization was performed with different initial amounts of enzymes. Figure 4c and 4d show that the immobilized amounts of laccase and HRP are partially functions of enzyme concentration. The amounts of immobilized enzymes increased with increasing amounts up to 600 mg/g of support. The maximum amounts of immobilized laccase on rGOFe3O4-M1, rGO, and Fe3O4 were 418, 258, and 123 mg/g of support, respectively, whereas those of immobilized HRP were 376, 227, and 99.4 mg/g of support, respectively. Here, rGO-Fe3O4M1 composite particles had approximately 1.7 and 3.8-fold higher enzyme loading potentials than those of pure rGO and Fe3O4 particles, respectively. The high IYs of laccase and HRP observed on rGO-Fe3O4-M1 and rGO particles were likely due to strong hydrophobic (noncovalent) interactions between these particles and the enzymes as was described previously for HRP and oxalate oxidase on chemically reduced GO particles, which is supported by zeta potential of synthesized rGO-Fe3O4-M1 particles (Figure S14).4 The loading of laccase and HRP on magnetic composite (rGO-Fe3O4-M1) particles was much higher than those of previous reports on laccase immobilization,13,18,39-42 and HRP immobilization.21,22,32 The compositions and morphologies of magnetic composite particles have a significant influence on the IYs and IEs of T. versicolor laccase as shown in Table S2. Spherical particles have been used mostly for

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immobilization in addition to those with hierarchical, tubular, and wormhole frameworks. A maximum loading of 492 mg/g of support was observed on carbon-based magnetic composite particles,13 but they resulted in a significantly lower IY of 19.6% compared to the IY of 91.1% on rGO-Fe3O4-M1 particles observed herein. Similarly, rGO-Fe3O4-M1 had an approximately 16-fold higher HRP loading capacity than that of the magnetic chemically rGO (Fe3O4/CRGO) nano-composite particles, which had a reported maximum loading of 23.3 mg/g of support.32 Additionally, the rGO-Fe3O4-M1 particles resulted in approximately 21- and 49-fold higher HRP loading capacities than those of Fe3O4 modified core-shell (Fe3O4@poly-dopamine),22 and coreshell-shell structures (Fe3O4@SiO2@poly-methyl methacrylate-co-4-vinylphenylbornoic acid),21 respectively. The spherical and porous nature of synthesized composite rGO-Fe3O4-M1 particles resulted in a significantly higher IY and IE than those of the sheeted rGO and non-porous Fe3O4 particles for the immobilization of laccase and HRP. Thus, comparatively, rGO-Fe3O4-M1 particles are much better for enzyme immobilization than similar composite particles. Fe3O4/CRGO spherical particles have been used for the immobilization of bovine serum albumin (BSA), lysozyme, and glucose oxidase, but exhibited a maximum enzyme loading of only 60 mg/g of support.32 Similarly, rGO-Fe3O4-M1 particles were more efficient in enzyme loading (418 mg/g of support) than that of the core-shell Fe3O4@GO particles (182 mg/g of support).17

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Figure 5. Analysis of immobilized laccase on rGO-Fe3O4-M1: a) TEM, b) HR-TEM, and c) mapping images of iron, oxygen, carbon, sulfur, and nitrogen.

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Instrumental analysis of immobilized laccase Initially, circular dichroism (CD) analysis was performed to assess structural changes after immobilization on rGO-Fe3O4-M1 and pure rGO. These results indicated that immobilized laccase on rGO-Fe3O4-M1 had similar secondary structure CD spectra to those of free laccase, whereas after immobilization on rGO, there was a significant shift in CD spectra (Figure S15). This change may lead to a loss in enzyme activity after immobilization on rGO. Laccase immobilization on rGO-Fe3O4-M1 was further evaluated with TEM, HR-TEM, and elemental mapping (C, Fe, N, O, and S) analysis (Table S3 and Figure 5). Additionally, immobilization was evaluated through Fourier transform infrared (FTIR) analysis (Figure S16a), whereby peaks in the range of 554-600 cm-1 show the vibration of Fe-O.16,32 Additional peaks were observed at 1589 and 1387 cm-1, which corresponds to symmetric and anti-symmetric stretching of carboxylic groups. Hydroxyl, epoxy, and alkoxy stretching vibrations were observed at 3424, 1231, and 1051 cm-1, respectively.4 A broad peak observed at about 1550–1650 cm-1 corresponds to the C=O stretching (amide I band, 1650 cm-1) and N-H bending (amide II band, 1550 cm-1) vibrations of the protein, with an additional peak of carboxylic C=O stretching at 1739 cm-1, which indicates laccase immobilization.5,32 Furthermore, fluorescein isothiocyanate (FITC)labeled laccase confocal laser scanning microscopy (CLSM) and TGA results suggested efficient immobilization of laccase on rGO-Fe3O4-M1 (Figure S16b-d). In the TGA, the high loading of laccase on rGO-Fe3O4-M1 was correlated with a significant reduction in the relative weight of the particles to 30.9%, whereas pure rGO-Fe3O4-M1 particles exhibited less reduction to 65.0%.

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Figure 6. Activity of free and immobilized laccase at different: a) pH values and b) temperatures. To determine the optimum pH value of free or immobilized enzyme, the reaction was performed under standard assay conditions in various buffers (50 mM): glycine-HCl (pH 2.0-2.5), sodium-citrate (pH 3.0-4.0), sodium-acetate (4.5-5.5), and sodium-phosphate (pH 6.07.0). Further, the optimum temperature of free or immobilized enzyme was analyzed under standard assay conditions over the range of 25-70 oC.

Characterization of immobilized laccase The essential requirement of efficient immobilization is to retain high enzyme activity after immobilization. The specific activity of immobilized laccase on rGO-Fe3O4-M1 (1,610 U/mg protein) was higher than that of the free enzyme (1,440 U/mg protein).15 The activities of free and immobilized laccase at different pH values and temperatures are shown in Figure 6. Both free and immobilized laccase on rGO-Fe3O4-M1, rGO, and Fe3O4 exhibited maximum activity at pH 3.0 (Figure 6a). The immobilized laccase retained high activity and was more resistible than the free enzyme over a pH range of 4.0-7.0. The optimum temperature of free and immobilized laccase on rGO and Fe3O4 particles was observed at 40 °C. Interestingly, a shift in optimum temperature to 45 °C was observed for laccase immobilized on rGO-Fe3O4-M1. Here, the immobilized laccase retained higher activity than that of the free enzyme over the temperature range of 45-70 °C (Figure 6b). Among the supports, rGO-Fe3O4-M1 resulted in the highest 18


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residual laccase activity over a broader range of pH values and temperatures, indicating that rGO-Fe3O4-M1 is better for retaining enzyme activity than the pure particles. The spherical and porous morphology of rGO-Fe3O4-M1 may have played a role in its higher stability than the sheeted rGO and spherical Fe3O4 particles as previously suggested for magnetic composite porous particles of hierarchical,13 tubular,39 and wormhole framework structures.41

Table 1. Kinetic parameters of free and immobilized laccase laccase

Km (µM)

kcat (s-1)

kcat/Km (s-1 µM-1)

free

29.3±2.2

2080±84

71.0±5.2

rGO-Fe3O4-M1

30.3±2.5

2140±92

70.6±5.0

rGO

47.8±6.0

1670±79

35.0±3.2

Fe3O4

64.6±5.0

1250±66

19.4±1.8

Kinetic studies The kinetic parameters (Km and Vmax) of free laccase were changed after immobilization on the different particles (Table 1). Specifically, the apparent Km values of free and immobilized laccase on rGO-Fe3O4-M1, rGO, and Fe3O4 were 29.3, 30.3, 47.8 and 64.6 µM, respectively. Interestingly, the Km values were quite similar for both free and immobilized laccase on rGOFe3O4-M1. On the other hand, the higher Km values of laccase immobilized on rGO and Fe3O4 suggested a lower affinity towards the substrate 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) after immobilization, which may have been due to mass transfer limitation. Significantly increased Km values (up to 90-fold) were previously observed after immobilization on various support materials.13,37,40,41,43 In these studies, increases in the Km were observed due to strong attachments of enzymes to supports, which resulted in either structural changes or

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hindrance in substrate transfer towards active sites. The apparent Vmax value of immobilized laccase on rGO-Fe3O4-M1 was increased to 1945 µmol/min/mg protein compared to 1890 µmol/min/mg protein of the free enzyme, whereas it decreased on rGO and Fe3O4 to 1520 and 1140 µmol/min/mg protein, respectively (Figure S17).15 Catalytic efficiency (kcat/Km) values of free (71.0 s-1µM-1) and immobilized laccase on rGO-Fe3O4-M1 (70.6 s-1µM-1) were quite similar. However, they were reduced to 35.0 and 19.4 s-1µM-1 for laccase immobilized on rGO and Fe3O4, respectively. The laccase immobilized on rGO-Fe3O4-M1 had approximately 2.0- and 3.6-fold higher catalytic efficiencies compared to those of laccase immobilized on rGO and Fe3O4 particles, respectively. In comparison with previous reports on T. versicolor laccase immobilization, this particular magnetic composite with rGO (rGO-Fe3O4-M1) is much better in retaining high catalytic efficiency to levels similar to those of the free enzyme than magnetic particles composites with either silica,18,39,41,42 or chitosan.40

Enzyme stability and reusability Figure 7a shows the stability of free and immobilized laccase at 25 °C. The stability of laccase was significantly improved after immobilization. rGO-Fe3O4-M1, rGO, and Fe3O4 immobilized laccase had residual activities of 83.2, 48.5, and 42.0% of initial activity after 96 h, respectively, whereas the free enzyme lost more than 93% of its initial activity under the same conditions. The half-life (t1/2) at 25 °C was 16.9, 81.5, 88.8, and 257 h for free enzyme and immobilized laccases on Fe3O4, rGO, and rGO-Fe3O4-M1, respectively (Table S4). Immobilized laccase on rGOFe3O4-M1 resulted in the highest stability after immobilization at pH 3.

20


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Figure 7. Stability and reusability of free and immobilized enzyme: a) stability at 25 °C, b) storage stability at 4 °C, and c) reusability. Stability was determined by measuring residual enzyme activity of free (0.01 mg/mL) or immobilized laccase (0.2 mg of particle/mL) under standard assay conditions in 50 mM sodium-citrate buffer (pH 3) at regular intervals of incubation (25 °C) for up to 240 h. Similarly, storage stability was determined under standard assay conditions for incubation (4 °C) up to 30 days. Further, the reusability of immobilized laccase (0.5 mg of particle/mL) was monitored by oxidizing ABTS under standard assay conditions at room temperature. The initial activity of free or immobilized enzyme was considered as 100% residual activity. 21



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Interestingly, immobilization of T. versicolor laccase on the iron minerals goethite and lepidocrocite did not result in any improvements in the stability after immobilization at pH 3.6.38 Similarly, the stability of HRP at 25 ºC was enhanced after immobilization onto rGO-Fe3O4-M1, rGO, and Fe3O4 particles with residual activities of 88.6, 75.4, and 51.3% of the initial activity at 240 h, respectively. Free HRP lost 63.2% of its residual activity at pH 7.4 in phosphate buffer (Figure S18a). Furthermore, storage stability of both free and immobilized laccase on rGOFe3O4-M1 was evaluated at 4 °C (Figure 7b). Free and immobilized laccase on composite rGOFe3O4-M1 retained 12.2 and 90.1% of residual activity after incubation for 30 days, respectively. However, laccase immobilized on pure rGO and Fe3O4 particles resulted in significantly lower stability than that of rGO-Fe3O4-M1-immobilized laccase with residual activities of 38.5 and 31.9%, respectively. Additionally, the storage stability of laccase immobilized on rGO-Fe3O4M1 observed herein is significantly higher than that reported for laccase immobilized on tubular halloysite nanotubes.44 Similarly, the storage stability for HRP was examined after storage at 4 ºC in phosphate buffer for 30 days (Figure S18b). Free and immobilized HRP on rGO-Fe3O4M1, rGO, and Fe3O4 particles retained 21.6, 84.2, 44.9 and 36.8% of the initial activity under the similar conditions. The reusability of immobilized laccase on the different particles is shown in Figure 7c. The laccase immobilized on rGO-Fe3O4-M1, rGO, and Fe3O4 retained 92.6, 73.9, and 21.3% of their initial activities after 10 cycles of use for the oxidation of ABTS, respectively. Compared to previously reported laccase immobilization systems, rGO-Fe3O4-M1 has higher reusability as presented in Table S2. Interestingly, these spherical and porous particles fare much better in reusability than tubular magnetic composite particles, where immobilized laccase lost about 35%

22


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of residual activity after only 6 cycles.39 Additionally, immobilized lipase on chemically modified yolk-shell silica spheres lost 39.7% of activity after only the first cycle,45 and immobilized β-glucuronidase on graphene/γ-Fe2O3 hybrid aerogels lost 59% of activity after 5 cycles of reuse.46 Similarly, HRP immobilized onto rGO-Fe3O4-M1, rGO, and Fe3O4 retained 89.8, 71.8, and 11.7% of their initial activities after 10 cycles of reuse for the oxidation of phenol, respectively (Figure S18c). The magnetic nature of rGO-Fe3O4-M1 composite particles may also allow for easy separation of the immobilized enzyme from the reaction mixture (Figure S19).

Oxidation of phenolic compounds Laccase has a broad range of specificity towards diverse groups of phenolic and non-phenolic compounds, including mediators such as ABTS.27-30 The relative oxidation activities towards different phenolic compounds were evaluated for free and immobilized laccase on rGO-Fe3O4M1, rGO, and Fe3O4 particles. After immobilization, laccase exhibited higher oxidation abilities towards different phenolic compounds as compared with that of free enzyme (Table S5). Among these immobilized laccases, that immobilized on rGO-Fe3O4-M1 exhibited approximately 115, 117, 134, and 192% higher oxidation activities towards 2,6-dimethoxy phenol (DMP), guaiacol, pyrogallol and 3,4-Dihydroxy-L-phenylalanine (L-DOPA), respectively. In a previous study, laccase immobilized on Sepharose as a support resulted in up to 46% lower oxidation potentials for 2,6-dichloro-indophenol, pyrogallol, and catechol compared to those of the free enzyme under similar conditions.47

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Table 2. Acute toxicity of particles towards V. fischeri particles

parametera

EC50 value (µg/mL)

EC50-15min

770 ± 60

EC50-30min

720 ± 57

EC50-15min

250 ± 20

EC50-30min

235 ± 19

rGO-Fe3O4-M1

Fe3O4 a

EC50–15min and EC50–30min were determined based on the bioluminescence after 15 and 30

min of incubation, respectively.

Acute toxicity Pure Fe3O4 magnetic particles are mostly biofriendly.48-50 However, they are toxic towards V. fishcheri and Daphnia magma.48 In addition, the toxicity values of modified or coated Fe3O4 particles are highly variable from moderate toxic to non-toxic in different model organisms or human cells.48-50 Similarly, rGO is less toxic than pure GO.51,52 Therefore, the synthesis of more biocompatible particles is required to achieve a broad range of biotechnological applications. The acute toxicity (EC50) values of rGO-Fe3O4-M1 were 773 and 725 µg/mL towards the V. fischeri after 15 and 30 min of incubation, respectively (Table 2, Figure S20). Under similar conditions, the EC50 values of pure Fe3O4 particles were 250 and 235 µg/mL, respectively. A similar EC50 value of 240 µg/mL was previously reported for Fe3O4 particles at 5 min of incubation.48 The significantly higher EC50 values for rGO-Fe3O4-M1 observed herein highlight the more biocompatible nature of these synthesized composite particles than that of the pure Fe3O4 particles.

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CONCLUSIONS A novel type of spherical and porous composites (rGO-Fe3O4-M1, -M2, and -M3) were synthesized to dually benefit from rGO and magnetic materials as supports for enzyme immobilization. Among the composite particles, rGO-Fe3O4-M1, which contained 71% Fe, resulted in significantly higher immobilization efficiency and loading than those of pure GO and Fe3O4 particles. Significant improvements in laccase stability, oxidation of phenolic compounds, and electrochemical properties were observed after immobilization on rGO-Fe3O4-M1 in addition to high reusability. Low acute toxicity towards V. fischeri of rGO-Fe3O4-M1 composite particles further suggested that they are more environmentally friendly than comparable commercial particles. Taken together, the data presented herein provide strong evidence that rGO-Fe3O4-M1 composite particles could be used as support for the immobilization of various enzymes.

EXPERIMENTAL SECTION Materials Commercial Fe3O4 particles (2650TS) were purchased from Nanostructured and Amorphous Materials, Inc. (Houston, TX, USA). Laccase (T. versicolor), HRP (Armoracia rusticana), ABTS, DMP, FITC, guaiacol, L-DOPA, and pyrogallol were purchased from Sigma-Aldrich (St. Louis, MO, USA). V. fischeri was purchased from Modern Water (New Castle, DE, USA). All other chemicals and reagents used in the experiments were analytical grade from commercial sources, unless otherwise stated.

Particle synthesis Three different rGO-Fe3O4 composite particles with varying Fe3O4 content were prepared by a one-pot spray pyrolysis process. The GO was synthesized through a modified Hummer’s method. The obtained GO was redispersed in distilled water and then exfoliated to generate GO 25



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sheets by ultrasonication. Next, 500 mL of the exfoliated GO suspension (1 mg/mL) was used to the spraying solution. To prepare the Fe3O4-rGO composite powder, 500 mL of the exfoliated GO suspension (1 mg/mL) was added to different amounts of iron chloride hexahydrate. The GO containing iron chloride hexahydrate was then dispersed in distilled water by ultrasonication and magnetic stirring. Next, different amounts of iron chloride hexahydrate were dissolved into the GO colloidal solution. A quartz reactor with a length of 1200 mm and diameter of 50 mm was used for this process.2 The droplets containing iron chloride and GO nanosheets transformed into the rGO-Fe3O4-M composite particles within the tubular reactor maintained at 800 °C. The flow rate of Ar used as a carrier gas was 10 L/min, and the corresponding residence time of the particles inside the reactor was 10 s. Pure rGO and Fe3O4 particles were also prepared from the spray solutions with GO colloidal solution and iron chloride hexahydrate, respectively, under the same preparation conditions as used to prepare the rGO-Fe3O4composite particles.

Characterization of prepared samples The crystal structures of the powders were determined by XRD (X’pert PRO MPD, PANalytical, the Netherlands) using Cu Kα radiation (λ = 1.5418 Å). Moreover, the morphological features were investigated by field-emission SEM (FE-SEM; Hitachi S-4800, Japan) and TEM (JEM2100F, JEOL, Tokyo, Japan) at an accelerating voltage of 200 kV. The XPS spectra of the particles were investigated using X-ray photoelectron spectroscopy (XPS, K-alpha) with Al Kα radiation (1486.6 eV). The magnetic property was measured at room temperature using a vibrating sample magnetometer (VSM, Lake Shore Cryotronics, Model 7300). TGA (Seiko Exstar 6000 TG/DTA 6100, Japan) analysis was performed in air at a heating rate of 20°C/min in order to determine the amount of rGO in the composite particles. The mean particle size and relative standard deviation of the rGO-Fe3O4-M1 composite particles were measured by dynamic light scattering analysis (Malvern, Nano ZS90). The specific surface areas of the microspheres were calculated by BET analysis of the nitrogen adsorption measurements (TriStar 3000, Micromeritics, USA).

Enzyme immobilization Particles (10 mg) were mixed with enzymes (1 mg protein) in 1 mL buffered solution (50 mM) with different pH values, and incubated for 4 h with shaking of 60 rpm at 4 °C. After the 26


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adsorption of enzymes, particles were separated by centrifugation at 10,000 rpm for 15 min at 4 °C. The protein concentration of washed solution was measured by the Bradford method as described previously.15 IY and IE were calculated as follows: IY (%) = amount of immobilized enzyme / amount of enzyme added × 100

(1)

IE (%) = total activity of immobilized enzyme / total activity of free enzyme × 100

(2)

Enzyme activities Laccase activity was determined spectrophotometrically by an increase in absorbance at 420 nm using ABTS (1 mM) as the substrate.15 HRP assays were performed using phenol (0.6 mM) and H2O2 (1.57 mM) as substrates and 4-AAP as the chromogen in phosphate buffer (0.1 M) at pH 7.4 Enzyme activities are expressed as international units (IU), where one IU represents the amount of enzyme that forms 1 µmol/min of products under standard assay conditions.15

Time profile and loading To determine the IE, enzyme immobilization on different particles was monitored over a period of 12 h. Furthermore, the enzyme loading capacity of the particles was evaluated by increasing the concentration of the enzyme up to 600 mg/g of support in the reaction under optimum conditions. On the basis of high IY and IE, laccase was used for further studies.

Characterization of immobilized laccase The activities of free and immobilized laccase on the particles were determined in different pH buffers (50 mM) ranging from pH 2.0-7.0 and at temperatures ranging from 25-70 °C using standard assay conditions. Kinetic parameters (Km and Vmax) were determined with varying concentrations of ABTS (0.001-2.0 mM) in 50 mM sodium-citrate buffer at 25 °C. Kinetic parameters (Km and Vmax) values for the substrate (ABTS) were obtained by non-linear regression analysis using GraphPad Prism 5 (San Diego, CA, USA).15

Stability and reusability of immobilized enzymes Enzyme stability was investigated by incubating free and immobilized laccase or HRP at 25 °C under standard assay conditions. Samples were withdrawn at regular intervals over 240 h to measure the residual activity. Furthermore, storage stability of HRP and laccase was evaluated at 27



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4 °C over a period of 30 days. The reusability of immobilized laccase and HRP was assessed by carrying out the oxidation of ABTS and phenol, respectively, under standard assay conditions.4,15 The abilities of free and immobilized laccase to oxidize phenolic compounds were analyzed by spectrophotometric methods at 25 °C under standard assay conditions.

Toxicity analysis The acute toxicity of the synthesized rGO-Fe3O4-M1 and Fe3O4 commercial particles were assessed using the 81.9% toxicity test protocol with a Microtox Model 500 Analyzer (Modern Water). rGO-Fe3O4-M1 (1.0 mg/mL) and Fe3O4 (0.5 mg/mL) were prepared in distilled water, and the osmolarity of each sample was adjusted with the osmolarity adjusting solution provided by the manufacturer.53,54 The endpoint measured by the Microtox assay detects the decrease in the intensity of light emitted by the luminescent marine bacterium V. fischeri after 15 and 30 min of exposure. Here, the EC50–15min and EC50-30min average values denote the effective concentration (EC) of effluent that caused a 50% reduction in the luminescence of the bacteria after incubations of 15 and 30 min, respectively.

Instrumentation Absorption spectra were recorded on a UV/Vis spectrophotometer (JENWAY Scientific, UK). FTIR spectroscopy (JASCO FT-IR 300E spectrometer, Japan) and CLSM images were taken with the FV-1000 Olympus confocal microscope (Olympus, Tokyo, Japan). CD analysis was performed using a CD detector (Chirascan-plus, Applied Photophysics, UK). The decomposition characteristics of synthesized rGO-Fe3O4 composite particles and those with immobilized laccase were determined using TGA analysis. ■ ASSOCIATED CONTENT Supporting Information Detailed about one-pot and continuous spray pyrolysis process, properties of synthesized particles, and comparative analysis of laccase immobilization, thermal denaturation, kinetic, oxidation of phenolic compounds, CD, CV, elemental mapping, FTIR, CLSM, SEAD, SEM, TEM, TGA, XRD, magnetic separation, N2 adsorption and desorption isotherms, Zeta potential, 28


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and acute toxicity data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected], (Jung-Kul Lee) Fax: (+82) 2-458-3504 E-mail: [email protected] (Yun Chan Kang) Fax: (+82) 2-928-3584

NOTES The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the Energy Efficiency & Resources Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP), granted financial resource from the Ministry of Trade, Industry & Energy, Republic of Korea (20153030091450). This research was also supported by a grant from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031955) funded by the Ministry of Science, ICT and Future Planning, Republic of Korea.

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Reduced Graphene Oxide Particles for Efficient Enzyme Immobilization

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