- Email: [email protected]
Effects of acoustic wave resonance oscillation on immobilized enzyme
Applied Surface Science 294 (2014) 66–70
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Effects of acoustic wave resonance oscillation on immobilized enzyme Hiroshi Nishiyama, Tomoya Watanabe, Yasunobu Inoue ∗ Department of Materials Science and Technology, Nagaoka University of Technology, Nagaoka 940-2188, Japan
a r t i c l e
i n f o
Article history: Received 30 August 2013 Received in revised form 3 December 2013 Accepted 4 December 2013 Available online 14 December 2013 Keywords: Acoustic wave Resonance oscillation Enzyme Catalysis Surface
a b s t r a c t In aiming at developing a new method to artificially activate enzyme catalysts immobilized on surface, the effects of resonance oscillation of bulk acoustic waves were studied. Glucose oxidase (GOD) was immobilized by a covalent coupling method on a ferroelectric lead zirconate titanate (PZT) device that was able to generate thickness-extensional resonance oscillation (TERO). Glucose oxidation by the GOD enzyme was studied in a microreactor. The generation of TERO immediately increased the catalytic activity of immobilized GOD by a factor of 2–3. With turn-off of TERO, no significant activity decrease occurred, and 80–90% of the enhanced activity was maintained while the reaction proceeded. The almost complete reversion of the activity to the original low level before TERO generation was observed when the immobilized GOD was exposed to a glucose substrate-free solution. These results indicated that the presence of glucose substrate was essential for TERO-induced GOD activation and preservation of the increased activity level. The influences of reaction temperature, glucose concentration, pH, and rf electric power on the TERO activation showed that TERO strengthened the interactions of the immobilized enzyme with glucose substrate and hence promoted the formation of an activation complex. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The immobilization of enzymes on solid surfaces has the advantage of separating the enzyme from the reactant and product solutions. However, poor catalytic performance is often the result. The activation of immobilized enzymes is thus a key issue, and the establishment of a method for artificial activation of enzyme catalysis is strongly desirable. Acoustic waves such as surface acoustic waves (SAWs) and bulk acoustic resonance oscillations (ROs) that can be generated on a single domain ferroelectric crystal by imposing radio frequency (rf) electric power are characteristic of periodic (dynamic) lattice distortion [1,2]. These acoustic waves have been applied to thin film metal catalysts deposited on ferroelectric substrates [3], and it has clearly been demonstrated that acoustic waves activate metalcatalyzed gas phase reactions by lowering the activation energy of the reactions [4–11]. For instance, significant effects were observed for the thickness-extensional mode RO (TERO) with the lattice vibration mode perpendicular to the surface, which enhanced the catalytic activity of a Pd catalyst for ethanol oxidation by a factor of 1880 [12]. For ethanol decomposition on a Ag catalyst, TERO
∗ Corresponding author. Tel.: +81 258 47 9832; fax: +81 258 47 9830. E-mail address: [email protected] (Y. Inoue). 0169-4332/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsusc.2013.12.016
increased the activity for ethylene production without a significant influence on acetaldehyde production, thus increasing reaction selectivity for ethylene production from 58 to 96% [13]. Photoelectron emission spectroscopy studies [14,15] demonstrated that dynamic lattice vibration modes due to TERO changed the work function of the catalyst metal surfaces, with which the catalyst activation mechanism was associated [6–15]. There have been few applications of acoustic waves to the activation of catalyzed-liquid phase reactions, due to the belief that contact of acoustic waves with a thick and dense liquid phase would result in a significant attenuation of distortion energy. Recently, however, we combined the acoustic wave technique to a microreactor system consisting of a very thin liquid layer and examined the effects of SAWs and ROs on a scandium triflate [Sc(OTf)3 ] catalyst for the liquid phase aldol condensation reaction of benzaldehyde and acetophenone to chalcone [16]. The propagation of Rayleigh SAW with vertical lattice displacement caused a 3-fold enhancement of the activity of the [Sc(OTf)3 ] catalyst coordinated on the SAW-propagation path, decreasing the activation energy of the reaction from 39 to 19 kJ mol−1 . With the effects of RO, TERO increased the activity by a factor of 8.2 and decreased the activation energy of the aldol condensation reaction by 70%. The dynamic lattice distortion vertical to surface is also useful for the activation of liquid phase catalytic reactions. From the viewpoint of acoustic wave applications to liquid phase catalytic reactions, it is of particular interest to see how
H. Nishiyama et al. / Applied Surface Science 294 (2014) 66–70
the catalytic properties of a bulky immobilized enzyme, i.e., a supermolecule, are influenced by the TERO-induced-dynamic lattice distortion of substrates. The present study was undertaken to examine the acoustic wave TERO effects on the oxidation of glucose on immobilized glucose oxidase. We found that acoustic wave TERO induces interesting properties of not only accelerating the immobilized enzyme reaction, but also maintaining the activated states. 2. Experimental A ferroelectric lead zirconate titanate (PZT) polycrystalline ceramic device (25 mm in diameter) covered with 0.4 mm thick brass as electrodes to introduce radio frequency electric power was obtained from Murata Manufacturing Co., Ltd. The PZT had a resonance frequency of 2.2 ± 0.3 kHz and a lattice vibration of the thickness-extensional mode resonance oscillation (TERO) that was normal to the surface. For enzyme immobilization, a covalent-bond method was employed as follows. The ferroelectric device was first dipped into 0.5 vol% 3-aminopropylethoxysilane in toluene for 2 h, dried, and then immersed in 2.5 vol% glutaraldehyde aqueous solution for 2 h. After washing with distilled and ion-exchanged water, the device was immersed at pH 7.0 in a phosphate buffer solution of GOD enzyme (Aspergillus niger, molecular weight: 1.6 × 105 g mol−1 , Wako Pure Chemical Co., Ltd.) at a concentration of 1000 U ml−1 . Glucose oxidase from Aspergillus niger catalyzes the oxidation of -d-glucose by molecular oxygen to ␦gluconolactone, which hydrolyzes spontaneously to gluconic acid and hydrogen peroxide. Fig. 1 shows a flow-type microreactor, a reaction system for glucose oxidation on an immobilized GOD, and a radio frequency electric power system for TERO generation. The microreactor was composed of two separable units. The upper unit had an entrance and exit for reactant and product solutions, respectively, and an electrode pin for the introduction of radio frequency electric power. The lower unit had a connection pin for electric power introduction. A GOD-immobilized PZT device was placed between the two units and fixed tightly with Viton rings. The depth of the liquid layer was 130 m. The radio frequency electric power was generated from a function analyzer (NF Corporation, Multifunction Generator WF1974), and amplified by an amplifier
Fig. 1. A schematic representation of a microreactor, a reaction system, and a radio frequency electric power supply for TERO generation.
67
(NF Corporation, High Speed Power Amplifier, 4005), and then introduced to a GOD-immobilized RO device. -d-Glucose (denoted as glucose for simplicity) (Nakarai Tesque Chemical Co.) substrate in a 0.1 M phosphate buffer solution was introduced to the microreactor by a syringe pump, allowed to flow on the immobilized GOD catalyst, and introduced to an electrochemical analyzer (BAS, ALS600D) to measure the amount of H2 O2 produced by the glucose oxidation reaction. The analyzer chronoamperometrically monitored H2 O2 using Pt electrodes for both counter and action electrodes and an Ag/AgCl/Cl electrode for a reference electrode. The catalytic activity of the immobilized GOD enzyme was determined from the reaction rate of H2 O2 production. The enzyme reaction conditions examined were as follows: glucose concentration range 0.5–10 mM, total flow rate 10–50 L min−1 , pH 5.5–8.5, and reaction temperature 278–300 K. The frequency was scanned in the range 1.5–4.0 kHz to generate resonance oscillation with a scan speed of 1 s−1 . The applied rf electric power was 0.1–4.0 W.
3. Results and discussion To examine the influence of TERO on the reactant solution, TERO was applied to an aqueous glucose solution without immobilized GOD. No production of H2 O2 was observed, confirming that TERO did not produce H2 O2 without the enzyme catalyst. Next, TERO was applied to a GOD enzyme-dissolved glucose reactant solution. The catalytic reaction proceeded, but the generation of TERO caused little enhancement of the catalytic reaction. This indicated that TERO had no ability to enhance the catalytic activity of free GOD enzyme dissolved in the solution. Thus, we can rule out the possibility of TERO-induced sonic wave effects on the reaction, such as cavitation in the liquid phase and/or at the surrounding wall of the microreactor, producing a local hot zone of high pressures and temperatures. Fig. 2 shows changes in the enzyme activity of immobilized GOD with a cycle of TERO-on and TERO-off. In the figure, the original activity without TERO, the activity during TERO-on, and the activity with TERO-off following TERO-on, are defined as Voff(1) , Von , and Voff(2) , respectively. The value of Voff(1) was attained at around 0.26 nmol min−1 at 283 K. The generation of TERO immediately increased the activity, and Von was around 0.71 nmol min−1 , i.e., 2.7 times larger activity with TERO-on. After turning TERO off, no significant activity decrease was observed. The activity, Voff(2) , remained at around 0.62 nmol min−1 . TERO generation in the second run provided nearly the same performance observed in the first run. Again, the activity decrease with TERO-off was small as in the first run. At a temperature of 300 K, changes in the activity with TERO-on and TERO-off were similar to those observed at 283 K except for a smaller activity enhancement with TERO-on. One might point out that TERO only has some preparation advantages of promoting the bonding of enzyme and glutaraldehyde in view of the irreversible behavior of TERO effects. To examine this possibility, TERO was applied once to an immobilized GOD in a GOD-free phosphoric solution, and then the catalytic activity of the enzyme for glucose oxidation reaction was evaluated. The activity was nearly the same as Voff(1) for an immobilized GOD, and thus it should be noted that little activity enhancement with TERO occurred in the absence of glucose substrate. In other words, the presence of glucose substrate was essential for the activation of immobilized GOD by TERO. The characteristic features of TERO effects were not only the considerable activation of an immobilized GOD enzyme, but also the continuation of the TERO-induced high activity after the TERO was turned off. In a previous study on the liquid phase aldol condensation reaction on a [Sc(OTf)3 ] acid catalyst coordinated on a
68
H. Nishiyama et al. / Applied Surface Science 294 (2014) 66–70
Fig. 3. Recovery of TERO-enhanced activity by exposure to glucose-free buffer solution. Immobilized GOD was exposed to flowing glucose-free buffer solution for a period of tc . The vacant and filled squares show the conditions of TERO-off and TERO-on, respectively.
Fig. 2. Changes in the catalytic activity of immobilized GOD for glucose oxidation at (a) 283 K and (b) 300 K. The first activity with TERO-off before TERO-on (), the activity with TERO-on (), and the activity with TERO-off after TERO-on ( ) are defined as Voff(1) , Von and Voff(2) , respectively.
ferroelectric device, the TERO increased the catalytic activity by a factor of 8.2, and the enhanced activity decreased to the original low level with TERO-off. Thus, the irreversible behavior of the immobilized GOD enzyme was intrinsically different from the reversible case of the [Sc(OTf)3 ] acid catalyst. In photoelectron emission microscopy (PEEM) studies of metal surfaces [14,15], the TERO-induced dynamic lattice distortion vertical to the surface was demonstrated to have the ability of changing the work functions of the metal surfaces and hence to induce significant effects on electron transfers between the metal surfaces and adsorbed species. Changes in the catalytic activity of the [Sc(OTf)3 ] acid catalyst with TERO-on were associated with changes in the electronic states of the [Sc(OTf)3 ] acid catalyst, which explained the reversible activity changes in the [Sc(OTf)3 ] catalyst with TERO-on and TERO-off. The irreversible behavior observed for the immobilized GOD enzyme suggests that the activation mechanism by TERO is different in this enzyme. To examine the reversibility of the catalytic performance, the GOD catalyst with enhanced activity was brought into contact with glucose-free solution. As shown in Fig. 3, the activity decreased considerably after contact with time, tc . To evaluate the recovery, a recovery coefficient, R, was defined as the ratio of activity difference of [Von − Voff(1) ] to that of [Von − Voff(2) ]. Fig. 4 shows the relationship of recovery coefficient R with tc in buffer solutions with and without glucose substrate. For the solution involving glucose, the recovery coefficient R was nearly unchanged even after prolonged contact. The experiment using the glucose-free solution was conducted in both the absence and the presence of TERO. For the glucose-free solution without TERO, the R value sharply increased from 0.22 to 0.70 for tc = 5 min, followed by a gradual increase, approaching nearly 1.0 for a long contact time. In order to observe the TERO
effects on the recovery process, TERO was applied while the activated GOD enzyme was brought into contact with glucose-free buffer solution. The recovery profile was nearly the same as the case without TERO, but it trended slightly faster in the range of shorter contact time by TERO. Fig. 5 shows the activation factor, Fa = [Von /Voff(1) ], as a function of electric power applied for the generation of TERO. The value of Fa was small for a small rf power, gradually increased with increasing power, and approached a saturated level. In the TERO effects on the gaseous ethanol oxidation and ethanol decomposition over metal catalyst surfaces such as Ag and Pd, the correlations of activation factor Fa vs. applied electric power showed that activity increases became larger with increasing power. The higher the electric power was, the higher was the efficiency for the enhancement of the catalytic activity. The correlations were accounted for in terms of work function changes in metal catalyst surfaces. The electric power dependence of GOD enzyme catalytic activity that approached a saturated level in a higher power range was different from the behavior of gas phase catalytic reactions on metal surfaces and suggested a small contribution of the electronic effects of metal electrodes on the activation of the immobilized enzyme catalyst.
Fig. 4. Changes in the recovery coefficient R with time exposed to glucosecontaining buffer solution () and to glucose-free buffer solutions under the conditions of TERO-off () and TERO-on ( ).
H. Nishiyama et al. / Applied Surface Science 294 (2014) 66–70
69
Fig. 5. Activation ratio Fa as a function of rf electric power for TERO generation.
Fig. 7. Temperature dependence of glucose oxidation on immobilized GOD with TERO-off and TERO-on.
Fig. 6 shows changes in the three enzyme activities of Voff(1) , Von , and Voff(2) with pH of the reactant solution. Upon increasing from pH = 5.6, the Voff(1) activity increased remarkably, passed through a maximum at around pH = 7.0 then sharply decreased. Similar changes in the activity of Von and Voff(2) with pH were observed. The activation factor Fa was 2.1, 2.1 and 2.3, and the ratio of Voff(2) /Von was 0.82, 0.80 and 0.79 at pH = 5.6, 7.0 and 8.0, respectively, indicating that these values were nearly independent of pH. Fig. 7 shows the temperature dependence of the glucose oxidation reaction on immobilized GOD. The apparent activation energy for the glucose oxidation reaction decreased from 82.8 kJ mo1−1 with TERO-off to 71.7 kJ mol−1 with TERO-on, which showed that TERO effectively lowered the energy barrier for the GOD-catalyzed glucose oxidation reaction. Fig. 8 shows the dependence of the three enzyme activities of Voff(1) , Von , and Voff(2) on the concentration of glucose substrate. The activity Voff(1) increased significantly in a low concentration of glucose, followed by a gradual increase in a moderate concentration region, and approached a saturated level at a high glucose concentration of 10 mM. Changes in the Von and Voff(2) with glucose concentration were similar to that observed for Voff(1) , but the activities themselves were considerably higher than Voff(1) over the range of glucose concentrations. As for the correlation of the activity Voff(1) and Von with glucose concentration, Fig. 9 shows the Lineweaver–Burk plots of V−1 vs.
[S]−1 where V is activity and [S] is the concentration of glucose. The experimental points with Voff(1) and Von fit well using a linear correlation, which confirms that the reaction proceeds according to the Michaelis–Menten mechanism, in which the enzyme activity with glucose concentration is given by
Fig. 6. Influence of pH on the catalytic activity of immobilized GOD with TERO-off and TERO-on.
V=
Vmax [S] [S]+Km
(1)
where Vmax is the reaction at an infinite concentration of glucose substrate, [S] is the concentration of glucose substrate, and Km is the Michaelis–Menten constant represented by (k−1 + k2 )/k1 where k1 and k−1 are the adsorption constant of glucose to and the desorption constant from immobilized GOD, respectively, and k2 is the rate constant for the decomposition of the GOD-glucose complex. Previously, we employed an in situ measurement method to evaluate the amount of glucose molecules adsorbed on immobilized GOD while the catalytic reaction took place [17] and demonstrated that a pseudo-equilibrium between the adsorbed glucose and glucose dissolved in the solution was established, which verified the validity of the Michaelis–Menten mechanism. The results also showed that almost all of the adsorbed glucose participated equivalently in the catalytic reaction. Taking these results into consideration, it is likely that the activation due to TERO occurs for nearly all of the active sites. It is interesting to see that Vmax increased from 1.8 to 4.1 nmol min−1 , and Km decreased from 0.44 to 0.15 mM with
Fig. 8. Glucose concentration dependence of catalytic activity on immobilized GOD with TERO-off and TERO-on.
70
H. Nishiyama et al. / Applied Surface Science 294 (2014) 66–70
Fig. 9. Lineweaver–Burk plots of the reciprocal of activity, V−1 , and glucose concentration [S]−1 .
TERO-on. An increase in Vmax and decrease in Km indicate that TERO enhances interactions between the enzyme and glucose substrate, and strengthens the binding of the glucose substrate to the immobilized GOD. This suggests that the vertical dynamic lattice vibration due to TERO induces relaxation to the bulky supermolecular structure stiffened by immobilization and increases the extent of freedom for the glucose substrate to be coordinated with the GOD enzyme. This plausibly promotes the formation of an active site. The conversion of activated states to the original states was very slow even under glucose-free conditions in view of the nature of an immobilized enzyme, and thus it follows that the formed active site structures can be preserved as long as glucose molecules are present in the solution. Glucose oxidase from Aspergillus niger is a homopolymer, consisting of two identical subunits, each of which has one molecule of tightly bound coenzyme, FAD, which works as a redox carrier in catalysis [18]. The glucose oxidase-catalyzed reaction proceeds according to a ping–pong mechanism [19] as follows: GO(Ox) + glucose → GO(Red) + gluconolactone GO(Red) + O2 → GO(Ox) + H2 O2 where GO(Ox) and GO(Red) are the enzyme with FAD in the oxidized and reduced forms, respectively. In this process, the TERO effects on the reaction rate increases are certainly associated with the activation of the first step of the reductive half-reaction, since the activated states of the enzyme were preserved after TEROoff in the presence of glucose molecules. This indicates that the TERO effects are responsible for the formation of active site structures by the interactions between the glucose molecule and the enzyme. The glucose substrate is bound to free GOD enzyme by the removal of both a water molecule in the enzyme and a proton
from protonated histidine, followed by hydride and proton transfer from glucose to FAD and His516, respectively [18]. Wohlfahrt et al. [20] proposed an active site structure in which the glucose molecule is coordinated with oxidized FAD and two histidines, His559 and His516. This active site structure was suggested for the free enzyme without immobilization, and it seems questionable whether the structure is maintained for the present immobilized GOD. The previous XPS results with GOD immobilized by a covalent method showed that the N 1s peak intensity was significantly increased by GOD addition, which clearly indicated the linkage of GOD with glutaraldehyde-covered surface in the immobilization [17]. However, it is certain that the dynamic lattice distortion due to TERO provides the surface-bound enzyme with some modes of relaxation and freedom, which produces conditions somewhat similar to those of the free enzyme and permits the enhancement of interactions between the glucose molecule and the enzyme. In conclusion, the present study demonstrates that acoustic wave TERO with vertical dynamic lattice displacement significantly activates the immobilized GOD enzyme by promotion of the formation of a substrate-enzyme complex. The characteristic feature is the preservation of activated activity at an 80% level without TERO, indicative of the specific formation of active site structures of the immobilized enzyme. These TERO effects promise applications to a variety of immobilized enzyme reaction systems that will be advantageous for commercial applications. Acknowledgement This work was supported by JSPS KAKENHI Grant Number 23246137. References [1] B.A. Auld, Acoustic Fields and Waves in Solids, vol. II, Wiley & Sons, Inc., 1973, pp. 163. [2] T. Ikeda, Fundamentals of Piezoelectricity, vol. 117, Oxford University Press, New York, 1990, pp. 83. [3] Y. Inoue, Surf. Sci. Rep. 62 (2007) 305–336. [4] Y. Inoue, M. Matsukawa, K. Sato, J. Am. Chem. Soc. 111 (1989) 8965–8966. [5] H. Nishiyama, N. Saito, Y. Watanabe, Y. Inoue, Faraday Discuss. 107 (1997) 425–434. [6] S. Kelling, T. Mitrelias, Y. Matsumoto, V.P. Ostanin, D.A. King, J. Chem. Phys. 107 (1997) 5609–5612. [7] S. Kelling, T. Mitrelias, Y. Matsumoto, V.P. Ostanin, D.A. King, Faraday Discuss. 107 (1997) 435–444. [8] S. Kelling, S. Cerasari, H.H. Rotermund, G. Ertl, D.A. King, Chem. Phys. Lett. 293 (1998) 325–330. [9] H. Nishiyama, Y. Inoue, Surf. Sci. 594 (2005) 156–162. [10] N. Saito, Y. Inoue, J. Phys. Chem. B 107 (2003) 2040–2045. [11] Y. Yukawa, N. Saito, H. Nishiyama, Y. Inoue, J. Phys. Chem. B 108 (2004) 20199–20203. [12] N. Saito, Y. Ohkawara, Y. Watanabe, Y. Inoue, Appl. Surf. Sci. 121/122 (1997) 343–346. [13] N. Saito, H. Nishiyama, K. Sato, Y. Inoue, Chem. Phys. Lett. 297 (1998) 72–76. [14] H. Nishiyama, Y. Inoue, J. Phys. Chem. B 107 (2003) 8738–8741. [15] H. Nishiyama, Y. Inoue, Surf. Sci. 600 (2006) 2644–2649. [16] H. Nishiyama, R. Asari, Y. Inoue, Phys. Chem. Chem. Phys. 12 (2010) 5970–5973. [17] Y. Inoue, Y. Kato, K. Sato, J. Chem. Soc. Faraday Trans. 88 (1992) 449–454. [18] V. Leskovac, S. Trivic, G. Wohlfahrt, J. Kandrac, D. Pericin, Int. J. Biochem. Cell Biol. 37 (2005) 731–750. [19] Q. Su, J.P. Klinman, Biochemistry 38 (1999) 8572–8581. [20] G. Wohlfahrt, S. Witt, J. Hendle, D. Schoburg, H.M. Kalisz, H.J. Hecht, Acta Crystallogr. D 55 (1999) 969–977.
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Effects of acoustic wave resonance oscillation on immobilized enzyme Hiroshi Nishiyama, Tomoya Watanabe, Yasunobu Inoue ∗ Department of Materials Science and Technology, Nagaoka University of Technology, Nagaoka 940-2188, Japan
a r t i c l e
i n f o
Article history: Received 30 August 2013 Received in revised form 3 December 2013 Accepted 4 December 2013 Available online 14 December 2013 Keywords: Acoustic wave Resonance oscillation Enzyme Catalysis Surface
a b s t r a c t In aiming at developing a new method to artificially activate enzyme catalysts immobilized on surface, the effects of resonance oscillation of bulk acoustic waves were studied. Glucose oxidase (GOD) was immobilized by a covalent coupling method on a ferroelectric lead zirconate titanate (PZT) device that was able to generate thickness-extensional resonance oscillation (TERO). Glucose oxidation by the GOD enzyme was studied in a microreactor. The generation of TERO immediately increased the catalytic activity of immobilized GOD by a factor of 2–3. With turn-off of TERO, no significant activity decrease occurred, and 80–90% of the enhanced activity was maintained while the reaction proceeded. The almost complete reversion of the activity to the original low level before TERO generation was observed when the immobilized GOD was exposed to a glucose substrate-free solution. These results indicated that the presence of glucose substrate was essential for TERO-induced GOD activation and preservation of the increased activity level. The influences of reaction temperature, glucose concentration, pH, and rf electric power on the TERO activation showed that TERO strengthened the interactions of the immobilized enzyme with glucose substrate and hence promoted the formation of an activation complex. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The immobilization of enzymes on solid surfaces has the advantage of separating the enzyme from the reactant and product solutions. However, poor catalytic performance is often the result. The activation of immobilized enzymes is thus a key issue, and the establishment of a method for artificial activation of enzyme catalysis is strongly desirable. Acoustic waves such as surface acoustic waves (SAWs) and bulk acoustic resonance oscillations (ROs) that can be generated on a single domain ferroelectric crystal by imposing radio frequency (rf) electric power are characteristic of periodic (dynamic) lattice distortion [1,2]. These acoustic waves have been applied to thin film metal catalysts deposited on ferroelectric substrates [3], and it has clearly been demonstrated that acoustic waves activate metalcatalyzed gas phase reactions by lowering the activation energy of the reactions [4–11]. For instance, significant effects were observed for the thickness-extensional mode RO (TERO) with the lattice vibration mode perpendicular to the surface, which enhanced the catalytic activity of a Pd catalyst for ethanol oxidation by a factor of 1880 [12]. For ethanol decomposition on a Ag catalyst, TERO
∗ Corresponding author. Tel.: +81 258 47 9832; fax: +81 258 47 9830. E-mail address: [email protected] (Y. Inoue). 0169-4332/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsusc.2013.12.016
increased the activity for ethylene production without a significant influence on acetaldehyde production, thus increasing reaction selectivity for ethylene production from 58 to 96% [13]. Photoelectron emission spectroscopy studies [14,15] demonstrated that dynamic lattice vibration modes due to TERO changed the work function of the catalyst metal surfaces, with which the catalyst activation mechanism was associated [6–15]. There have been few applications of acoustic waves to the activation of catalyzed-liquid phase reactions, due to the belief that contact of acoustic waves with a thick and dense liquid phase would result in a significant attenuation of distortion energy. Recently, however, we combined the acoustic wave technique to a microreactor system consisting of a very thin liquid layer and examined the effects of SAWs and ROs on a scandium triflate [Sc(OTf)3 ] catalyst for the liquid phase aldol condensation reaction of benzaldehyde and acetophenone to chalcone [16]. The propagation of Rayleigh SAW with vertical lattice displacement caused a 3-fold enhancement of the activity of the [Sc(OTf)3 ] catalyst coordinated on the SAW-propagation path, decreasing the activation energy of the reaction from 39 to 19 kJ mol−1 . With the effects of RO, TERO increased the activity by a factor of 8.2 and decreased the activation energy of the aldol condensation reaction by 70%. The dynamic lattice distortion vertical to surface is also useful for the activation of liquid phase catalytic reactions. From the viewpoint of acoustic wave applications to liquid phase catalytic reactions, it is of particular interest to see how
H. Nishiyama et al. / Applied Surface Science 294 (2014) 66–70
the catalytic properties of a bulky immobilized enzyme, i.e., a supermolecule, are influenced by the TERO-induced-dynamic lattice distortion of substrates. The present study was undertaken to examine the acoustic wave TERO effects on the oxidation of glucose on immobilized glucose oxidase. We found that acoustic wave TERO induces interesting properties of not only accelerating the immobilized enzyme reaction, but also maintaining the activated states. 2. Experimental A ferroelectric lead zirconate titanate (PZT) polycrystalline ceramic device (25 mm in diameter) covered with 0.4 mm thick brass as electrodes to introduce radio frequency electric power was obtained from Murata Manufacturing Co., Ltd. The PZT had a resonance frequency of 2.2 ± 0.3 kHz and a lattice vibration of the thickness-extensional mode resonance oscillation (TERO) that was normal to the surface. For enzyme immobilization, a covalent-bond method was employed as follows. The ferroelectric device was first dipped into 0.5 vol% 3-aminopropylethoxysilane in toluene for 2 h, dried, and then immersed in 2.5 vol% glutaraldehyde aqueous solution for 2 h. After washing with distilled and ion-exchanged water, the device was immersed at pH 7.0 in a phosphate buffer solution of GOD enzyme (Aspergillus niger, molecular weight: 1.6 × 105 g mol−1 , Wako Pure Chemical Co., Ltd.) at a concentration of 1000 U ml−1 . Glucose oxidase from Aspergillus niger catalyzes the oxidation of -d-glucose by molecular oxygen to ␦gluconolactone, which hydrolyzes spontaneously to gluconic acid and hydrogen peroxide. Fig. 1 shows a flow-type microreactor, a reaction system for glucose oxidation on an immobilized GOD, and a radio frequency electric power system for TERO generation. The microreactor was composed of two separable units. The upper unit had an entrance and exit for reactant and product solutions, respectively, and an electrode pin for the introduction of radio frequency electric power. The lower unit had a connection pin for electric power introduction. A GOD-immobilized PZT device was placed between the two units and fixed tightly with Viton rings. The depth of the liquid layer was 130 m. The radio frequency electric power was generated from a function analyzer (NF Corporation, Multifunction Generator WF1974), and amplified by an amplifier
Fig. 1. A schematic representation of a microreactor, a reaction system, and a radio frequency electric power supply for TERO generation.
67
(NF Corporation, High Speed Power Amplifier, 4005), and then introduced to a GOD-immobilized RO device. -d-Glucose (denoted as glucose for simplicity) (Nakarai Tesque Chemical Co.) substrate in a 0.1 M phosphate buffer solution was introduced to the microreactor by a syringe pump, allowed to flow on the immobilized GOD catalyst, and introduced to an electrochemical analyzer (BAS, ALS600D) to measure the amount of H2 O2 produced by the glucose oxidation reaction. The analyzer chronoamperometrically monitored H2 O2 using Pt electrodes for both counter and action electrodes and an Ag/AgCl/Cl electrode for a reference electrode. The catalytic activity of the immobilized GOD enzyme was determined from the reaction rate of H2 O2 production. The enzyme reaction conditions examined were as follows: glucose concentration range 0.5–10 mM, total flow rate 10–50 L min−1 , pH 5.5–8.5, and reaction temperature 278–300 K. The frequency was scanned in the range 1.5–4.0 kHz to generate resonance oscillation with a scan speed of 1 s−1 . The applied rf electric power was 0.1–4.0 W.
3. Results and discussion To examine the influence of TERO on the reactant solution, TERO was applied to an aqueous glucose solution without immobilized GOD. No production of H2 O2 was observed, confirming that TERO did not produce H2 O2 without the enzyme catalyst. Next, TERO was applied to a GOD enzyme-dissolved glucose reactant solution. The catalytic reaction proceeded, but the generation of TERO caused little enhancement of the catalytic reaction. This indicated that TERO had no ability to enhance the catalytic activity of free GOD enzyme dissolved in the solution. Thus, we can rule out the possibility of TERO-induced sonic wave effects on the reaction, such as cavitation in the liquid phase and/or at the surrounding wall of the microreactor, producing a local hot zone of high pressures and temperatures. Fig. 2 shows changes in the enzyme activity of immobilized GOD with a cycle of TERO-on and TERO-off. In the figure, the original activity without TERO, the activity during TERO-on, and the activity with TERO-off following TERO-on, are defined as Voff(1) , Von , and Voff(2) , respectively. The value of Voff(1) was attained at around 0.26 nmol min−1 at 283 K. The generation of TERO immediately increased the activity, and Von was around 0.71 nmol min−1 , i.e., 2.7 times larger activity with TERO-on. After turning TERO off, no significant activity decrease was observed. The activity, Voff(2) , remained at around 0.62 nmol min−1 . TERO generation in the second run provided nearly the same performance observed in the first run. Again, the activity decrease with TERO-off was small as in the first run. At a temperature of 300 K, changes in the activity with TERO-on and TERO-off were similar to those observed at 283 K except for a smaller activity enhancement with TERO-on. One might point out that TERO only has some preparation advantages of promoting the bonding of enzyme and glutaraldehyde in view of the irreversible behavior of TERO effects. To examine this possibility, TERO was applied once to an immobilized GOD in a GOD-free phosphoric solution, and then the catalytic activity of the enzyme for glucose oxidation reaction was evaluated. The activity was nearly the same as Voff(1) for an immobilized GOD, and thus it should be noted that little activity enhancement with TERO occurred in the absence of glucose substrate. In other words, the presence of glucose substrate was essential for the activation of immobilized GOD by TERO. The characteristic features of TERO effects were not only the considerable activation of an immobilized GOD enzyme, but also the continuation of the TERO-induced high activity after the TERO was turned off. In a previous study on the liquid phase aldol condensation reaction on a [Sc(OTf)3 ] acid catalyst coordinated on a
68
H. Nishiyama et al. / Applied Surface Science 294 (2014) 66–70
Fig. 3. Recovery of TERO-enhanced activity by exposure to glucose-free buffer solution. Immobilized GOD was exposed to flowing glucose-free buffer solution for a period of tc . The vacant and filled squares show the conditions of TERO-off and TERO-on, respectively.
Fig. 2. Changes in the catalytic activity of immobilized GOD for glucose oxidation at (a) 283 K and (b) 300 K. The first activity with TERO-off before TERO-on (), the activity with TERO-on (), and the activity with TERO-off after TERO-on ( ) are defined as Voff(1) , Von and Voff(2) , respectively.
ferroelectric device, the TERO increased the catalytic activity by a factor of 8.2, and the enhanced activity decreased to the original low level with TERO-off. Thus, the irreversible behavior of the immobilized GOD enzyme was intrinsically different from the reversible case of the [Sc(OTf)3 ] acid catalyst. In photoelectron emission microscopy (PEEM) studies of metal surfaces [14,15], the TERO-induced dynamic lattice distortion vertical to the surface was demonstrated to have the ability of changing the work functions of the metal surfaces and hence to induce significant effects on electron transfers between the metal surfaces and adsorbed species. Changes in the catalytic activity of the [Sc(OTf)3 ] acid catalyst with TERO-on were associated with changes in the electronic states of the [Sc(OTf)3 ] acid catalyst, which explained the reversible activity changes in the [Sc(OTf)3 ] catalyst with TERO-on and TERO-off. The irreversible behavior observed for the immobilized GOD enzyme suggests that the activation mechanism by TERO is different in this enzyme. To examine the reversibility of the catalytic performance, the GOD catalyst with enhanced activity was brought into contact with glucose-free solution. As shown in Fig. 3, the activity decreased considerably after contact with time, tc . To evaluate the recovery, a recovery coefficient, R, was defined as the ratio of activity difference of [Von − Voff(1) ] to that of [Von − Voff(2) ]. Fig. 4 shows the relationship of recovery coefficient R with tc in buffer solutions with and without glucose substrate. For the solution involving glucose, the recovery coefficient R was nearly unchanged even after prolonged contact. The experiment using the glucose-free solution was conducted in both the absence and the presence of TERO. For the glucose-free solution without TERO, the R value sharply increased from 0.22 to 0.70 for tc = 5 min, followed by a gradual increase, approaching nearly 1.0 for a long contact time. In order to observe the TERO
effects on the recovery process, TERO was applied while the activated GOD enzyme was brought into contact with glucose-free buffer solution. The recovery profile was nearly the same as the case without TERO, but it trended slightly faster in the range of shorter contact time by TERO. Fig. 5 shows the activation factor, Fa = [Von /Voff(1) ], as a function of electric power applied for the generation of TERO. The value of Fa was small for a small rf power, gradually increased with increasing power, and approached a saturated level. In the TERO effects on the gaseous ethanol oxidation and ethanol decomposition over metal catalyst surfaces such as Ag and Pd, the correlations of activation factor Fa vs. applied electric power showed that activity increases became larger with increasing power. The higher the electric power was, the higher was the efficiency for the enhancement of the catalytic activity. The correlations were accounted for in terms of work function changes in metal catalyst surfaces. The electric power dependence of GOD enzyme catalytic activity that approached a saturated level in a higher power range was different from the behavior of gas phase catalytic reactions on metal surfaces and suggested a small contribution of the electronic effects of metal electrodes on the activation of the immobilized enzyme catalyst.
Fig. 4. Changes in the recovery coefficient R with time exposed to glucosecontaining buffer solution () and to glucose-free buffer solutions under the conditions of TERO-off () and TERO-on ( ).
H. Nishiyama et al. / Applied Surface Science 294 (2014) 66–70
69
Fig. 5. Activation ratio Fa as a function of rf electric power for TERO generation.
Fig. 7. Temperature dependence of glucose oxidation on immobilized GOD with TERO-off and TERO-on.
Fig. 6 shows changes in the three enzyme activities of Voff(1) , Von , and Voff(2) with pH of the reactant solution. Upon increasing from pH = 5.6, the Voff(1) activity increased remarkably, passed through a maximum at around pH = 7.0 then sharply decreased. Similar changes in the activity of Von and Voff(2) with pH were observed. The activation factor Fa was 2.1, 2.1 and 2.3, and the ratio of Voff(2) /Von was 0.82, 0.80 and 0.79 at pH = 5.6, 7.0 and 8.0, respectively, indicating that these values were nearly independent of pH. Fig. 7 shows the temperature dependence of the glucose oxidation reaction on immobilized GOD. The apparent activation energy for the glucose oxidation reaction decreased from 82.8 kJ mo1−1 with TERO-off to 71.7 kJ mol−1 with TERO-on, which showed that TERO effectively lowered the energy barrier for the GOD-catalyzed glucose oxidation reaction. Fig. 8 shows the dependence of the three enzyme activities of Voff(1) , Von , and Voff(2) on the concentration of glucose substrate. The activity Voff(1) increased significantly in a low concentration of glucose, followed by a gradual increase in a moderate concentration region, and approached a saturated level at a high glucose concentration of 10 mM. Changes in the Von and Voff(2) with glucose concentration were similar to that observed for Voff(1) , but the activities themselves were considerably higher than Voff(1) over the range of glucose concentrations. As for the correlation of the activity Voff(1) and Von with glucose concentration, Fig. 9 shows the Lineweaver–Burk plots of V−1 vs.
[S]−1 where V is activity and [S] is the concentration of glucose. The experimental points with Voff(1) and Von fit well using a linear correlation, which confirms that the reaction proceeds according to the Michaelis–Menten mechanism, in which the enzyme activity with glucose concentration is given by
Fig. 6. Influence of pH on the catalytic activity of immobilized GOD with TERO-off and TERO-on.
V=
Vmax [S] [S]+Km
(1)
where Vmax is the reaction at an infinite concentration of glucose substrate, [S] is the concentration of glucose substrate, and Km is the Michaelis–Menten constant represented by (k−1 + k2 )/k1 where k1 and k−1 are the adsorption constant of glucose to and the desorption constant from immobilized GOD, respectively, and k2 is the rate constant for the decomposition of the GOD-glucose complex. Previously, we employed an in situ measurement method to evaluate the amount of glucose molecules adsorbed on immobilized GOD while the catalytic reaction took place [17] and demonstrated that a pseudo-equilibrium between the adsorbed glucose and glucose dissolved in the solution was established, which verified the validity of the Michaelis–Menten mechanism. The results also showed that almost all of the adsorbed glucose participated equivalently in the catalytic reaction. Taking these results into consideration, it is likely that the activation due to TERO occurs for nearly all of the active sites. It is interesting to see that Vmax increased from 1.8 to 4.1 nmol min−1 , and Km decreased from 0.44 to 0.15 mM with
Fig. 8. Glucose concentration dependence of catalytic activity on immobilized GOD with TERO-off and TERO-on.
70
H. Nishiyama et al. / Applied Surface Science 294 (2014) 66–70
Fig. 9. Lineweaver–Burk plots of the reciprocal of activity, V−1 , and glucose concentration [S]−1 .
TERO-on. An increase in Vmax and decrease in Km indicate that TERO enhances interactions between the enzyme and glucose substrate, and strengthens the binding of the glucose substrate to the immobilized GOD. This suggests that the vertical dynamic lattice vibration due to TERO induces relaxation to the bulky supermolecular structure stiffened by immobilization and increases the extent of freedom for the glucose substrate to be coordinated with the GOD enzyme. This plausibly promotes the formation of an active site. The conversion of activated states to the original states was very slow even under glucose-free conditions in view of the nature of an immobilized enzyme, and thus it follows that the formed active site structures can be preserved as long as glucose molecules are present in the solution. Glucose oxidase from Aspergillus niger is a homopolymer, consisting of two identical subunits, each of which has one molecule of tightly bound coenzyme, FAD, which works as a redox carrier in catalysis [18]. The glucose oxidase-catalyzed reaction proceeds according to a ping–pong mechanism [19] as follows: GO(Ox) + glucose → GO(Red) + gluconolactone GO(Red) + O2 → GO(Ox) + H2 O2 where GO(Ox) and GO(Red) are the enzyme with FAD in the oxidized and reduced forms, respectively. In this process, the TERO effects on the reaction rate increases are certainly associated with the activation of the first step of the reductive half-reaction, since the activated states of the enzyme were preserved after TEROoff in the presence of glucose molecules. This indicates that the TERO effects are responsible for the formation of active site structures by the interactions between the glucose molecule and the enzyme. The glucose substrate is bound to free GOD enzyme by the removal of both a water molecule in the enzyme and a proton
from protonated histidine, followed by hydride and proton transfer from glucose to FAD and His516, respectively [18]. Wohlfahrt et al. [20] proposed an active site structure in which the glucose molecule is coordinated with oxidized FAD and two histidines, His559 and His516. This active site structure was suggested for the free enzyme without immobilization, and it seems questionable whether the structure is maintained for the present immobilized GOD. The previous XPS results with GOD immobilized by a covalent method showed that the N 1s peak intensity was significantly increased by GOD addition, which clearly indicated the linkage of GOD with glutaraldehyde-covered surface in the immobilization [17]. However, it is certain that the dynamic lattice distortion due to TERO provides the surface-bound enzyme with some modes of relaxation and freedom, which produces conditions somewhat similar to those of the free enzyme and permits the enhancement of interactions between the glucose molecule and the enzyme. In conclusion, the present study demonstrates that acoustic wave TERO with vertical dynamic lattice displacement significantly activates the immobilized GOD enzyme by promotion of the formation of a substrate-enzyme complex. The characteristic feature is the preservation of activated activity at an 80% level without TERO, indicative of the specific formation of active site structures of the immobilized enzyme. These TERO effects promise applications to a variety of immobilized enzyme reaction systems that will be advantageous for commercial applications. Acknowledgement This work was supported by JSPS KAKENHI Grant Number 23246137. References [1] B.A. Auld, Acoustic Fields and Waves in Solids, vol. II, Wiley & Sons, Inc., 1973, pp. 163. [2] T. Ikeda, Fundamentals of Piezoelectricity, vol. 117, Oxford University Press, New York, 1990, pp. 83. [3] Y. Inoue, Surf. Sci. Rep. 62 (2007) 305–336. [4] Y. Inoue, M. Matsukawa, K. Sato, J. Am. Chem. Soc. 111 (1989) 8965–8966. [5] H. Nishiyama, N. Saito, Y. Watanabe, Y. Inoue, Faraday Discuss. 107 (1997) 425–434. [6] S. Kelling, T. Mitrelias, Y. Matsumoto, V.P. Ostanin, D.A. King, J. Chem. Phys. 107 (1997) 5609–5612. [7] S. Kelling, T. Mitrelias, Y. Matsumoto, V.P. Ostanin, D.A. King, Faraday Discuss. 107 (1997) 435–444. [8] S. Kelling, S. Cerasari, H.H. Rotermund, G. Ertl, D.A. King, Chem. Phys. Lett. 293 (1998) 325–330. [9] H. Nishiyama, Y. Inoue, Surf. Sci. 594 (2005) 156–162. [10] N. Saito, Y. Inoue, J. Phys. Chem. B 107 (2003) 2040–2045. [11] Y. Yukawa, N. Saito, H. Nishiyama, Y. Inoue, J. Phys. Chem. B 108 (2004) 20199–20203. [12] N. Saito, Y. Ohkawara, Y. Watanabe, Y. Inoue, Appl. Surf. Sci. 121/122 (1997) 343–346. [13] N. Saito, H. Nishiyama, K. Sato, Y. Inoue, Chem. Phys. Lett. 297 (1998) 72–76. [14] H. Nishiyama, Y. Inoue, J. Phys. Chem. B 107 (2003) 8738–8741. [15] H. Nishiyama, Y. Inoue, Surf. Sci. 600 (2006) 2644–2649. [16] H. Nishiyama, R. Asari, Y. Inoue, Phys. Chem. Chem. Phys. 12 (2010) 5970–5973. [17] Y. Inoue, Y. Kato, K. Sato, J. Chem. Soc. Faraday Trans. 88 (1992) 449–454. [18] V. Leskovac, S. Trivic, G. Wohlfahrt, J. Kandrac, D. Pericin, Int. J. Biochem. Cell Biol. 37 (2005) 731–750. [19] Q. Su, J.P. Klinman, Biochemistry 38 (1999) 8572–8581. [20] G. Wohlfahrt, S. Witt, J. Hendle, D. Schoburg, H.M. Kalisz, H.J. Hecht, Acta Crystallogr. D 55 (1999) 969–977.