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Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres
Materials Science and Engineering C 33 (2013) 1438–1445
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres Hong Wu, Yanpeng Liang, Jiafu Shi, Xiaoli Wang, Dong Yang, Zhongyi Jiang ⁎ Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
a r t i c l e
i n f o
Article history: Received 7 March 2012 Received in revised form 6 November 2012 Accepted 13 December 2012 Available online 22 December 2012 Keywords: Titania Covalent binding Immobilized enzyme Biocatalysis Stability Kinetic parameters
a b s t r a c t In this study, a novel approach combing the chelation and covalent binding was explored for facile and efficient enzyme immobilization. The unique capability of titania to chelate with catecholic derivatives at ambient conditions was utilized for titania surface functionalization. The functionalized titania was then used for enzyme immobilization. Titania submicrospheres (500–600 nm) were synthesized by a modified sol–gel method and functionalized with carboxylic acid groups through a facile chelation method by using 3-(3,4-dihydroxyphenyl) propionic acid as the chelating agent. Then, catalase (CAT) was covalently immobilized on these functionalized titania submicrospheres through 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. The immobilized CAT retained 65% of its free form activity with a loading capacity of 100–150 mg/g titania. The pH stability, thermostability, recycling stability and storage stability of the immobilized CAT were evaluated. A remarkable enhancement in enzyme stability was achieved. The immobilized CAT retained 90% and 76% of its initial activity after 10 and 16 successive cycles of decomposition of hydrogen peroxide, respectively. Both the Km and the Vmax values of the immobilized CAT (27.4 mM, 13.36 mM/min) were close to those of the free CAT (25.7 mM, 13.46 mM/min). © 2012 Elsevier B.V. All rights reserved.
1. Introduction Enzymes are versatile biocatalysts that exhibit a number of unique advantages over conventional chemical catalysts, such as high activity, selectivity and specificity [1]. Till now, enzymes have been found in numerous applications including polymer synthesis [2], biomaterials [3], bioconversion [4], bioseparation [5], biosensors [6], etc. Development of immobilized enzymes has attracted considerable attention for many years to facilitate the reaction process and reduce the operation cost. High catalytic activity and loading capacity, easy separation and reusability are the essential requirements for immobilized enzymes [7]. Currently, adsorption, covalent binding, entrapment, encapsulation and cross-linking, or their combinations, are still the prevalent methods utilized for enzyme immobilization [8]. Compared with other immobilization methods, the covalent binding which involves the formation of covalent bonds between the reactive groups on the support and the functional groups on the enzymes offers the strongest linkage between the enzyme and the support [9]. Immobilized enzyme leaching out of reaction media and the consequent contamination to the product are thus minimized [10]. In addition, the recycling stability of immobilized enzyme is significantly enhanced. For example, catalase was covalently immobilized on nanofibers and retained 70% of its initial activity after 10 batch cycles ⁎ Corresponding author. Tel./fax: +86 22 23500086. E-mail address: [email protected] (Z. Jiang). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.12.048
[11]. The diffusion limitation will be reduced or eliminated when enzyme molecules are covalently immobilized on the carrier's outer surface. However, the control for covalent immobilization process is usually caustic in the past: it should meet the temperature and pH requirements of the reaction, and should be mild enough to avoid enzyme denaturation as well. Recently, there are a multitude of soft methods for covalent immobilization including the widely used EDC/NHS chemistry method [12]. Inorganic carriers usually possess high mechanical properties, thermal stability and resistance against microbial attack and organic solvents. Many kinds of inorganic solids have been used for the covalent immobilization of enzymes such as Au, SiO2, Fe3O4 and TiO2 [13–16]. Most of these inorganic carriers couldn't react with enzymes directly without surface functionalization. Therefore, surface functionalization of the carrier is necessary prior to covalent attachment of enzymes. Lü et al. synthesized epoxy group functionalized cubic mesoporous silica for covalent attachment of Penicillin G acylase. The initial specific activity of such immobilized enzyme was higher than that of enzyme immobilized by physical adsorption. The immobilized enzyme retained 72% of its initial activity after 10 batches [14]. Liu X. synthesized the magnetic particles functionalized by expoxy group for lipase immobilization. The loading capacity was 68.3 mg lipase/g support, and the activity retention of lipase was 60.4% [15]. Immobilization of catalase on modified nanofiber membranes led to an enhanced thermostability [17]. α-Amylase was covalently immobilized onto acid chloride group functionalized glass beads. The loading capacity was found to be
H. Wu et al. / Materials Science and Engineering C 33 (2013) 1438–1445
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was vigorously stirred at room temperature for 30 min to allow a complete chemisorption of catecholic salt onto the TiO2 particle surface. The low pH condition prevented the catechol end groups of 3-(3,4dihydroxyphenyl) propionic acid from oxidation. The modified TiO2 submicrospheres were collected and rinsed thoroughly with deionized water. The as-synthesized carboxylic-functionalized titania (TiO2– COOH) submicrospheres were dried at 80 °C for 24 h.
25.2 mg/g glass support. The free enzyme lost all its activity within 15 days. Immobilized enzyme lost only 20% of activity in 25 days [18]. Although the above covalent immobilization commonly led to enhanced stability, the functionalization process prior to immobilization was tedious and time-consuming, usually involving multiple steps including particle surface activation with organosilane, chemical reactions, polymerization and grafting reactions. Moreover, high temperature, addition of organic solvents and nitrogen protection were often required during the functionalization process. It has been found that catechol and catecholic derivatives could chelate with transition metal oxide such as titania and iron oxide to form a stable salt chelate complex at room temperature rapidly [19–23]. The titania support surface could be easily modified under mild conditions at an ambient temperature through the simple chelation process. In addition, TiO2-based materials had excellent pH and corrosion resistance, superior mechanical strength, outstanding antimicrobial performance, and in particular good biocompatibility [24]. Herein, a novel and facile approach was presented to synthesize carboxyl functionalized titania submicrospheres for enzyme immobilization. Titania submicrospheres were synthesized by a modified sol–gel method and then functionalized with carboxylic acid groups via the chelation approach using 3-(3,4-dihydroxyphenyl) propionic acid as carboxylation reagent. Catalase (CAT), as a model enzyme, was covalently anchored to the functionalized titania submicrospheres via EDC/NHS coupling reaction. The immobilized catalase was used to decompose hydrogen peroxide to water and oxygen. The activity and stability of the immobilized enzyme were investigated and compared with those of free enzyme counterpart.
Covalent immobilization of CAT onto the TiO2–COOH submicrospheres was performed via EDC/NHS coupling reaction [26]. The immobilization procedure consisted of two steps: EDC and NHS activation followed by coupling reaction of enzyme. First, 50.0 mg of TiO2–COOH submicrospheres were dispersed in 10.0 ml of MES buffer solution (50 mM, pH 6.5) under ultrasonic treatment for 15 min. Then, NHS (1.5 mmol) and EDC (0.3 mmol) were added to the above suspension. The mixture was vigorously stirred at room temperature for 1 h. The NHS-activated TiO2–COOH submicrospheres were collected by centrifugation and washed with MES buffer solution (50 mM, pH 6.5) thoroughly to remove unreacted NHS and EDC. Second, the NHS-activated TiO2–COOH submicrospheres were dispersed in 10.0 ml of the CAT solution (5 mg/ml) prepared with MES buffer solution (50 mM, pH 6.5). The enzyme coupling reaction was allowed to proceed for 4 h at room temperature under magnetic stirring. The catalase-titania conjugates (TiO2–CAT) were separated by centrifugation and washed thoroughly with deionized water. The immobilized enzyme was lyophilized and stored at −20 °C.
2. Materials and methods
2.4. Enzyme loading capacity and activity assay
2.1. Materials
The amount of CAT immobilized was determined by measuring the initial and final concentrations of CAT within the enzyme and washing solutions using Coomassie Brilliant Blue reagent, following the Bradford's method [27]. The loading capacity was determined according to Eq. (1):
Catalase (hydrogen peroxide oxidoreductase; 2.85×104 units/mg protein; EC.1.11.1.6) from bovine liver, 2-(N-morpholino) ethanesulfonic acid sodium salt (MES) and Tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma. 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Medpep Co., Ltd. Acetone, ethylene glycol, tetrabutyl titanate (TBT, >98%), hydrogen peroxide (H2O2, 30%), acetic acid, sodium acetate, sodium dihydrogen phosphate dehydrate (NaH2PO4·2H2O) and sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O) were purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). 3-(3,4-Dihydroxyphenyl) propionic acid (98%) was purchased from Alfa Aesar. 2.2. Synthesis of the carboxyl acid functionalized titania submicrospheres Titania (TiO2) submicrospheres were synthesized by a modified sol–gel method as described in the literature [25]. 0.02 mol tetrabutoxytitanium (TBT, 6.8 ml) was added to 100 ml ethylene glycol in a round flask purged with nitrogen gas. The solution was magnetically stirred rapidly for 20 h at 30 °C. The resultant transparent solution was poured into acetone solution containing ~ 0.3 wt.% of water under vigorous stirring for 30 min. After aging for about 1 h, the white precipitate was collected by centrifugation at 3058 g for 5 min, washed with distilled water and ethanol alternatively to remove the residue ethylene glycol. The obtained TiO2 submicrospheres with a size of 500–600 nm were dried at 80 °C for 24 h. 3-(3,4-Dihydroxyphenyl) propionic acid was used as the carboxylation reagent for the surface modification of the TiO2 submicrospheres. A facile chemical adsorption procedure was conducted [19–23]. The TiO2 submicrospheres were suspended in an aqueous HCl solution (pH 2.0) under ultrasonic treatment for 0.5 h. Then, the TiO2 submicrospheres were filtered and added into an excessive 3-(3,4-dihydroxyphenyl) propionic acid aqueous solution (4 mg/ml, pH 2.0). The suspension
2.3. Immobilization of enzyme on functionalized titania submicrospheres
M
mg enzyme ðm−C 1 V 1 Þ ¼ g support W
ð1Þ
where M represented the loading capacity; m was the amount of CAT introduced into the immobilization medium; C1 and V1 were the enzyme concentration and volume of the washing solution, respectively; W was the weight of the TiO2–COOH submicrospheres. The enzyme concentration (C1) was determined via UV–vis standard curve (correlation coefficient R2 = 0.998). The activity of free and immobilized CAT was determined by measuring the decrease in the absorbance of hydrogen peroxide at 240 nm in 5 min due to the decomposition of H2O2 [28].
2 H2 O2
Catalase
→ 2 H2 O þ O2 :
ð2Þ
The immobilized CAT (1 mg) (or free enzyme (0.1 ml, 1 mg/ml)) was added into the H2O2 solution (17.64 mM) prepared in 50 ml phosphate buffer solution (50 mM, pH 7) under agitation. The decrease in absorbance at 240 nm was recorded after 5 min. One unit will decompose 1.0 μmol of H2O2 per min at pH 7.0 at 25 °C, measured by the decrease rate of A240 [29]. The relative activity of immobilized enzyme was defined as the ratio of its activity to the free form activity under the equal amount of enzyme (Eq. (3)).
relative activityð% Þ ¼
immobilized enzyme activity 100: f ree enzyme activity
ð3Þ
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The activation energy (Ed) for CAT thermal denaturation was determined by applying the Arrhenius equation Eq. (9):
2.5. Kinetic parameters (Km and Vmax) Kinetic parameters, Km and Vmax, for the free and immobilized enzymes were determined using the Michaelis–Menten equation, given by Eq. (4) 1 K 1 1 ¼ m þ V V max ½S V max
ð4Þ
where V was the initial reactive rate, [S] was the initial substrate concentration, Vmax was the maximum reaction rate attained at infinite initial substrate concentration and Km was the Michaelis–Menten constant. To determine the Km and Vmax, the activity assay was applied for different hydrogen peroxide concentrations from 5 mM to 35 mM. The amount of free CAT added to the reaction system was 0.1 mg, and correspondingly, 1 mg solid with ~ 0.1 mg immobilized CAT was used to keep the amounts of enzyme identically. Enzyme activity was determined at pH 7.0 at 25 °C. Kinetic parameters for both the free and the immobilized CAT were calculated accordingly. 2.6. Effect of pH and temperature on enzyme activity The effects of pH and temperature on the activity for free and immobilized CAT were studied by changing the reaction pH value from 3.0 to 9.0 (sodium acetate buffer pH 3.0–5.0; MES buffer pH 6.0; phosphate buffer pH 7.0–8.0; Tris–HCl buffer pH 9.0) at room temperature (25 °C) and the temperature from 20 °C to 70 °C at pH 7.0. Optimum reaction conditions (pH and temperature) were determined accordingly. The activity found at each specific pH (or temperature) was compared with the activity at optimum pH (or temperature) in terms of relative activity (Eq. (5)) [30]. relatively activityð% Þ enzyme activity at the specific pHðor temperatureÞ ¼ 100: ð5Þ enzyme activity at the optimum pHðor temperatureÞ
2.7. Thermostability The thermostability of free and immobilized CAT was examined by measuring the residual activity of enzyme incubated at different temperatures (20 °C to 75 °C) in phosphate buffer solution (50 mM, pH 7.0) for 2 h. Activity of enzymes was measured at 25 °C and pH 7.0. The relative activity of enzyme was defined as the ratio of its activity after incubation to the activity under the optimal condition (Eq. (6)) [4]. relatively activityð% Þ ¼
enzyme activity af ter incubation enzyme activity at the optimum temperature 100: ð6Þ
The thermal denaturation kinetic of CAT was described as first order [31,32]. The thermal denaturation constants (kd) were calculated from the first order exponential approach [32] (Eq. (7)): Acat =Acat;0 ¼ expð−kd t Þ
ð7Þ
where Acat,0 was the CAT activity before incubation, Acat was the CAT activity after incubation at a certain temperature for a certain time and t was the incubation time. The half-life (t1/2) value for CAT thermal denaturation was determined from Eq. (8): t 1=2 ¼ ln2=kd :
ð8Þ
lnkd ¼ −Ed =RT þ lnC:
ð9Þ
The Ed was calculated by the plot of log denaturation rate constants (ln kd) versus reciprocal of the absolute temperature (T) from Eq. (10): Slope ¼ −Ed =R:
ð10Þ
The change in enthalpy (ΔH° kJ mol −1) for CAT thermal denaturation was determined using the Eq. (11): 0
ΔH ¼ Ed −RT
ð11Þ
where R was the gas constant (8.3145 J mol −1 K −1) and T was the corresponding absolute temperature [33]. 2.8. Recycling stability The immobilized CAT (1 mg) was removed after each reaction batch by centrifugation, and washed with phosphate buffer solution (50 mM, pH 7.0) to remove any residual substrate and then added to the next reaction cycle. The recycling stability of immobilized CAT was explored by measuring the enzyme activity in each successive reaction cycle and expressed by recycling efficiency (Eq. (12)). th
recycling efficiencyð%Þ ¼
enzyme activity in the n cycle enzyme activity in the 1st cycle 100:
ð12Þ
2.9. Storage stability Free CAT and immobilized CAT were stored in phosphate buffer solution (50 mM, pH 7.0) at 4 °C for a certain period of time. The storage stability was compared in terms of storage efficiency defined as the ratio of the activity of free or immobilized enzyme after storage to their initial activity (Eq. (13)). storage efficiencyð%Þ ¼
enzyme activity after storage 100 initial enzyme activity
ð13Þ
3. Results and discussion 3.1. Characterization of the support and immobilized enzyme Surface modification of Ti and TiO2 via chelation of catechol had been well-documented in the previous literatures [19,21–23,34,35]. Herein, the titania particles prepared through the sol–gel method were functionalized with caboxyl acid groups by using 3-(3,4dihydroxyphenyl) propionic acid as the chelating agent. The morphology of the pristine and functionalized titania submicrospheres was observed by transmission electron microscope (TEM) (Fig. 1(a–c)). The average particle sizes of the TiO2 submicrospheres, TiO2–COOH submicrospheres and those with immobilized enzyme were found to be in the range of 500–600 nm. There was no obvious change in size and morphology after covalent immobilization of CAT. Thermo gravimetric analysis (TGA) was performed to determine the modification percentage, that is, the 3-(3,4-dihydroxyphenyl) propionic acid content on the titania. The weight loss in the temperature range of 100–800 °C attributed to the decomposition of organic modifier was found to be about 10 wt.%, and then the modification percentage could be calculated to be about 0.55 mmol (\COOH)/g TiO2–COOH particles.
H. Wu et al. / Materials Science and Engineering C 33 (2013) 1438–1445
(a)
(b)
(c)
(d)
1441
2935 2868 1080
Intensity
TiO2 1474 TiO2 -COOH
1438
1540
1650 TiO2 -CAT
3000
2500
2000
1500
1000
Wave number / cm-1
Fig. 1. TEM photographs of TiO2 (a), TiO2–COOH (b) and TiO2–CAT (c); FITR spectra of TiO2, TiO2–COOH and TiO2–CAT (d).
The chemical structure of TiO2, TiO2–COOH and TiO2–CAT was determined by Fourier transform infrared spectra (FTIR). As shown in Fig. 1(d), the absorption bands at 1086, 2868 and 2935 cm −1 for TiO2 were assigned to the C\C\O stretching vibration, symmetric and asymmetric \CH2\ stretching vibrations of ethylene glycol, respectively. The characteristic peaks of ethylene glycol disappeared after carboxyl functionalization [36]. The new bands located at 1474 and 1438 cm −1 were assigned to the C_C benzene ring characteristic vibrations [37]. For the TiO2–CAT, the main characteristic peak of amide I band at 1650 cm −1 was caused by C_O stretching vibrations of peptide linkages, whereas, the amide II band at 1540 cm −1 was attributed to the combination of N–H bending and C–N stretching [38–40]. The appearance of the above bands indicated the successful immobilization of CAT on the TiO2–COOH submicrospheres. EDX mapping was conducted with NHS activated TiO2–COOH and TiO2–CAT and the results were shown in Fig. 2. The nitrogen element in the NHS activated TiO2–COOH and TiO2–COOH was originated from the NHS and CAT molecules, respectively. The N/Ti atomic ratio of the CAT (0.1077) was higher than that of the NHS activated TiO2–COOH (0.0663), indicating that the CAT was indeed attached onto the supports. 3.2. Immobilization procedure, loading efficiency and activity CAT was covalently immobilized on the TiO2–COOH submicrospheres via EDC/NHS coupling reaction. The immobilization process was illustrated in Fig. 3. First, 3-(3,4-dihydroxyphenyl) propionic acid with two phenolic hydroxyl groups and a carboxyl group was used as both the chelating and carboxylation reagent. A chelation occurred between the titania and the phenolic hydroxyl groups at room temperature in a few minutes, thus rendering the titania surface with carboxyl groups. Second,
the TiO2–COOH submicrospheres were activated with EDC and NHS to yield an ester intermediate. Finally, the CAT molecules were immobilized on the activated titania surface via covalent bonds. EDC was a zero-length cross-linker widely used in protein conjugations. The coupling reaction process of CAT on the TiO2–COOH submicrospheres could be achieved by a two-step procedure [41]. EDC could react with the carboxyl group on the surface of the TiO2–COOH submicrospheres, forming an O-acylisourea intermediate which was very unstable and susceptible to hydrolysis. The O-acylisourea intermediate could react with an amine group to produce a stable amide bond [42]. But the instability of the O-acylisourea intermediate resulted in a low coupling efficiency. The addition of NHS could stabilize the O-acylisourea intermediate by converting it to an amine-reactive NHS ester (succinimidyl ester). The coupling efficiency could thus be increased by 10–20 folds [43,44]. A control experiment was conducted without adding EDC/NHS during enzyme immobilization. The support exhibited no catalytic activity through the enzyme assays, confirming that the carboxylic acid groups on the TiO2–COOH could not react with CAT at room temperature. In summary, the TiO2–COOH submicrospheres were first reacted with EDC and NHS to yield an ester intermediate. After the excess NHS and EDC were washed with deionized water, the activated supports were then reacted with CAT. The activity of immobilized CAT was 1.85 × 10 4 units/mg protein. It was found that the immobilized CAT retained 65% of its original activity. The loading capacity determined by Bradford's method was 100–150 mg/g titania submicrospheres. 3.3. Kinetic parameters for free and immobilized enzyme Kinetic parameters were measured for both free and immobilized CAT at 25 °C, pH 7.0. The Michaelis constant (Km) and the maximum
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calcium alginate and found that the Km was 2.5 times greater than that of free enzyme [48]. The Km value of the bovine liver CAT increased by 3 times after covalent immobilization on Eupergit C [49]. In this study, the Km and Vmax values for free and immobilized CAT were 25.7 mM and 13.46 mM/min, and 27.4 mM and 13.36 mM/min, respectively. The Km value reflected the affinity of the enzyme toward substrate. The lower value of Km indicated the higher affinity between enzyme and substrate [50]. The comparable Km values for free and covalent immobilized CAT (25.7 mM vs. 27.4 mM) indicated almost the identical affinity towards the hydrogen peroxide substrate molecules. The Vmax value reflected the intrinsic characteristic of the immobilized enzyme which could be affected by diffusion constrains [29,49]. Additional diffusional resistance often occurred in the entrapment/encapsulation method. In contrast, since covalently immobilized enzyme molecules were located on the outer surface of the solid supports, the inner diffusion limitation would be much reduced or even eliminated. It can be derived that the three-dimensional conformation and active sites of the enzyme were well-preserved in the present study. 3.4. Effect of pH and temperature on enzyme activity The effect of pH on activity was studied at different pH values (3.0 to 9.0) at room temperature (25 °C). The relative activity of free and immobilized CAT was shown in Fig. 5(a). The immobilized CAT and the free CAT displayed similar pH profiles. Both free and immobilized CAT showed maximum activity at pH 7.0. The immobilized CAT displayed higher relative activity than its free counterpart under identical pH conditions. The optimum reaction temperature of CAT at pH 7.0 shifted from 30 °C to 40 °C after immobilization (Fig. 5(b)). The immobilized CAT exhibited enhanced resistance to denaturation under higher temperatures. This enhanced stability might be attributed to the following reason: the immobilized enzyme was in a more favorable microenvironment than free enzyme [7,45,51]. 3.5. Thermostability Fig. 2. The EDX analysis of NHS activated TiO2–COOH (a) and TiO2–CAT (b).
reaction rate (Vmax) were calculated from Lineweaver–Burk plots as shown in Fig. 4. The values of Km and Vmax were listed in Table 1. The value of Km was the same order of magnitude as those reported by other investigators [45–47]. The Michaelis constant (Km) commonly increased upon immobilization, and sometimes this increase was remarkable. Boon L. Tee et al. used EDC and sulfo-NHS for covalent binding of thermostable α-amylase to
The results of thermostability tests of free and immobilized CAT incubated at various temperatures and pH 7.0 were shown in Fig. 6(a). The highest activity for immobilized CAT was achieved at 30 °C, completely consistent with that of free CAT. With the increase of incubation temperature, the activity of free CAT decreased more sharply than that of immobilized CAT. Approximately 40% of the activity for free CAT was retained after being incubated at 50 °C for 2 h whereas the immobilized CAT retained about 60% of its activity. The free CAT lost activity entirely after incubation at 75 °C for 2 h. In contrast, the immobilized CAT retained about 6% of its activity
Fig. 3. Schematic representation of the immobilization approach.
H. Wu et al. / Materials Science and Engineering C 33 (2013) 1438–1445
0.5
1/V (mM/min)-1
(a)
0.6
0.4
100
Y = 0.07427 + 1.91213 * X 2 R =0.99488 Km =25.7mM
Relative activity (%)
(a)
1443
0.3 0.2
80 60 40 20
immobilized CAT free CAT
0.1 0 0.0 0.00
0.05
0.10
1/[S]
0.6 0.5
4
5
6
7
8
9
pH value
(b) 100
Y = 0.07486 + 2.04995 * X 2 R =0.99343 Km =27.4mM
0.4 0.3 0.2
80 60 40 immobilized CAT free CAT
20
0.1 0.0 0.00
3
0.25
0.8 0.7
1/V (mM/min)-1
0.20
(mM)-1
Relative activity (%)
(b)
0.15
0.05
0.10
0.15
0.20
0.25
0.30
20
Fig. 4. Typical Lineweaver–Burk plots for free CAT (a) and immobilized CAT (b) (25 °C, pH 7.0).
after the same treatment. Generally, increased temperature leads to an increase in the thermal motion of the CAT molecules, resulting in a disruption of the covalent bonds in enzyme structure and thus further leading to enzyme denaturation [52]. For immobilized CAT, the formation of covalent bonds between the CAT and the carrier would reduce conformational flexibility and thermal vibrations, thus preventing protein unfolding and denaturation [10]. The enhanced thermostability of the immobilized CAT might be caused by the stabilization via the multipoint covalent attachment of the enzyme to the support [1]. The values of thermal denaturation constants (kd) and half-lives (t1/2) for CAT were listed in Table 2. As the incubation temperature increased, the thermal denaturation constants (kd) values of free and immobilized CAT both increased. On the contrary, the half-life (t1/2) values decreased. This result indicated that, for both free and immobilized CAT, the higher incubation temperature the quicker denaturation rate became. The kd values for immobilized CAT were lower than that for free CAT, while the t1/2 values were higher than that for free CAT at the same incubation temperature. Therefore, the immobilized CAT was more stable than free CAT when incubated in the same temperature.
Table 1 Kinetic parameters for free and immobilized CAT (25 °C, pH 7.0). CAT
Km (mM)
Vmax (mM/min)
Free Immobilized
25.7 ± 0.28 27.4 ± 0.22
13.46 ± 0.78 13.36 ± 0.62
30
40
50
60
70
Temperature (oC)
1/[S] (mM)-1
Fig. 5. Effect of (a) pH value (25 °C) and (b) temperature (pH 7.0) on the activity of free and immobilized CAT.
The ΔH° values for both free and immobilized CAT were listed in Table 3. Both the ΔH° values for free and immobilized CAT decreased with the increased incubation temperature, indicating that less energy was required to denature CAT at higher incubation temperature. Therefore, both free and immobilized CAT were easy to denature at high incubation temperature. However, at the same temperature, the ΔH° values for immobilized CAT were higher than that for free CAT. This result indicated that the immobilized CAT required more energy to denature than free CAT. In other words, the immobilized CAT exhibited more activity than free CAT at the same incubation temperature. 3.6. Recycling stability The recycling stability was very important for immobilized enzymes in view of their potential industrial applications. The recycling stability of the immobilized CAT was investigated by conducting successive batches of H2O2 decomposition (Fig. 6(b)). The immobilized CAT retained 90% and 76% of its initial activity after 10 and 16 batches, respectively. This high recycling stability was most probably due to the strong and stable covalent bonds between the enzyme and the support, preventing enzyme leakage during reaction and separation processes. The result was pretty promising in the potential industrial applications. 3.7. Storage stability Free and immobilized CAT were stored in phosphate buffer (50 mM, pH 7.0) at 4 °C, and the activity measurements were carried
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(a) Relative activity (%)
100
Table 3 The change in enthalpy (ΔH°) for the thermal denaturation of free and immobilized CAT.
immobilized CAT free CAT
Temperature (°C)
80
40 50 60 70 75
60 40
20
30
40 50 60 Temperature (oC)
70
80
(b) Relative activity (%)
100 80
Immobilized CAT
81.796 81.713 81.630 81.547 81.505
82.596 82.513 82.430 82.347 82.305
out for a period of 60 days (Fig. 6(c)). After 60 days of storage, the free CAT lost almost all its activity while the immobilized CAT still remained as high as 35% of its initial activity. It was worth mentioning that the enhanced storage stability was much more pronounced after 20 days of storage. The immobilized CAT remained about 52% of its original activity, and the free CAT only remained 20% of its original activity. Covalent immobilization definitely helped the enzyme to maintain a stable position in comparison to its free counterpart. 4. Conclusions
60 40 20 0 0
2
4
6
8
10
12
14
16
Recycling number
(c) 100
Relative activity (%)
Free CAT
20 0
immobilized enzyme free enzyme
80
A novel immobilization approach combining the metal chelation and covalent binding was presented. Titania submicrospheres were functionalized with carboxyl acid groups by facile chelation before enzyme immobilization. CAT was then immobilized via EDC/NHS coupling reaction. Only a slight change in the kinetic parameters was found after immobilization. The immobilized CAT retained 65% of its free form enzyme activity and 90% of initial activity after 10 successive reaction cycles, showing potential applications in catalysis and biosensors. In addition, besides the carboxyl groups, titania can also be functionalized with other various groups, such as amine groups, amino groups and hydroxyl groups, by the well-defined chelation chemistry. Therefore, the current approach can be extended to the immobilization of the many kinds of enzymes, such as lipase and yeast alcohol dehydrogenase (YADH), for various biomaterial-related applications. Acknowledgment
60
The authors thank the financial support from the National Science Foundation of China (21076145), the Natural Science Foundation of Tianjin (09JCYBJC06700), the Program for New Century Excellent Talents in University (NCET-10-0623), the National Basic Research Program of China (2009CB724705), National Science Fund for Distinguished Young Scholars (21125627), the Program of Introducing Talents of Discipline to Universities (B06006), and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.
40 20 0 -20
0
10
20
30
40
50
60 References
Storage time (day) Fig. 6. Thermal stability of free and immobilized CAT incubated at different temperatures and pH 7.0 for 2 h (a), recycling stability of immobilized CAT at pH 7.0, 25 °C (b) and storage stability of free and immobilized CAT in phosphate buffer (50 mM, pH 7.0) at 4 °C (c).
Table 2 Thermal denaturation kinetic parameters of free and immobilized CAT. Temperature (°C)
40 50 60 70 75
ΔH° (kJ mol−1)
kd (h−1)
t1/2 (h)
Free CAT
Immobilized CAT
Free CAT
Immobilized CAT
0.1215 0.4732 1.3173 1.9798 –
0.0489 0.2569 0.8141 1.1317 1.4225
5.70 1.46 0.53 0.35 –
14.16 2.70 0.85 0.61 0.49
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Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres Hong Wu, Yanpeng Liang, Jiafu Shi, Xiaoli Wang, Dong Yang, Zhongyi Jiang ⁎ Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
a r t i c l e
i n f o
Article history: Received 7 March 2012 Received in revised form 6 November 2012 Accepted 13 December 2012 Available online 22 December 2012 Keywords: Titania Covalent binding Immobilized enzyme Biocatalysis Stability Kinetic parameters
a b s t r a c t In this study, a novel approach combing the chelation and covalent binding was explored for facile and efficient enzyme immobilization. The unique capability of titania to chelate with catecholic derivatives at ambient conditions was utilized for titania surface functionalization. The functionalized titania was then used for enzyme immobilization. Titania submicrospheres (500–600 nm) were synthesized by a modified sol–gel method and functionalized with carboxylic acid groups through a facile chelation method by using 3-(3,4-dihydroxyphenyl) propionic acid as the chelating agent. Then, catalase (CAT) was covalently immobilized on these functionalized titania submicrospheres through 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. The immobilized CAT retained 65% of its free form activity with a loading capacity of 100–150 mg/g titania. The pH stability, thermostability, recycling stability and storage stability of the immobilized CAT were evaluated. A remarkable enhancement in enzyme stability was achieved. The immobilized CAT retained 90% and 76% of its initial activity after 10 and 16 successive cycles of decomposition of hydrogen peroxide, respectively. Both the Km and the Vmax values of the immobilized CAT (27.4 mM, 13.36 mM/min) were close to those of the free CAT (25.7 mM, 13.46 mM/min). © 2012 Elsevier B.V. All rights reserved.
1. Introduction Enzymes are versatile biocatalysts that exhibit a number of unique advantages over conventional chemical catalysts, such as high activity, selectivity and specificity [1]. Till now, enzymes have been found in numerous applications including polymer synthesis [2], biomaterials [3], bioconversion [4], bioseparation [5], biosensors [6], etc. Development of immobilized enzymes has attracted considerable attention for many years to facilitate the reaction process and reduce the operation cost. High catalytic activity and loading capacity, easy separation and reusability are the essential requirements for immobilized enzymes [7]. Currently, adsorption, covalent binding, entrapment, encapsulation and cross-linking, or their combinations, are still the prevalent methods utilized for enzyme immobilization [8]. Compared with other immobilization methods, the covalent binding which involves the formation of covalent bonds between the reactive groups on the support and the functional groups on the enzymes offers the strongest linkage between the enzyme and the support [9]. Immobilized enzyme leaching out of reaction media and the consequent contamination to the product are thus minimized [10]. In addition, the recycling stability of immobilized enzyme is significantly enhanced. For example, catalase was covalently immobilized on nanofibers and retained 70% of its initial activity after 10 batch cycles ⁎ Corresponding author. Tel./fax: +86 22 23500086. E-mail address: [email protected] (Z. Jiang). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.12.048
[11]. The diffusion limitation will be reduced or eliminated when enzyme molecules are covalently immobilized on the carrier's outer surface. However, the control for covalent immobilization process is usually caustic in the past: it should meet the temperature and pH requirements of the reaction, and should be mild enough to avoid enzyme denaturation as well. Recently, there are a multitude of soft methods for covalent immobilization including the widely used EDC/NHS chemistry method [12]. Inorganic carriers usually possess high mechanical properties, thermal stability and resistance against microbial attack and organic solvents. Many kinds of inorganic solids have been used for the covalent immobilization of enzymes such as Au, SiO2, Fe3O4 and TiO2 [13–16]. Most of these inorganic carriers couldn't react with enzymes directly without surface functionalization. Therefore, surface functionalization of the carrier is necessary prior to covalent attachment of enzymes. Lü et al. synthesized epoxy group functionalized cubic mesoporous silica for covalent attachment of Penicillin G acylase. The initial specific activity of such immobilized enzyme was higher than that of enzyme immobilized by physical adsorption. The immobilized enzyme retained 72% of its initial activity after 10 batches [14]. Liu X. synthesized the magnetic particles functionalized by expoxy group for lipase immobilization. The loading capacity was 68.3 mg lipase/g support, and the activity retention of lipase was 60.4% [15]. Immobilization of catalase on modified nanofiber membranes led to an enhanced thermostability [17]. α-Amylase was covalently immobilized onto acid chloride group functionalized glass beads. The loading capacity was found to be
H. Wu et al. / Materials Science and Engineering C 33 (2013) 1438–1445
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was vigorously stirred at room temperature for 30 min to allow a complete chemisorption of catecholic salt onto the TiO2 particle surface. The low pH condition prevented the catechol end groups of 3-(3,4dihydroxyphenyl) propionic acid from oxidation. The modified TiO2 submicrospheres were collected and rinsed thoroughly with deionized water. The as-synthesized carboxylic-functionalized titania (TiO2– COOH) submicrospheres were dried at 80 °C for 24 h.
25.2 mg/g glass support. The free enzyme lost all its activity within 15 days. Immobilized enzyme lost only 20% of activity in 25 days [18]. Although the above covalent immobilization commonly led to enhanced stability, the functionalization process prior to immobilization was tedious and time-consuming, usually involving multiple steps including particle surface activation with organosilane, chemical reactions, polymerization and grafting reactions. Moreover, high temperature, addition of organic solvents and nitrogen protection were often required during the functionalization process. It has been found that catechol and catecholic derivatives could chelate with transition metal oxide such as titania and iron oxide to form a stable salt chelate complex at room temperature rapidly [19–23]. The titania support surface could be easily modified under mild conditions at an ambient temperature through the simple chelation process. In addition, TiO2-based materials had excellent pH and corrosion resistance, superior mechanical strength, outstanding antimicrobial performance, and in particular good biocompatibility [24]. Herein, a novel and facile approach was presented to synthesize carboxyl functionalized titania submicrospheres for enzyme immobilization. Titania submicrospheres were synthesized by a modified sol–gel method and then functionalized with carboxylic acid groups via the chelation approach using 3-(3,4-dihydroxyphenyl) propionic acid as carboxylation reagent. Catalase (CAT), as a model enzyme, was covalently anchored to the functionalized titania submicrospheres via EDC/NHS coupling reaction. The immobilized catalase was used to decompose hydrogen peroxide to water and oxygen. The activity and stability of the immobilized enzyme were investigated and compared with those of free enzyme counterpart.
Covalent immobilization of CAT onto the TiO2–COOH submicrospheres was performed via EDC/NHS coupling reaction [26]. The immobilization procedure consisted of two steps: EDC and NHS activation followed by coupling reaction of enzyme. First, 50.0 mg of TiO2–COOH submicrospheres were dispersed in 10.0 ml of MES buffer solution (50 mM, pH 6.5) under ultrasonic treatment for 15 min. Then, NHS (1.5 mmol) and EDC (0.3 mmol) were added to the above suspension. The mixture was vigorously stirred at room temperature for 1 h. The NHS-activated TiO2–COOH submicrospheres were collected by centrifugation and washed with MES buffer solution (50 mM, pH 6.5) thoroughly to remove unreacted NHS and EDC. Second, the NHS-activated TiO2–COOH submicrospheres were dispersed in 10.0 ml of the CAT solution (5 mg/ml) prepared with MES buffer solution (50 mM, pH 6.5). The enzyme coupling reaction was allowed to proceed for 4 h at room temperature under magnetic stirring. The catalase-titania conjugates (TiO2–CAT) were separated by centrifugation and washed thoroughly with deionized water. The immobilized enzyme was lyophilized and stored at −20 °C.
2. Materials and methods
2.4. Enzyme loading capacity and activity assay
2.1. Materials
The amount of CAT immobilized was determined by measuring the initial and final concentrations of CAT within the enzyme and washing solutions using Coomassie Brilliant Blue reagent, following the Bradford's method [27]. The loading capacity was determined according to Eq. (1):
Catalase (hydrogen peroxide oxidoreductase; 2.85×104 units/mg protein; EC.1.11.1.6) from bovine liver, 2-(N-morpholino) ethanesulfonic acid sodium salt (MES) and Tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma. 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Medpep Co., Ltd. Acetone, ethylene glycol, tetrabutyl titanate (TBT, >98%), hydrogen peroxide (H2O2, 30%), acetic acid, sodium acetate, sodium dihydrogen phosphate dehydrate (NaH2PO4·2H2O) and sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O) were purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). 3-(3,4-Dihydroxyphenyl) propionic acid (98%) was purchased from Alfa Aesar. 2.2. Synthesis of the carboxyl acid functionalized titania submicrospheres Titania (TiO2) submicrospheres were synthesized by a modified sol–gel method as described in the literature [25]. 0.02 mol tetrabutoxytitanium (TBT, 6.8 ml) was added to 100 ml ethylene glycol in a round flask purged with nitrogen gas. The solution was magnetically stirred rapidly for 20 h at 30 °C. The resultant transparent solution was poured into acetone solution containing ~ 0.3 wt.% of water under vigorous stirring for 30 min. After aging for about 1 h, the white precipitate was collected by centrifugation at 3058 g for 5 min, washed with distilled water and ethanol alternatively to remove the residue ethylene glycol. The obtained TiO2 submicrospheres with a size of 500–600 nm were dried at 80 °C for 24 h. 3-(3,4-Dihydroxyphenyl) propionic acid was used as the carboxylation reagent for the surface modification of the TiO2 submicrospheres. A facile chemical adsorption procedure was conducted [19–23]. The TiO2 submicrospheres were suspended in an aqueous HCl solution (pH 2.0) under ultrasonic treatment for 0.5 h. Then, the TiO2 submicrospheres were filtered and added into an excessive 3-(3,4-dihydroxyphenyl) propionic acid aqueous solution (4 mg/ml, pH 2.0). The suspension
2.3. Immobilization of enzyme on functionalized titania submicrospheres
M
mg enzyme ðm−C 1 V 1 Þ ¼ g support W
ð1Þ
where M represented the loading capacity; m was the amount of CAT introduced into the immobilization medium; C1 and V1 were the enzyme concentration and volume of the washing solution, respectively; W was the weight of the TiO2–COOH submicrospheres. The enzyme concentration (C1) was determined via UV–vis standard curve (correlation coefficient R2 = 0.998). The activity of free and immobilized CAT was determined by measuring the decrease in the absorbance of hydrogen peroxide at 240 nm in 5 min due to the decomposition of H2O2 [28].
2 H2 O2
Catalase
→ 2 H2 O þ O2 :
ð2Þ
The immobilized CAT (1 mg) (or free enzyme (0.1 ml, 1 mg/ml)) was added into the H2O2 solution (17.64 mM) prepared in 50 ml phosphate buffer solution (50 mM, pH 7) under agitation. The decrease in absorbance at 240 nm was recorded after 5 min. One unit will decompose 1.0 μmol of H2O2 per min at pH 7.0 at 25 °C, measured by the decrease rate of A240 [29]. The relative activity of immobilized enzyme was defined as the ratio of its activity to the free form activity under the equal amount of enzyme (Eq. (3)).
relative activityð% Þ ¼
immobilized enzyme activity 100: f ree enzyme activity
ð3Þ
1440
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The activation energy (Ed) for CAT thermal denaturation was determined by applying the Arrhenius equation Eq. (9):
2.5. Kinetic parameters (Km and Vmax) Kinetic parameters, Km and Vmax, for the free and immobilized enzymes were determined using the Michaelis–Menten equation, given by Eq. (4) 1 K 1 1 ¼ m þ V V max ½S V max
ð4Þ
where V was the initial reactive rate, [S] was the initial substrate concentration, Vmax was the maximum reaction rate attained at infinite initial substrate concentration and Km was the Michaelis–Menten constant. To determine the Km and Vmax, the activity assay was applied for different hydrogen peroxide concentrations from 5 mM to 35 mM. The amount of free CAT added to the reaction system was 0.1 mg, and correspondingly, 1 mg solid with ~ 0.1 mg immobilized CAT was used to keep the amounts of enzyme identically. Enzyme activity was determined at pH 7.0 at 25 °C. Kinetic parameters for both the free and the immobilized CAT were calculated accordingly. 2.6. Effect of pH and temperature on enzyme activity The effects of pH and temperature on the activity for free and immobilized CAT were studied by changing the reaction pH value from 3.0 to 9.0 (sodium acetate buffer pH 3.0–5.0; MES buffer pH 6.0; phosphate buffer pH 7.0–8.0; Tris–HCl buffer pH 9.0) at room temperature (25 °C) and the temperature from 20 °C to 70 °C at pH 7.0. Optimum reaction conditions (pH and temperature) were determined accordingly. The activity found at each specific pH (or temperature) was compared with the activity at optimum pH (or temperature) in terms of relative activity (Eq. (5)) [30]. relatively activityð% Þ enzyme activity at the specific pHðor temperatureÞ ¼ 100: ð5Þ enzyme activity at the optimum pHðor temperatureÞ
2.7. Thermostability The thermostability of free and immobilized CAT was examined by measuring the residual activity of enzyme incubated at different temperatures (20 °C to 75 °C) in phosphate buffer solution (50 mM, pH 7.0) for 2 h. Activity of enzymes was measured at 25 °C and pH 7.0. The relative activity of enzyme was defined as the ratio of its activity after incubation to the activity under the optimal condition (Eq. (6)) [4]. relatively activityð% Þ ¼
enzyme activity af ter incubation enzyme activity at the optimum temperature 100: ð6Þ
The thermal denaturation kinetic of CAT was described as first order [31,32]. The thermal denaturation constants (kd) were calculated from the first order exponential approach [32] (Eq. (7)): Acat =Acat;0 ¼ expð−kd t Þ
ð7Þ
where Acat,0 was the CAT activity before incubation, Acat was the CAT activity after incubation at a certain temperature for a certain time and t was the incubation time. The half-life (t1/2) value for CAT thermal denaturation was determined from Eq. (8): t 1=2 ¼ ln2=kd :
ð8Þ
lnkd ¼ −Ed =RT þ lnC:
ð9Þ
The Ed was calculated by the plot of log denaturation rate constants (ln kd) versus reciprocal of the absolute temperature (T) from Eq. (10): Slope ¼ −Ed =R:
ð10Þ
The change in enthalpy (ΔH° kJ mol −1) for CAT thermal denaturation was determined using the Eq. (11): 0
ΔH ¼ Ed −RT
ð11Þ
where R was the gas constant (8.3145 J mol −1 K −1) and T was the corresponding absolute temperature [33]. 2.8. Recycling stability The immobilized CAT (1 mg) was removed after each reaction batch by centrifugation, and washed with phosphate buffer solution (50 mM, pH 7.0) to remove any residual substrate and then added to the next reaction cycle. The recycling stability of immobilized CAT was explored by measuring the enzyme activity in each successive reaction cycle and expressed by recycling efficiency (Eq. (12)). th
recycling efficiencyð%Þ ¼
enzyme activity in the n cycle enzyme activity in the 1st cycle 100:
ð12Þ
2.9. Storage stability Free CAT and immobilized CAT were stored in phosphate buffer solution (50 mM, pH 7.0) at 4 °C for a certain period of time. The storage stability was compared in terms of storage efficiency defined as the ratio of the activity of free or immobilized enzyme after storage to their initial activity (Eq. (13)). storage efficiencyð%Þ ¼
enzyme activity after storage 100 initial enzyme activity
ð13Þ
3. Results and discussion 3.1. Characterization of the support and immobilized enzyme Surface modification of Ti and TiO2 via chelation of catechol had been well-documented in the previous literatures [19,21–23,34,35]. Herein, the titania particles prepared through the sol–gel method were functionalized with caboxyl acid groups by using 3-(3,4dihydroxyphenyl) propionic acid as the chelating agent. The morphology of the pristine and functionalized titania submicrospheres was observed by transmission electron microscope (TEM) (Fig. 1(a–c)). The average particle sizes of the TiO2 submicrospheres, TiO2–COOH submicrospheres and those with immobilized enzyme were found to be in the range of 500–600 nm. There was no obvious change in size and morphology after covalent immobilization of CAT. Thermo gravimetric analysis (TGA) was performed to determine the modification percentage, that is, the 3-(3,4-dihydroxyphenyl) propionic acid content on the titania. The weight loss in the temperature range of 100–800 °C attributed to the decomposition of organic modifier was found to be about 10 wt.%, and then the modification percentage could be calculated to be about 0.55 mmol (\COOH)/g TiO2–COOH particles.
H. Wu et al. / Materials Science and Engineering C 33 (2013) 1438–1445
(a)
(b)
(c)
(d)
1441
2935 2868 1080
Intensity
TiO2 1474 TiO2 -COOH
1438
1540
1650 TiO2 -CAT
3000
2500
2000
1500
1000
Wave number / cm-1
Fig. 1. TEM photographs of TiO2 (a), TiO2–COOH (b) and TiO2–CAT (c); FITR spectra of TiO2, TiO2–COOH and TiO2–CAT (d).
The chemical structure of TiO2, TiO2–COOH and TiO2–CAT was determined by Fourier transform infrared spectra (FTIR). As shown in Fig. 1(d), the absorption bands at 1086, 2868 and 2935 cm −1 for TiO2 were assigned to the C\C\O stretching vibration, symmetric and asymmetric \CH2\ stretching vibrations of ethylene glycol, respectively. The characteristic peaks of ethylene glycol disappeared after carboxyl functionalization [36]. The new bands located at 1474 and 1438 cm −1 were assigned to the C_C benzene ring characteristic vibrations [37]. For the TiO2–CAT, the main characteristic peak of amide I band at 1650 cm −1 was caused by C_O stretching vibrations of peptide linkages, whereas, the amide II band at 1540 cm −1 was attributed to the combination of N–H bending and C–N stretching [38–40]. The appearance of the above bands indicated the successful immobilization of CAT on the TiO2–COOH submicrospheres. EDX mapping was conducted with NHS activated TiO2–COOH and TiO2–CAT and the results were shown in Fig. 2. The nitrogen element in the NHS activated TiO2–COOH and TiO2–COOH was originated from the NHS and CAT molecules, respectively. The N/Ti atomic ratio of the CAT (0.1077) was higher than that of the NHS activated TiO2–COOH (0.0663), indicating that the CAT was indeed attached onto the supports. 3.2. Immobilization procedure, loading efficiency and activity CAT was covalently immobilized on the TiO2–COOH submicrospheres via EDC/NHS coupling reaction. The immobilization process was illustrated in Fig. 3. First, 3-(3,4-dihydroxyphenyl) propionic acid with two phenolic hydroxyl groups and a carboxyl group was used as both the chelating and carboxylation reagent. A chelation occurred between the titania and the phenolic hydroxyl groups at room temperature in a few minutes, thus rendering the titania surface with carboxyl groups. Second,
the TiO2–COOH submicrospheres were activated with EDC and NHS to yield an ester intermediate. Finally, the CAT molecules were immobilized on the activated titania surface via covalent bonds. EDC was a zero-length cross-linker widely used in protein conjugations. The coupling reaction process of CAT on the TiO2–COOH submicrospheres could be achieved by a two-step procedure [41]. EDC could react with the carboxyl group on the surface of the TiO2–COOH submicrospheres, forming an O-acylisourea intermediate which was very unstable and susceptible to hydrolysis. The O-acylisourea intermediate could react with an amine group to produce a stable amide bond [42]. But the instability of the O-acylisourea intermediate resulted in a low coupling efficiency. The addition of NHS could stabilize the O-acylisourea intermediate by converting it to an amine-reactive NHS ester (succinimidyl ester). The coupling efficiency could thus be increased by 10–20 folds [43,44]. A control experiment was conducted without adding EDC/NHS during enzyme immobilization. The support exhibited no catalytic activity through the enzyme assays, confirming that the carboxylic acid groups on the TiO2–COOH could not react with CAT at room temperature. In summary, the TiO2–COOH submicrospheres were first reacted with EDC and NHS to yield an ester intermediate. After the excess NHS and EDC were washed with deionized water, the activated supports were then reacted with CAT. The activity of immobilized CAT was 1.85 × 10 4 units/mg protein. It was found that the immobilized CAT retained 65% of its original activity. The loading capacity determined by Bradford's method was 100–150 mg/g titania submicrospheres. 3.3. Kinetic parameters for free and immobilized enzyme Kinetic parameters were measured for both free and immobilized CAT at 25 °C, pH 7.0. The Michaelis constant (Km) and the maximum
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calcium alginate and found that the Km was 2.5 times greater than that of free enzyme [48]. The Km value of the bovine liver CAT increased by 3 times after covalent immobilization on Eupergit C [49]. In this study, the Km and Vmax values for free and immobilized CAT were 25.7 mM and 13.46 mM/min, and 27.4 mM and 13.36 mM/min, respectively. The Km value reflected the affinity of the enzyme toward substrate. The lower value of Km indicated the higher affinity between enzyme and substrate [50]. The comparable Km values for free and covalent immobilized CAT (25.7 mM vs. 27.4 mM) indicated almost the identical affinity towards the hydrogen peroxide substrate molecules. The Vmax value reflected the intrinsic characteristic of the immobilized enzyme which could be affected by diffusion constrains [29,49]. Additional diffusional resistance often occurred in the entrapment/encapsulation method. In contrast, since covalently immobilized enzyme molecules were located on the outer surface of the solid supports, the inner diffusion limitation would be much reduced or even eliminated. It can be derived that the three-dimensional conformation and active sites of the enzyme were well-preserved in the present study. 3.4. Effect of pH and temperature on enzyme activity The effect of pH on activity was studied at different pH values (3.0 to 9.0) at room temperature (25 °C). The relative activity of free and immobilized CAT was shown in Fig. 5(a). The immobilized CAT and the free CAT displayed similar pH profiles. Both free and immobilized CAT showed maximum activity at pH 7.0. The immobilized CAT displayed higher relative activity than its free counterpart under identical pH conditions. The optimum reaction temperature of CAT at pH 7.0 shifted from 30 °C to 40 °C after immobilization (Fig. 5(b)). The immobilized CAT exhibited enhanced resistance to denaturation under higher temperatures. This enhanced stability might be attributed to the following reason: the immobilized enzyme was in a more favorable microenvironment than free enzyme [7,45,51]. 3.5. Thermostability Fig. 2. The EDX analysis of NHS activated TiO2–COOH (a) and TiO2–CAT (b).
reaction rate (Vmax) were calculated from Lineweaver–Burk plots as shown in Fig. 4. The values of Km and Vmax were listed in Table 1. The value of Km was the same order of magnitude as those reported by other investigators [45–47]. The Michaelis constant (Km) commonly increased upon immobilization, and sometimes this increase was remarkable. Boon L. Tee et al. used EDC and sulfo-NHS for covalent binding of thermostable α-amylase to
The results of thermostability tests of free and immobilized CAT incubated at various temperatures and pH 7.0 were shown in Fig. 6(a). The highest activity for immobilized CAT was achieved at 30 °C, completely consistent with that of free CAT. With the increase of incubation temperature, the activity of free CAT decreased more sharply than that of immobilized CAT. Approximately 40% of the activity for free CAT was retained after being incubated at 50 °C for 2 h whereas the immobilized CAT retained about 60% of its activity. The free CAT lost activity entirely after incubation at 75 °C for 2 h. In contrast, the immobilized CAT retained about 6% of its activity
Fig. 3. Schematic representation of the immobilization approach.
H. Wu et al. / Materials Science and Engineering C 33 (2013) 1438–1445
0.5
1/V (mM/min)-1
(a)
0.6
0.4
100
Y = 0.07427 + 1.91213 * X 2 R =0.99488 Km =25.7mM
Relative activity (%)
(a)
1443
0.3 0.2
80 60 40 20
immobilized CAT free CAT
0.1 0 0.0 0.00
0.05
0.10
1/[S]
0.6 0.5
4
5
6
7
8
9
pH value
(b) 100
Y = 0.07486 + 2.04995 * X 2 R =0.99343 Km =27.4mM
0.4 0.3 0.2
80 60 40 immobilized CAT free CAT
20
0.1 0.0 0.00
3
0.25
0.8 0.7
1/V (mM/min)-1
0.20
(mM)-1
Relative activity (%)
(b)
0.15
0.05
0.10
0.15
0.20
0.25
0.30
20
Fig. 4. Typical Lineweaver–Burk plots for free CAT (a) and immobilized CAT (b) (25 °C, pH 7.0).
after the same treatment. Generally, increased temperature leads to an increase in the thermal motion of the CAT molecules, resulting in a disruption of the covalent bonds in enzyme structure and thus further leading to enzyme denaturation [52]. For immobilized CAT, the formation of covalent bonds between the CAT and the carrier would reduce conformational flexibility and thermal vibrations, thus preventing protein unfolding and denaturation [10]. The enhanced thermostability of the immobilized CAT might be caused by the stabilization via the multipoint covalent attachment of the enzyme to the support [1]. The values of thermal denaturation constants (kd) and half-lives (t1/2) for CAT were listed in Table 2. As the incubation temperature increased, the thermal denaturation constants (kd) values of free and immobilized CAT both increased. On the contrary, the half-life (t1/2) values decreased. This result indicated that, for both free and immobilized CAT, the higher incubation temperature the quicker denaturation rate became. The kd values for immobilized CAT were lower than that for free CAT, while the t1/2 values were higher than that for free CAT at the same incubation temperature. Therefore, the immobilized CAT was more stable than free CAT when incubated in the same temperature.
Table 1 Kinetic parameters for free and immobilized CAT (25 °C, pH 7.0). CAT
Km (mM)
Vmax (mM/min)
Free Immobilized
25.7 ± 0.28 27.4 ± 0.22
13.46 ± 0.78 13.36 ± 0.62
30
40
50
60
70
Temperature (oC)
1/[S] (mM)-1
Fig. 5. Effect of (a) pH value (25 °C) and (b) temperature (pH 7.0) on the activity of free and immobilized CAT.
The ΔH° values for both free and immobilized CAT were listed in Table 3. Both the ΔH° values for free and immobilized CAT decreased with the increased incubation temperature, indicating that less energy was required to denature CAT at higher incubation temperature. Therefore, both free and immobilized CAT were easy to denature at high incubation temperature. However, at the same temperature, the ΔH° values for immobilized CAT were higher than that for free CAT. This result indicated that the immobilized CAT required more energy to denature than free CAT. In other words, the immobilized CAT exhibited more activity than free CAT at the same incubation temperature. 3.6. Recycling stability The recycling stability was very important for immobilized enzymes in view of their potential industrial applications. The recycling stability of the immobilized CAT was investigated by conducting successive batches of H2O2 decomposition (Fig. 6(b)). The immobilized CAT retained 90% and 76% of its initial activity after 10 and 16 batches, respectively. This high recycling stability was most probably due to the strong and stable covalent bonds between the enzyme and the support, preventing enzyme leakage during reaction and separation processes. The result was pretty promising in the potential industrial applications. 3.7. Storage stability Free and immobilized CAT were stored in phosphate buffer (50 mM, pH 7.0) at 4 °C, and the activity measurements were carried
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H. Wu et al. / Materials Science and Engineering C 33 (2013) 1438–1445
(a) Relative activity (%)
100
Table 3 The change in enthalpy (ΔH°) for the thermal denaturation of free and immobilized CAT.
immobilized CAT free CAT
Temperature (°C)
80
40 50 60 70 75
60 40
20
30
40 50 60 Temperature (oC)
70
80
(b) Relative activity (%)
100 80
Immobilized CAT
81.796 81.713 81.630 81.547 81.505
82.596 82.513 82.430 82.347 82.305
out for a period of 60 days (Fig. 6(c)). After 60 days of storage, the free CAT lost almost all its activity while the immobilized CAT still remained as high as 35% of its initial activity. It was worth mentioning that the enhanced storage stability was much more pronounced after 20 days of storage. The immobilized CAT remained about 52% of its original activity, and the free CAT only remained 20% of its original activity. Covalent immobilization definitely helped the enzyme to maintain a stable position in comparison to its free counterpart. 4. Conclusions
60 40 20 0 0
2
4
6
8
10
12
14
16
Recycling number
(c) 100
Relative activity (%)
Free CAT
20 0
immobilized enzyme free enzyme
80
A novel immobilization approach combining the metal chelation and covalent binding was presented. Titania submicrospheres were functionalized with carboxyl acid groups by facile chelation before enzyme immobilization. CAT was then immobilized via EDC/NHS coupling reaction. Only a slight change in the kinetic parameters was found after immobilization. The immobilized CAT retained 65% of its free form enzyme activity and 90% of initial activity after 10 successive reaction cycles, showing potential applications in catalysis and biosensors. In addition, besides the carboxyl groups, titania can also be functionalized with other various groups, such as amine groups, amino groups and hydroxyl groups, by the well-defined chelation chemistry. Therefore, the current approach can be extended to the immobilization of the many kinds of enzymes, such as lipase and yeast alcohol dehydrogenase (YADH), for various biomaterial-related applications. Acknowledgment
60
The authors thank the financial support from the National Science Foundation of China (21076145), the Natural Science Foundation of Tianjin (09JCYBJC06700), the Program for New Century Excellent Talents in University (NCET-10-0623), the National Basic Research Program of China (2009CB724705), National Science Fund for Distinguished Young Scholars (21125627), the Program of Introducing Talents of Discipline to Universities (B06006), and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.
40 20 0 -20
0
10
20
30
40
50
60 References
Storage time (day) Fig. 6. Thermal stability of free and immobilized CAT incubated at different temperatures and pH 7.0 for 2 h (a), recycling stability of immobilized CAT at pH 7.0, 25 °C (b) and storage stability of free and immobilized CAT in phosphate buffer (50 mM, pH 7.0) at 4 °C (c).
Table 2 Thermal denaturation kinetic parameters of free and immobilized CAT. Temperature (°C)
40 50 60 70 75
ΔH° (kJ mol−1)
kd (h−1)
t1/2 (h)
Free CAT
Immobilized CAT
Free CAT
Immobilized CAT
0.1215 0.4732 1.3173 1.9798 –
0.0489 0.2569 0.8141 1.1317 1.4225
5.70 1.46 0.53 0.35 –
14.16 2.70 0.85 0.61 0.49
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