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Horseradish Peroxidase Immobilized on Graphene Oxide: Physical Properties and Applications in Phenolic Compound Removal Feng Zhang,† Bin Zheng,† Jiali Zhang,‡ Xuelei Huang,† Hui Liu,† Shouwu Guo,*,‡ and Jingyan Zhang*,† State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China UniVersity of Science and Technology, Shanghai, 200237, People’s Republic of China, and National Key Laboratory of Micro/Nano Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of the Ministry of Education, Research Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong UniVersity, Shanghai 200240, People’s Republic of China ReceiVed: February 4, 2010; ReVised Manuscript ReceiVed: March 24, 2010
Composition, morphology, and surface characteristics of solid substrates play critical roles in regulating immobilized enzyme activity. Grapheme oxide (GO), a novel nanostructured material, has been illustrated as an ideal enzyme immobilization substrate due to its unique chemical and structural properties. Physical properties and catalytic activity of GO immobilized horseradish peroxidase (HRP) and its application in phenolic compound removal are described in the present study. HRP loading on GO was found to be much higher than that on reported substrates. The GO immobilized HRP showed improved thermal stability and a wide active pH range, attractive for practical applications. The removal of phenolic compounds from aqueous solution using the GO immobilized HRP was explored with seven phenolic compounds as model substrates. The GO immobilized HRP exhibited overall a high removal efficiency to several phenolic compounds in comparison to soluble HRP, especially for 2,4-dimetheoxyphenol and 2-chlorphenol, the latter a major component of industrial wastewater. Introduction Enzyme immobilization has been proven an efficient way to enhance the catalytic performance of enzymes.1–3 Immobilized enzymes, in comparison with soluble enzymes, demonstrate numerous advantages, such as high thermal and storage stability and easy separation from reaction mixture. Thus, immobilized enzymes have found a wide range of applications in industrial biocatalysis,4 biosensors,5 diagnostics,6 and other biotechnologies.7,8 Researches have illustrated the composition, morphology, and surface characteristics of solid substrates play critical roles in regulating enzyme activity at solid substrate surfaces.9 Ideally, the solid substrate should prevent enzyme aggregation and denaturation, but would not perturb the native conformation of the enzymes in an undesired way. So far, a variety of materials, such as glass,10,11 polymers,12 macroporous materials,13 and nanoscaled materials,14 have been employed as substrates for enzyme immobilization. Among them, nanoscaled materials, due to their small size, large specific surface area, and desired aqueous suspending ability, have exhibited advantages over traditional bulk materials and received a great deal of attention in recent years.15–17 However, in use of nanometer scaled materials we encounter difficulty in characterizing the immobilized enzyme molecules on them, which limits the deep understanding of enzyme-surface interactions and further designing of solid support on a rational basis to optimize the immobilized enzyme performance. We illustrated previously that individual graphene oxide (GO) sheet had an atomically flat surface, and could serve as an ideal solid substrate for enzyme * To whom correspondence should be addressed. E-mail: jyzhang@ ecust.edu.cn and
[email protected]. † East China University of Science and Technology. ‡ Shanghai Jiao Tong University.
immobilization.18 We demonstrated that enzyme molecules could be directly immobilized on GO without using any coupling reagent due to the intrinsic surface functional groups of GO. In addition, with the atomically flat surface of GO, the loading density and the conformation of the immobilized enzyme molecules on GO were able to be studied in situ by using atomic force microscopy (AFM). The AFM images clearly demonstrated that enzyme molecules were randomly distributed on the GO surface, and increasing enzyme loading did not affect the catalytic activity of the immobilized enzymes. The advantages of using GO as a solid substrate for enzyme immobilization stimulated us to explore the properties and potential applications of the GO immobilized enzymes. We describe herein the physical and catalytic properties, and applications of enzyme immobilized on GO. In this work, horseradish peroxide (HRP) was used as a model enzyme, due to its ability to catalyze the oxidation of a variety of substrates including phenolic compounds, and has been employed in phenolic compound removal.19–21 The optimum pH, reusability, and thermal and storage stabilities of the immobilized HRP on GO were studied. The application of the immobilized HRP in the removal of phenolic compound was explored and compared with that of soluble HRP. We found removal efficiency, stability, and thermal stability of GO immobilized HRP were improved in comparison to those of soluble HRP. Experimental Section Chemicals and Materials. HRP (E.C. 1.11.1.7) was purchased from YuanJu Bio-Tech Co. Ltd. (Shanghai, China), and stored at -20 °C before use. The stock solution of HRP of 1 mg mL-1 was prepared in phosphate buffer and stored at 4 °C. All other chemicals including seven phenolic compounds, phenol, 4-methoxyphenol, 2-methoxyphenol, 3-aminophenol,
10.1021/jp101073b 2010 American Chemical Society Published on Web 04/06/2010
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Figure 1. Tapping mode AFM images of GO (a) on mica surface and (b) HRP bound GO. The scale bars equal 800 (a) and 200 nm (b).
catechol, 2-cholorolphenol, and 2,4-dimetheoxyphenol were of analytical grade and purchased from SinoPharm Chemical Reagent Co. Lid. and were used as purchased. A stock solution (0.1 M) of each phenolic compound was prepared with phosphate buffer and stored at 4 °C. A diluted solution of hydrogen peroxide for enzyme activity assay was freshly prepared. GO was prepared with natural graphite powder through a modified Hummer’s method.22–24 The aqueous suspension of GO was stored at room temperature on the lab bench, and used for characterizations and enzyme immobilization. Instruments. Electronic absorption spectra of the catalytic products and reactions were recorded on a Cary 50 spectrophotometer (Varian, USA). IR spectra of GO were recorded on a Perkin-Nicolet FT-IR 200 in the range of 4000-400 cm-1. Samples were run as KBr pellets. HPLC measurements were performed on LC-10AT (Shimadzu, Japan) with C18 column, and eluted with methanol (80%) and water (20%) at a flow rate of 0.8 mL min-1. Atomic force microscopic images of graphene oxide were taken on a Nanoscope MultiMode V scanning probe microscopy (SPM) system (Veeco, USA). The scanning rate was set usually at 0.7-1 Hz. The samples for AFM were prepared by dropping an aqueous suspension (∼0.02 mg mL-1) of the graphene oxide on a freshly cleaved mica surface and dried under vacuum at 80 °C. The surface functionalities of GO were characterized by FT-IR, and the morphology and structure of GO were characterized by AFM.24 The AFM image of GO immobilized enzyme was acquired under tapping mode in a liquid cell.25 Methods. HRP Immobilization on GO. In a typical immobilization experiment, 50 µg of GO (water suspension) was added to 1 mL of 0.1 M, pH 7, potassium phosphate buffer containing HRP with different concentrations.26 The mixtures were incubated for 30 min on ice with shaking, and then centrifuged (enzyme immobilization generally completes in about 15 min in this work, to ensure the completion of the immobilization the incubation time was maintained 30 min for all experiments). The solid GO and the supernatants were collected, respectively. The solid was washed three times with the buffer to remove nonspecific adsorbed HRP. The resulting immobilized enzymes were stored at -20 °C prior to use. The supernatant was used to measure the concentration of the residual enzyme to determine the enzyme loading on GO. Enzyme Loading Determination. The enzyme loading on GO is defined as the enzyme amount difference between the total enzyme used and the residual enzyme present in the supernatant after immobilization. The enzyme concentration in the supernatant was determined either by measuring the UV absorbance at 403 nm (Extinction coefficient, ε ) 1.02 × 105 M-1 cm-1) or by measuring its initial catalytic reaction rates with substrates.
The R/Z number was checked for the enzyme in supernatant to ensure the integrity of the HRP. Enzyme ActiWity. A colorimetric assay was used to evaluated the catalytic activity of the immobilized HRP.27 The immobilized HRP was added to the assay solution that contained 1 mL of 60 mM phenol, 14.38 mM 4-aminoantipine (4-AAP), and 1.21 mM hydrogen peroxide in 1 mL of 0.1 M, pH 7.0, phosphate buffer. The catalytic reaction was monitored by following the absorption of the red color product of the reaction (quinoneimine, extinction coefficient, 7210 M-1 cm-1) at 510 nm.28 One unit of the activity (U) of HRP was defined as the amount of HRP required to hydrolyze 1 µmol of hydrogen peroxide converted per minute under the conditions stated above. Phenolic Compound RemoWal. The removal of phenolic compounds was determined by measuring the residual phenolic compounds in supernatants with HPLC or the colorimetric method with potassium ferricyanide and 4-aminoantipyrine.29 For colorimetric assay, the sample (total 1 mL) containing 50 µL of clear supernatants of the reactions was mixed with 100 µL of potassium ferricyanide (83.4 mM), 100 µL of 4-AAP (20.8 mM), and 750 µL of 0.1 M phosphate buffer. After the color of the reaction mixture was developed completely over a few minutes, absorbance was measured at 505 nm against a blank (800 µL of phosphate buffer, 100 µL of ferricyanide solution, and 100 µL of AAP solutions). Absorbance values were converted to the concentrations of phenolic compounds by using a calibration curve, which was plotted according to the standard phenol concentration with the initial reaction catalytic rates. For samples measured by HPLC, 1 mL aliquots of the supernatant of the reaction mixture (1 mL of 60 mM phenolic compounds, 1.2 mM hydrogen peroxide in 1 mL of 0.1 M, pH 7.0, phosphate buffer) were extracted with 2 mL of CHCl3, then the organic layer was filtered and loaded on to the HPLC column. The phenolic compounds were eluted isocratically with methanol 80% and water 20% at a flow rate of 0.8 mL min-1. Results and Discussion Characterization of GO. Immobilization substrate GO was prepared according to literature procedure.22–24 The solid GO was dispersed into water forming a stable brown solution (0.51 mg mL-1) by sonication. The surface morphology of the asprepared GO was characterized by AFM. Figure 1a shows the AFM image of the prepared GO sheets which are ∼1 nm thick. The surface functionalities of GO were further characterized by IR.18,24 HRP Immobilization. The immobilization of HRP on GO was carried out by incubating GO solution with HRP in phosphate buffer at pH 7.0. Compared with many other
Regulating Immobilized Enzyme Activity
Figure 2. Effect of the total concentration of HRP on the HRP loading on GO. The concentration of GO was kept constant (50 µg). HRP were added to phosphate buffer (0.1 M, pH 7) containing GO, and the immobilization was performed at 0 °C for 30 min.
immobilization supports, the GO surface is enriched with oxygen containing functional groups, such as hydroxyl, carbonyl, and epoxy groups.24,30 Thus the enzyme immobilization can be performed directly with high enzyme loading, and the interaction between GO and HRP is mainly electrostatic interaction.18 The image of the immobilized HRP on GO (Figure 1b) was obtained by tapping mode in a liquid cell. From the image one can directly observe the loaded enzyme, which is not possible for most solid substrates. At a constant GO concentration, we found that HRP loading increased with the total enzyme increase as shown in Figure 2. When total HRP concentration was increased from 0.002 µM to 0.92 µM, HRP loading increased from 0.1 µg to 5.5 µg. The loading on GO (maximum 100 mg of HRP per gram of GO at pH 7) is much higher than that of many reported supports, for example, 9.6 mg/g on porous aminopropyl glass beads,31 16 mg/g on alkylamine glass,11 18 mg/g on aminopropyl glass beads,29 and 21 mg/g on alkylamine modified pore glass substrate.32 The high enzyme loading is attributed to the large surface area of GO and oxygen rich functional groups on the GO surface. Since the interaction between GO and the surface of protein is electrostatic interaction, the enzyme loading can be tuned by varying the pH value of the buffer. The stability studies (described below) of the immobilized HRP support that electrostatic interaction is strong enough to immobilize HRP. Physical Properties of the Immobilized HRP. The stability of the GO immobilized HRP in comparison with free HRP was examined under different pH. The optimum pH for the immobilized HRP is around 7.0 as shown in Figure 3, which is comparable to that of free HRP.33 However, the immobilized HRP retained about 36% activity at pH 10, while for free HRP only 10% activity was left at the same pH. Overall, the immobilized HRP exhibited higher activity at pH above 7 than that of free HRP, while it showed a slightly lower activity under acidic condition. Though immobilized HRP is more stable under certain pH, overall similar pH dependence between the immobilized HRP and free HRP suggests that the catalytic performance of the immobilized HRP on GO is not generally affected too much by the immobilization. Thermostability of the GO immobilized HRP was determined by measuring residual activities of the immobilized HRP samples that were incubated over different times in the buffer at temperatures of 40-60 °C. For comparison, free HRP samples were measured under the same conditions. As shown in Figure 4, there is no dramatic difference between free and immobilized HRP samples at 40 °C after 60 min of incubation, but the difference increased after more than 80 min of incubation. When
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Figure 3. Effect of pH on the activity of free and immobilized HRP: (9) immobilized HRP and (0) free HRP. All reactions were carried out at room temperature with different buffers (sodium acetate buffer was used for pH 4.0 and 5.0, potassium phosphate buffer for pH 6.0-8.0, and tris buffer for pH 9.0 or 10.0).
Figure 4. Time-dependent deactivation of HRP: (4) free HRP (40 °C), (2) immobilized HRP (40 °C), (0) free HRP (50 °C), and (9) immobilized HRP (50 °C). The activities were measured with HRP (0.25 U mL-1), phenol (60 mM), 4-AAP (14.4 mM), and H2O2 (1.2 mM).
the incubation temperature was raised to 50 °C, the immobilized HRP exhibited much higher thermal stability, about 72% activity was still maintained after 20 min of incubation, while only 28% activity was found in free HRP. This thermostabilty difference trend was maintained with the incubation time increased to 120 min, and there was still 50% activity left for the immobilized HRP. Under the same condition only 20% activity was observed for free HRP. Apparently the GO immobilized HRP is better protected from the conformational changes imposed by heat in comparison to free HRP.17,29,34,35 One attractive advantage of the immobilized enzyme is that it can be easily separated from the reaction system and reused, which greatly decreases the cost of the enzyme under practical application. After the first cycle of the catalytic reaction with the HRP immobilized on GO, the reaction mixture was centrifuged to remove the supernatant and the immobilized HRP was repeatedly washed and the activity was assayed again. The catalytic cycles versus the enzyme activities of the immobilized HRP were plotted in Figure 5. The activity of the immobilized HRP decreased with the increase of the number of cycles. After 7 cycles, enzyme activity dropped to 25% of its initial activity. The reusability of the immobilized enzyme is dependent on solid support. For many solid supports, more than 50% enzyme activity was lost after five cycles or even less than five
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Figure 5. Reuse capacity of immobilized HRP. Conditions: 0.25 U mL-1 HRP, 60 mM phenol, 14.4 mM 4-AAP, and 1.2 mM H2O2.
Figure 6. Stabilities of free and the immobilized HRP at 4 °C: (9) immobilized HRP and (0) free HRP. The conditions for catalytic assay were the same as described in Figure 5.
cycles.26,31 The activity decline of the immobilized HRP on GO probably can be attributed to the accumulation of the reaction products produced during the enzymatic reaction on the GO, which may cover the enzyme and affect the reaction of the next cycle.36 In addition, the repeated washings and reaction cycles may disturb the electrostatic interaction between GO and HRP, thus leading to the enzyme bleeding gradually from the GO support, which may also contribute to the activity reduction. The storage stability of free and immobilized HRP was compared in phosphate buffer (0.1 M, pH 7.0) at 4 °C for a predetermined period of time. As shown in Figure 6, under the same storage condition, the activity of soluble HRP dropped much faster than that of the immobilized HRP. The immobilized
Zhang et al. HRP retained about 56% of its original activity in the buffer solution after 40 days. In contrast, the free HRP only maintained about 12% of its original activity over the same period of time. The reason may be connected with improved resistance of the immobilized HRP to conformational changes in solution due to charged residues being neutralized by the interaction with solid substrate or less exposed after immobilization, and possible distortion effects imposed by aqueous medium on the active site of HRP decreased.37–40 Phenolic Compound Removal. The immobilized HRP exhibited a similar substrate affinity, but with a lower catalytic efficiency, which is similar to many other immobilized enzyme systems.33,41–43 We have found the activity drop was mainly caused by the HRP conformation change that occurred after immobilization.18 However, with the better stability and larger active pH range, it is possible that the immobilized HRP would show better removal efficiency for phenolic compounds. The ability of the immobilized HRP on GO to remove phenolic compounds was tested in aqueous solution. Seven phenolic compounds were employed as substrates as described in the Experimental Section. The same experiments were carried out in parallel with free HRP. Because the oxidation products of some phenolic compounds can form stable color products with 4-AAP, the colorimetric method was used to determine the concentration of the residual phenolic compounds along with the HPLC method. For comparison, the reaction time for all compounds was set to 30 min, although the higher removal efficiency for these substrates can be reached by prolonged exposure to HRP/H2O2 systems (data not shown). The overall removal efficiency under this condition for the immobilized HRP was higher or comparable to that of free HRP as summarized in Table 1. The highest removal efficiency was found with substrate 2,4-dimetheoxyphenol, for which the immobilized HRP is two times more efficient than free HRP. The immobilized HRP also exhibited a slightly higher removal efficiency for 2-cholorolphenol, which is a major component of industrial wastewater. Because the surface of the GO is negatively charged,44 the interaction between HRP and GO more likely occurs with the positively charged residues on the surface of HRP. Figure 7 shows the electrostatic surface potential of HRP. There is no large positively charged area, fewer than 10 positive charged residues located on the HRP surface. Thus the substrate binding site (shown in the left of Figure 7) is not likely to be blocked by the interaction between HRP with GO. This is consistent with the similar affinities of the immobilized and free HRP we
Figure 7. Electrostatic surface potential of HRP. Pictures were generated with the program DsVisualizer (PDB 1H5A). Red areas indicate positive charged residues, blue are negative charged residues.
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TABLE 1: Removal of Phenolic Compounds by Free and Immobilized HRPa removal efficiency (%)b substrate phenol 4-methoxyphenol 2-methoxyphenol 3-aminophenol catechol 2-cholorolphenol 2,4-dimetheoxyphenol
free HRP immobilized HRP 57.1 69.5 66.0c 67.4 83.4 16.1 17.6
64.0 69.0 68.0c 72.7 87.6 20.4 34.4
immobilized/free HRP ratiob
ratioc
1.12 0.99 -d 1.08 1.05 1.27 1.96
1.13 1.08 1.03 1.26 -d 1.37 -d
a
Starting phenolic compounds 60 mM, HRP 0.83 U, H2O2 1.2 mM. Determined by HPLC. c Determined by colorimetric assay. d Cannot be determined due to the instability of the oxidation products.
b
previously obtained. The crystal structure of the complex of HRP and substrate ferulic acid (3-(4-hydroxy-3-methoxyphenyl)2-propenoic acid) showed that the methoxy oxygen of ferulic acid is hydrogen bonded to the arginine 38 residue at the substrate binding site,45 which is helpful to stabilize the enzyme and substrate complex. That is likely true for o-methoxysubstituted phenol substrates for immobilized HRP in our case. It is also possible that the orientation/conformation change of HRP induced by immobilization might be favorable to the catalytic reaction with ortho-substituted phenols. Conclusion In this study, the physical and catalytic properties and the application in phenolic compound removal of the HRP immobilized on GO were investigated. Compared with soluble HRP, HRP immobilized on GO could retain the activity at a wider range of pH; it also exhibited better storage stabilitiy and thermostability. However, the reusability of the immobilized HRP was not satisfactory, possibly due to the enzyme leakage during the reaction cycle. Among the tested phenolic substrates, the immobilized HRP showed higher removal efficiency to 2-cholorolphenol and 2,4-dimetheoxyphenol. The reason could be the immobilization induced conformation changes that are favorable for these two substrates. The results illustrate that the GO immobilized enzyme systems should be promising in wastewater treatment, and in other enzyme catalytic protocols. Acknowledgment. This work was supported by the State Key Laboratory of Bioreactor Engineering, NSFC of P. R. China (Nos. 20671034 and 20774029), and the National “973 Program” (Nos. 2007CB936000 and 2010CB933900). References and Notes (1) Bornscheuer, U. T. Angew. Chem., Int. Ed. 2003, 42, 3336. (2) Betancor, L.; Luckarift, H. R. Trends Biotechnol. 2008, 26, 566. (3) Badalo, A.; Gomez, J. L.; Gomez, E.; Bastida, J.; Maximo, M. F. Chemosphere 2006, 63, 626. (4) Bahar, T.; Celibi, S. S. J. Appl. Polym. Sci. 1999, 72, 68. (5) Gavalas, V. G.; Chaniotakis, N. A. Anal. Chim. Acta 2000, 404, 67. (6) Malcata, F. X.; Hill, C. G., Jr.; Amundson, C. H. Biotechnol. Bioeng. 1991, 38, 854. (7) Lante, A. C. A.; Pasini, G. Ann. N.Y. Acad. Sci. 1992, 672, 558.
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