Enzyme and Microbial Technology 55 (2014) 1–6
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Immobilization of horseradish peroxidase in phospholipid-templated titania and its applications in phenolic compounds and dye removal Yanjun Jiang, Wei Tang, Jing Gao ∗ , Liya Zhou, Ying He School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China
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Article history: Received 13 September 2013 Received in revised form 15 November 2013 Accepted 18 November 2013 Keywords: Horseradish peroxidase Phospholipid-templated titania Enzyme encapsulation Phenolic compound Dye
a b s t r a c t In this study, horseradish peroxidase (HRP) was encapsulated in phospholipid-templated titania particles through the biomimetic titanification process and used for the treatment of wastewater polluted with phenolic compounds and dye. The encapsulated HRP exhibited improved thermal stability, a wide range of pH stability and high tolerance against inactivating agents. It was observed an increase in Km value for the encapsulated HRP (8.21 mM) when compared with its free counterpart. For practical applications in the removal of phenolic compounds and dye by the encapsulated HRP, the removal efficiency for phenol, 2chlorophenol, Direct Black-38 were 92.99%, 87.97%, and 79.72%, respectively, in the first treatment cycle. Additionally, the encapsulated HRP showed better removal efficiency than free HRP and a moderately good capability of reutilization. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Enzymes are able to catalyze many chemical processes under most benign experimental conditions [1]. In this way, enzymes could be excellent catalysts for a much more sustainable chemical industry [2]. However, enzymes have some limitations for nonbiological applications [3,4]. Thus, for many industrial applications, enzymes have to be immobilized, via very simple and cost-effective protocols, in order to improve the properties of enzymes, such as activity, stability, and selectivity [5,6]. Over the last several decades, three types of methodology for enzyme immobilization have been reported in scientific literatures: via binding to or encapsulation in an inorganic or organic polymer [7,8], or by cross-linking the enzyme molecules [9]. No single method has been emerged as the standard for enzyme immobilization and ongoing efforts are striving to optimize these methods to render them adequate for specific applications [10,11]. Compared to most organic polymers, the silica matrix exhibits higher mechanical strength, enhanced thermal stability, and negligible swelling in organic solvents. Thus, silica matrix made by sol–gel process, has emerged as a promising platform for encapsulation of enzymes to be used in biocatalysis, biosensors, and
Abbreviations: HRP, horseradish peroxidase. ∗ Corresponding author. Tel.: +86 22 60204293; fax: +86 22 60204294. E-mail address: [email protected] (J. Gao). 0141-0229/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.enzmictec.2013.11.005
biomedical fields [12,13]. However, the relative harsh reaction conditions and long aging time [14] of the traditional sol–gel process may induce the deactivation of enzymes. Thus, the biomimetic silicification process, which provides a rapid, low temperature method for silica precipitation, has emerged as a versatile tool for preparing excellent supports for enzyme immobilization. Until now, a remarkable diversity of enzymes has been encapsulated in bioinspired silicas, and it is reasonable to envisage that this technological impact of bioinspired encapsulation will continue to grow [5]. Although much of the research effort on biomimetic silicification has been taken, exploration of phospholipid-templated silica matrix seems to have received less attention. This approach can overcome the disadvantages of entrapment techniques, including the leaching of adsorbed biomolecules, the chemical degradation of the anchoring bond of covalently attached enzymes, and diffusion limitations of substrates and products. This is clearly confirmed by various authors [15–18]. Furthermore, this phospholipid-templated approach can eliminate specific enzyme–silica interactions during the silica formation process, and can produce more active biocatalyst than those prepared by trapping enzymes directly in silica hydrogels [16]. In comparison with silica-based materials, titania-based materials have attracted great interest in a number of fields [19–21], owing to their unique chemical and physical properties, including stable chemical structure [22], good biocompatibility, relative high conductivity, and environmentally benign nature [23]. Additionally, titania is an amphoteric oxide, allowing it to be an anion and
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cation exchanger at acidic and alkaline pH, whereas silica can only act as a cation exchanger [14,24]. Thus, the titania material has been investigated and proved to be an ideal enzyme carrier [25–27]. In the past years, the so-called biomimetic titanification, which typically refers to the mimic of biological synthesis process to form titania, has emerged as a versatile tool for preparing titania materials [28,29]. This facile and green process for titania synthesis can be controlled at near-neutral pH, ambient temperature within a short period [23,24,27]. Many proteins and peptides, such as silicatein [30], lysozyme [31], protamine [27], and R5 peptide [32] have been successfully employed for the formation of titania through the biomimetic titanification process [33]. In contrast to these reports, we report the first examination of using dodecylamine to catalyze the hydrolysis and subsequent polycondensation of a water-stable alkoxide-like conjugate of titanium to yield titania under ambient conditions for enzyme immobilization. To eliminate the specific enzyme-titania interactions, generate 3-D open mesoporosity for facile diffusion of substrates in biocatalysis process, and then produce more active biocatalyst, phospholipid was used as template. To the best of our knowledge, this is the first report that concerned the preparation of phospholipid-templated titania particles by using biomimetic titanification and as an efficient matrix for enzyme immobilization. Phenolic compounds and direct dyes are widely distributed in the wastewater of the textile industry and they can be highly harmful toward aquatic life and humans. [34,35]. Therefore, a number of techniques including adsorption, chemical oxidation, solvent extraction and biodegradation aimed at preferential removal of the phenolic compounds [36–38] and dyes [39,40] from wastewaters have been developed. Among these treatment technologies, the use of oxido-reductive enzymes such as horseradish peroxidase (HRP) to catalyze the removal of pollutes has become increasingly important [41–44]. Thus, in the present work, HRP was encapsulated in phospholipid-templated titania particles that induced by dodecylamine through the biomimetic titanification process. The effect of pH, thermal stability, tolerance against inactivating agents, and kinetic parameters of the encapsulated HRP were investigated. The removal of phenolic compounds (phenol and 2-chlorophenol) and dye (Direct Black-38) by the free and encapsulated HRP was also investigated. The results presented here not only open a novel avenue for immobilizing enzymes but also provide methods that can be readily adapted for a range of metal oxide synthesis. Additionally, the relatively low price and easy availability ensure dodecylamine as a promising titania-precipitating agent for largescale utilization. 2. Materials and methods 2.1. Materials Horseradish peroxidase (HRP, EC. 1.11.1.7, 150 U/mg) was purchased from Source leaves Biotechnology Co. (shanghai, China). Soybean lecithin, phenol, 2chlorophenol and H2 O2 30% (w/v) were purchased from Jiang Tian Chemical Technology Co. (Tianjin, China). Titanium (IV) bis (ammonium lactato) dihydroxide (Ti-BALDH) and Direct Black-38 were purchased from Sigma Chemical Co. (St. Louis, USA). Other chemicals were of analytical grade and were used as received without further purification. 2.2. Preparation of the encapsulated HRP After optimization in the preliminary experiment, a typical experimental procedure for encapsulation of HRP in titania was described as follows: A first solution of lactose (0.05 g) in 7.2 mL phosphate buffer pH 7.0 containing HRP (4.32 mg) was prepared at 37 ◦ C. It was added slowly under vigorous stirring to a second solution, also prepared at 37 ◦ C, composed of lecithin (0.7 g) and dodecylamine (0.05 g) in 5.2 g ethanol. Then 2 mL of Ti-BALDH (0.25 mol/L, pH 7.0) solution was then added slowly to the above mixture, the biomimetic titanification process was preceded for 15 min. A gentle stirring was maintained until the titania particles were formed. To remove
lecithin, the resulting particles were washed with Triton X-100 (7.5%) and distilled water, then the encapsulated HRP was obtained. Surface morphology of the encapsulated HRP was investigated through scanning electron microscopy (JSM-6700F, JEOL, Japan). Samples were dried by rinsing with anhydrous acetone, sputter-coated with gold prior to the examination. The diameters of the titania particles were determined by using a Brookhaven Instruments BI200SM dynamic light scattering (DLS) system. The enzymatic activity of HRP was measured by Worthington method [37]. One unit of HRP activity (U) was defined as the amount of HRP required to hydrolyze 1 mol of H2 O2 in 1 min at 25 ◦ C and pH 7.0. 2.3. The properties of the encapsulated HRP The effect of pH on the activity was evaluated by incubating free and encapsulated HRP with equal activity in phosphate buffers (0.1 M) for 2 h at pH 3.0–9.0 under 25 ◦ C. Then the HRPs were taken out and the residual activities were measured. The relative activity was calculated as the ratio between the activity at each pH and the maximum activity. The study on thermal stability of free and encapsulated HRP was carried out by measuring the residual activity incubated over different times in the phosphate buffer (0.1 M, pH 7.0) at 50 ◦ C and 60 ◦ C. The tolerance capacity of free and encapsulated HRP against inactivating chemicals was tested by incubating HRP in different denaturing solutions, namely 10 M of CaCl2 , CuCl2 , BaCl2 , MnCl2 , 8 mM of H2 O2 , 25% (v/v) methanol, and 25% (v/v) acetone (0.1 M PBS, pH 7.0) at 25 ◦ C for 30 min. The residual activities were measured by Worthington method. The kinetic model used in this study was based on the Michaelis–Menten equation (1/V vs 1/[S]). The experimental initial reaction rates (Vi) were determined from the plot of the consumption of H2 O2 as a function of time. The concentration of phenol was 0.17 mM, and the concentration of H2 O2 was varied from 0.7 mM to 2.0 mM in the reaction medium.
2.4. Enzymatic removal of phenolic compounds and dye Two phenolics (phenol and 2-chlorophenol) and one dye (Direct Black-38) were employed in this research. The phenol (8 mM), 2-chlorophenol (60 mM) and Direct Black-38 (120 mg/L) degradation were studied under the same H2 O2 concentration (8 mM) with the encapsulated HRP (130 U) at 25 ◦ C. The solution was stirred to ensure full contact of the substrates and the encapsulated HRP. The residual phenolic compunds were measured on the basis of colorimetric method with potassium ferricyanide and 4-aminoantipyrine with a UV–vis spectrophotometer at 505 nm in 100 mL round bottom flask [45]. Dye decolorization was measured based on the maximum absorbance of Direct Black-38 at 550 nm [9].
3. Results and discussion 3.1. Characterization of the encapsulated HRP With the objective of synthesizing titania materials as enzyme host from Ti-BALDH, we used lecithin/dodecylamine mixedmicelle as the template. Scheme 1 shows the building process of the phospholipid-templated titania particles and HRP encapsulation. In this process, lecithin and dodecylamine were anticipated to play three roles. First, the lecithin/dodecylamine mixed-micelle that worked as template for the titania formation could generate 3-D open mesoporosity for facile diffusion of substrates in biocatalysis process. Second, dodecylamine played the role of nucleophilic catalyst to promote the condensation of titania precursor in the synthesis process, without generating elevated pH, which was indeed advantageous to avoid enzyme denaturation. Third, the lecithin could protect the enzymes from unfriendly environment. Fig. 1 shows typical SEM image of the obtained HRP-containing titania particles (encapsulated HRP). It can be seen that the samples were formed by an agglomeration of microspheres, which was similar with the silica-based materials reported by various authors [15–18]. DLS results (Fig. S1) showed that the titania particles had an average size of about 190 nm. After optimization, the immobilization yield would be 70.51% and the enzyme activity recovery was 56.31%. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.enzmictec. 2013.11.005.
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Scheme 1. Schematic representation of the building process for the phospholipid-templated titania particles and enzyme encapsulation.
Fig. 1. SEM image of encapsulated HRP.
3.2. Effect of pH on the activity of free and encapsulated HRP The activities of free and encapsulated HRP were measured at different pH values. As shown in Fig. 2, free HRP reached a maximum activity at pH 6.0, whereas the optimal pH for encapsulated HRP rose to 7.0. It may be possible that the presence of lactose led to a near acidic microenvironment of enzyme, thus the HRP encapsulated in the titania particles experienced a lower pH in comparison with free HRP in buffer medium and thus the optimum pH was shifted to a higher level [46]. Additionally, the activity of encapsulated HRP was less sensitive to pH compared to its free counterpart. 80.55% and 86.68% of relative activity can be retained for encapsulated HRP under the acidic condition (pH 3.0) and
Fig. 3. Thermal stability of the free and encapsulated HRP. Initial activity of HRP was 1.106 U.
alkaline condition (pH 9.0), respectively, while only 39.58% and 28.84% of initial activity for free HRP was left at pH 3.0 and pH 4.0, respectively. The improvement in pH tolerance of the encapsulated HRP can be explained by the stabilizing and protective effect of the titania particles and immobilization technology [14].
3.3. Thermal stability of the free and encapsulated HRP
Fig. 2. Effect of pH on the activity of free and encapsulated HRP. Initial activity of HRP was 1.106 U, maximum activity of the free and encapsulated HRP were 0.996 U and 1.093 U after 2 h.
Thermal stability of the free and encapsulated HRP was determined by measuring residual activities of the samples incubated over different times in the phosphate buffer (0.1 M, pH 7.0) at 50 ◦ C and 60 ◦ C. The initial activity of HRP was taken as 100% and the results were shown in Fig. 3. It was observed that the activity of the encapsulated HRP decreased more slowly than free HRP. After 5 h of incubation at 50 ◦ C, free HRP retained only 27.42% of its initial activity. However, the encapsulated HRP maintained 78.91% of its initial activity. The same behavior was observed at 60 ◦ C. The encapsulated HRP retained 62.21% of initial activity after 5 h, while free enzyme only retained 16.18% of initial activity. These results demonstrated that the encapsulated HRP exhibited improved thermostability compared with free HRP. This can be explained by the protection of the titania and lactose (the stabilizing agent), which provided a suitable microenvironment that favored intramolecular stabilizing forces and consequently increasing the stability of HRP [16].
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Y. Jiang et al. / Enzyme and Microbial Technology 55 (2014) 1–6 Table 1 Kinetic parameters for free and encapsulated HRP.
Free HRP Encapsulated HRP
Vm (mM/min)
Km (mM)
2.53 0.53
1.98 8.21
HRP is commercially important for reactions in the elimination of pollutants such as phenol and aniline in wastewater treatments. In these reactions, H2 O2 , which can inhibit the activity of enzyme, is usually utilized as a substrate. Thus, the stability of HRP in H2 O2 solution was investigated. As shown in Fig. 4, about 93.91% of enzymatic activity for encapsulated HRP was retained after incubating 30 min in H2 O2 solution (8.0 M), while for free HRP, only 53.18% of initial activity was retained. These results indicated that immobilization can protect HRP from inactivation induced by hydrogen peroxide. 3.5. Kinetic parameters of free and encapsulated HRP Fig. 4. Tolerance of HRP against inactivating agents. Initial activity of HRP was 1.106 U, maximum activity of the free and encapsulated HRP were 1.057 U and 1.100 U after incubation 30 min.
3.4. Tolerance of HRP against inactivating agents The tolerance of free and encapsulated HRP against different inactivating chemicals was tested to foresee their potential effect in applications where such conditions may be present. As shown in Fig. 4, the residual activities of free and encapsulated HRP were measured after incubating 30 min with several chemical agents (BaCl2 , CuCl2 , CaCl2 , MnCl2 , H2 O2 , methanol, and acetone) at 25 ◦ C [47]. Compared to free HRP, less decrease in the activities was detected for the encapsulated HRP, which presented a residual activity approximately 34–44% higher than free HRP against CaCl2 , MnCl2 , methanol and acetone, 26–29% higher than free HRP against BaCl2 and CuCl2 . These results indicate that the HRP encapsulated in biomimetic titania exhibited a remarkably higher tolerance against inactivating agents.
The kinetics of the free and encapsulated HRP were investigated follows the Michaelis–Menten equation. The increase in Km value and the decrease in Vmax value indicated the reduction in the affinity of HRP for binding substrate after HRP immobilization. As seen in the Table 1, these results were similar with other investigations reporting significant affinity decreases for the immobilized biocatalyst [48]. Taken together, all these data indicated that the encapsulated HRP behaved as a lower affinity for the substrate than free HRP, which may be caused by the steric hindrance of the active site by the support, or the loss of enzyme flexibility necessary for substrate binding [47,49]. 3.6. Reusability of encapsulated HRP on phenolic compounds and dye removal The main purpose of enzyme immobilization is to increase the frequency of use in industrial applications, thus the cost associated could be largely reduced. The reusability of encapsulated HRP to treat phenolic compounds and Direct Black-38 was investigated in
Fig. 5. Reusability of encapsulated HRP on phenolic compounds and dye removal. Conditions: HRP content 130 U, H2 O2 8 mM 50 mL, (a) Phenol 8 mM 50 mL, (b) 2-chlorophenol 60 mM 50 mL, (c) Direct Black-38 120 mg/L 50 mL.
Y. Jiang et al. / Enzyme and Microbial Technology 55 (2014) 1–6
the present study. To distinguish between enzymatic degradation and physical adsorption by the support, a control experiment was carried out by using the titania particles without enzyme loading. As shown in Fig. 5a, the removal efficiency of phenol by free and encapsulated HRP was about 70.17% and 92.99%, respectively, in the first treatment cycle. After six consecutive operations, the phenol removal efficiency for encapsulated HRP was more than 50%. After eight repeated tests, the removal efficiency of 2-chlorophenol by encapsulated HRP decreased from 87.97% to 53.62% (Fig. 5b). The formation and accumulation of dark precipitates on the encapsulated HRP can be observed. As shown in Fig. 5c, the removal of Direct Black-38 by HRP was investigated. In the first treatment cycle, the decolorization efficiency of the free and encapsulated HRP was 46.82% and 79.72%, respectively. After 5 consecutive operations, each of 10 h duration, the decolorization efficiency was decreased to 51.58% for the encapsulated HRP. It must be pointed out that, the removal efficiency of titaina particles by physical adsorption was below 20% for all the tests, which indicated that the encapsulated HRP performed good activity and reusability. The loss of catalytic activity that occurred during the repetitive use can be attributed to enzyme inactivation as a result of radical attack [49]. Additionally, removal efficiency declined in subsequent cycles can be attributed to the mass transfer limitation or blockage of enzyme active sites due to the accumulating of polymerization products in the interior environment of the immobilized HRP particles [50]. 4. Conclusions In the current work, HRP was encapsulated in phospholipidtemplated titania particles through a biomimetic process and the properties of the immobilized HRP were investigated. The encapsulated HRP showed improved pH and thermal stabilities. The tolerance capacity against inactivating agents was also enhanced. The encapsulated HRP presented high efficiency in the repeated elimination of the phenolics and dye. Overall, the results of this study presented the phospholipid-templated titania encapsulation as a promising method for enzyme immobilization and the immobilized HRP can be easily used in industry applications. Acknowledgments This work was supported by the National Nature Science Foundation of China (Nos. 21006020, 21276060, and 21276062), the Application Basic Research Plan Key Basic Research Project of Hebei Province (11965150D) and the Natural Science Foundation of Tianjin (13JCYBJC18500). References [1] Sassolas A, Blum LJ, Leca-Bouvier BD. Immobilization strategies to develop enzymatic biosensors. Biotechnol Adv 2012;30:489–511. [2] Brady D, Jordaan J. Advances in enzyme immobilisation. Biotechnol Lett 2009;31:1639–50. [3] Hanefeld U, Gardossi L, Magner E. Understanding enzyme immobilization. Chem Soc Rev 2009;38:453–68. [4] Sheldon RA, van Pelt S. Enzyme immobilisation in biocatalysis: why, what and how. Chem Soc Rev 2013;42:6223–35. [5] Betancor L, Luckarift HR. Bioinspired enzyme encapsulation for biocatalysis. Trends Biotechnol 2008;26:566–72. [6] Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Technol 2007;40:1451–63. [7] Pramparo L, Stüber F, Font J, Fortuny A, Fabregat A, Bengoa C. Immobilisation of horseradish peroxidase on Eupergit® C for the enzymatic elimination of phenol. J Hazard Mater 2010;177:990–1000. [8] Yu JH, Ju HX. Preparation of porous titania sol–gel matrix for immobilization of horseradish peroxidase by a vapor deposition method. Anal Chem 2002;74:3579–83. [9] Bayramoglu G, Altintas B, Arica MY. Cross-linking of horseradish peroxidase adsorbed on polycationic films: utilization for direct dye degradation. Bioprocess Biosyst Eng 2012;35:1355–65.
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Immobilization of horseradish peroxidase in phospholipid-templated titania and its applications in phenolic compounds and dye removal Yanjun Jiang, Wei Tang, Jing Gao ∗ , Liya Zhou, Ying He School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China
a r t i c l e
i n f o
Article history: Received 13 September 2013 Received in revised form 15 November 2013 Accepted 18 November 2013 Keywords: Horseradish peroxidase Phospholipid-templated titania Enzyme encapsulation Phenolic compound Dye
a b s t r a c t In this study, horseradish peroxidase (HRP) was encapsulated in phospholipid-templated titania particles through the biomimetic titanification process and used for the treatment of wastewater polluted with phenolic compounds and dye. The encapsulated HRP exhibited improved thermal stability, a wide range of pH stability and high tolerance against inactivating agents. It was observed an increase in Km value for the encapsulated HRP (8.21 mM) when compared with its free counterpart. For practical applications in the removal of phenolic compounds and dye by the encapsulated HRP, the removal efficiency for phenol, 2chlorophenol, Direct Black-38 were 92.99%, 87.97%, and 79.72%, respectively, in the first treatment cycle. Additionally, the encapsulated HRP showed better removal efficiency than free HRP and a moderately good capability of reutilization. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Enzymes are able to catalyze many chemical processes under most benign experimental conditions [1]. In this way, enzymes could be excellent catalysts for a much more sustainable chemical industry [2]. However, enzymes have some limitations for nonbiological applications [3,4]. Thus, for many industrial applications, enzymes have to be immobilized, via very simple and cost-effective protocols, in order to improve the properties of enzymes, such as activity, stability, and selectivity [5,6]. Over the last several decades, three types of methodology for enzyme immobilization have been reported in scientific literatures: via binding to or encapsulation in an inorganic or organic polymer [7,8], or by cross-linking the enzyme molecules [9]. No single method has been emerged as the standard for enzyme immobilization and ongoing efforts are striving to optimize these methods to render them adequate for specific applications [10,11]. Compared to most organic polymers, the silica matrix exhibits higher mechanical strength, enhanced thermal stability, and negligible swelling in organic solvents. Thus, silica matrix made by sol–gel process, has emerged as a promising platform for encapsulation of enzymes to be used in biocatalysis, biosensors, and
Abbreviations: HRP, horseradish peroxidase. ∗ Corresponding author. Tel.: +86 22 60204293; fax: +86 22 60204294. E-mail address: [email protected] (J. Gao). 0141-0229/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.enzmictec.2013.11.005
biomedical fields [12,13]. However, the relative harsh reaction conditions and long aging time [14] of the traditional sol–gel process may induce the deactivation of enzymes. Thus, the biomimetic silicification process, which provides a rapid, low temperature method for silica precipitation, has emerged as a versatile tool for preparing excellent supports for enzyme immobilization. Until now, a remarkable diversity of enzymes has been encapsulated in bioinspired silicas, and it is reasonable to envisage that this technological impact of bioinspired encapsulation will continue to grow [5]. Although much of the research effort on biomimetic silicification has been taken, exploration of phospholipid-templated silica matrix seems to have received less attention. This approach can overcome the disadvantages of entrapment techniques, including the leaching of adsorbed biomolecules, the chemical degradation of the anchoring bond of covalently attached enzymes, and diffusion limitations of substrates and products. This is clearly confirmed by various authors [15–18]. Furthermore, this phospholipid-templated approach can eliminate specific enzyme–silica interactions during the silica formation process, and can produce more active biocatalyst than those prepared by trapping enzymes directly in silica hydrogels [16]. In comparison with silica-based materials, titania-based materials have attracted great interest in a number of fields [19–21], owing to their unique chemical and physical properties, including stable chemical structure [22], good biocompatibility, relative high conductivity, and environmentally benign nature [23]. Additionally, titania is an amphoteric oxide, allowing it to be an anion and
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cation exchanger at acidic and alkaline pH, whereas silica can only act as a cation exchanger [14,24]. Thus, the titania material has been investigated and proved to be an ideal enzyme carrier [25–27]. In the past years, the so-called biomimetic titanification, which typically refers to the mimic of biological synthesis process to form titania, has emerged as a versatile tool for preparing titania materials [28,29]. This facile and green process for titania synthesis can be controlled at near-neutral pH, ambient temperature within a short period [23,24,27]. Many proteins and peptides, such as silicatein [30], lysozyme [31], protamine [27], and R5 peptide [32] have been successfully employed for the formation of titania through the biomimetic titanification process [33]. In contrast to these reports, we report the first examination of using dodecylamine to catalyze the hydrolysis and subsequent polycondensation of a water-stable alkoxide-like conjugate of titanium to yield titania under ambient conditions for enzyme immobilization. To eliminate the specific enzyme-titania interactions, generate 3-D open mesoporosity for facile diffusion of substrates in biocatalysis process, and then produce more active biocatalyst, phospholipid was used as template. To the best of our knowledge, this is the first report that concerned the preparation of phospholipid-templated titania particles by using biomimetic titanification and as an efficient matrix for enzyme immobilization. Phenolic compounds and direct dyes are widely distributed in the wastewater of the textile industry and they can be highly harmful toward aquatic life and humans. [34,35]. Therefore, a number of techniques including adsorption, chemical oxidation, solvent extraction and biodegradation aimed at preferential removal of the phenolic compounds [36–38] and dyes [39,40] from wastewaters have been developed. Among these treatment technologies, the use of oxido-reductive enzymes such as horseradish peroxidase (HRP) to catalyze the removal of pollutes has become increasingly important [41–44]. Thus, in the present work, HRP was encapsulated in phospholipid-templated titania particles that induced by dodecylamine through the biomimetic titanification process. The effect of pH, thermal stability, tolerance against inactivating agents, and kinetic parameters of the encapsulated HRP were investigated. The removal of phenolic compounds (phenol and 2-chlorophenol) and dye (Direct Black-38) by the free and encapsulated HRP was also investigated. The results presented here not only open a novel avenue for immobilizing enzymes but also provide methods that can be readily adapted for a range of metal oxide synthesis. Additionally, the relatively low price and easy availability ensure dodecylamine as a promising titania-precipitating agent for largescale utilization. 2. Materials and methods 2.1. Materials Horseradish peroxidase (HRP, EC. 1.11.1.7, 150 U/mg) was purchased from Source leaves Biotechnology Co. (shanghai, China). Soybean lecithin, phenol, 2chlorophenol and H2 O2 30% (w/v) were purchased from Jiang Tian Chemical Technology Co. (Tianjin, China). Titanium (IV) bis (ammonium lactato) dihydroxide (Ti-BALDH) and Direct Black-38 were purchased from Sigma Chemical Co. (St. Louis, USA). Other chemicals were of analytical grade and were used as received without further purification. 2.2. Preparation of the encapsulated HRP After optimization in the preliminary experiment, a typical experimental procedure for encapsulation of HRP in titania was described as follows: A first solution of lactose (0.05 g) in 7.2 mL phosphate buffer pH 7.0 containing HRP (4.32 mg) was prepared at 37 ◦ C. It was added slowly under vigorous stirring to a second solution, also prepared at 37 ◦ C, composed of lecithin (0.7 g) and dodecylamine (0.05 g) in 5.2 g ethanol. Then 2 mL of Ti-BALDH (0.25 mol/L, pH 7.0) solution was then added slowly to the above mixture, the biomimetic titanification process was preceded for 15 min. A gentle stirring was maintained until the titania particles were formed. To remove
lecithin, the resulting particles were washed with Triton X-100 (7.5%) and distilled water, then the encapsulated HRP was obtained. Surface morphology of the encapsulated HRP was investigated through scanning electron microscopy (JSM-6700F, JEOL, Japan). Samples were dried by rinsing with anhydrous acetone, sputter-coated with gold prior to the examination. The diameters of the titania particles were determined by using a Brookhaven Instruments BI200SM dynamic light scattering (DLS) system. The enzymatic activity of HRP was measured by Worthington method [37]. One unit of HRP activity (U) was defined as the amount of HRP required to hydrolyze 1 mol of H2 O2 in 1 min at 25 ◦ C and pH 7.0. 2.3. The properties of the encapsulated HRP The effect of pH on the activity was evaluated by incubating free and encapsulated HRP with equal activity in phosphate buffers (0.1 M) for 2 h at pH 3.0–9.0 under 25 ◦ C. Then the HRPs were taken out and the residual activities were measured. The relative activity was calculated as the ratio between the activity at each pH and the maximum activity. The study on thermal stability of free and encapsulated HRP was carried out by measuring the residual activity incubated over different times in the phosphate buffer (0.1 M, pH 7.0) at 50 ◦ C and 60 ◦ C. The tolerance capacity of free and encapsulated HRP against inactivating chemicals was tested by incubating HRP in different denaturing solutions, namely 10 M of CaCl2 , CuCl2 , BaCl2 , MnCl2 , 8 mM of H2 O2 , 25% (v/v) methanol, and 25% (v/v) acetone (0.1 M PBS, pH 7.0) at 25 ◦ C for 30 min. The residual activities were measured by Worthington method. The kinetic model used in this study was based on the Michaelis–Menten equation (1/V vs 1/[S]). The experimental initial reaction rates (Vi) were determined from the plot of the consumption of H2 O2 as a function of time. The concentration of phenol was 0.17 mM, and the concentration of H2 O2 was varied from 0.7 mM to 2.0 mM in the reaction medium.
2.4. Enzymatic removal of phenolic compounds and dye Two phenolics (phenol and 2-chlorophenol) and one dye (Direct Black-38) were employed in this research. The phenol (8 mM), 2-chlorophenol (60 mM) and Direct Black-38 (120 mg/L) degradation were studied under the same H2 O2 concentration (8 mM) with the encapsulated HRP (130 U) at 25 ◦ C. The solution was stirred to ensure full contact of the substrates and the encapsulated HRP. The residual phenolic compunds were measured on the basis of colorimetric method with potassium ferricyanide and 4-aminoantipyrine with a UV–vis spectrophotometer at 505 nm in 100 mL round bottom flask [45]. Dye decolorization was measured based on the maximum absorbance of Direct Black-38 at 550 nm [9].
3. Results and discussion 3.1. Characterization of the encapsulated HRP With the objective of synthesizing titania materials as enzyme host from Ti-BALDH, we used lecithin/dodecylamine mixedmicelle as the template. Scheme 1 shows the building process of the phospholipid-templated titania particles and HRP encapsulation. In this process, lecithin and dodecylamine were anticipated to play three roles. First, the lecithin/dodecylamine mixed-micelle that worked as template for the titania formation could generate 3-D open mesoporosity for facile diffusion of substrates in biocatalysis process. Second, dodecylamine played the role of nucleophilic catalyst to promote the condensation of titania precursor in the synthesis process, without generating elevated pH, which was indeed advantageous to avoid enzyme denaturation. Third, the lecithin could protect the enzymes from unfriendly environment. Fig. 1 shows typical SEM image of the obtained HRP-containing titania particles (encapsulated HRP). It can be seen that the samples were formed by an agglomeration of microspheres, which was similar with the silica-based materials reported by various authors [15–18]. DLS results (Fig. S1) showed that the titania particles had an average size of about 190 nm. After optimization, the immobilization yield would be 70.51% and the enzyme activity recovery was 56.31%. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.enzmictec. 2013.11.005.
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Scheme 1. Schematic representation of the building process for the phospholipid-templated titania particles and enzyme encapsulation.
Fig. 1. SEM image of encapsulated HRP.
3.2. Effect of pH on the activity of free and encapsulated HRP The activities of free and encapsulated HRP were measured at different pH values. As shown in Fig. 2, free HRP reached a maximum activity at pH 6.0, whereas the optimal pH for encapsulated HRP rose to 7.0. It may be possible that the presence of lactose led to a near acidic microenvironment of enzyme, thus the HRP encapsulated in the titania particles experienced a lower pH in comparison with free HRP in buffer medium and thus the optimum pH was shifted to a higher level [46]. Additionally, the activity of encapsulated HRP was less sensitive to pH compared to its free counterpart. 80.55% and 86.68% of relative activity can be retained for encapsulated HRP under the acidic condition (pH 3.0) and
Fig. 3. Thermal stability of the free and encapsulated HRP. Initial activity of HRP was 1.106 U.
alkaline condition (pH 9.0), respectively, while only 39.58% and 28.84% of initial activity for free HRP was left at pH 3.0 and pH 4.0, respectively. The improvement in pH tolerance of the encapsulated HRP can be explained by the stabilizing and protective effect of the titania particles and immobilization technology [14].
3.3. Thermal stability of the free and encapsulated HRP
Fig. 2. Effect of pH on the activity of free and encapsulated HRP. Initial activity of HRP was 1.106 U, maximum activity of the free and encapsulated HRP were 0.996 U and 1.093 U after 2 h.
Thermal stability of the free and encapsulated HRP was determined by measuring residual activities of the samples incubated over different times in the phosphate buffer (0.1 M, pH 7.0) at 50 ◦ C and 60 ◦ C. The initial activity of HRP was taken as 100% and the results were shown in Fig. 3. It was observed that the activity of the encapsulated HRP decreased more slowly than free HRP. After 5 h of incubation at 50 ◦ C, free HRP retained only 27.42% of its initial activity. However, the encapsulated HRP maintained 78.91% of its initial activity. The same behavior was observed at 60 ◦ C. The encapsulated HRP retained 62.21% of initial activity after 5 h, while free enzyme only retained 16.18% of initial activity. These results demonstrated that the encapsulated HRP exhibited improved thermostability compared with free HRP. This can be explained by the protection of the titania and lactose (the stabilizing agent), which provided a suitable microenvironment that favored intramolecular stabilizing forces and consequently increasing the stability of HRP [16].
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Y. Jiang et al. / Enzyme and Microbial Technology 55 (2014) 1–6 Table 1 Kinetic parameters for free and encapsulated HRP.
Free HRP Encapsulated HRP
Vm (mM/min)
Km (mM)
2.53 0.53
1.98 8.21
HRP is commercially important for reactions in the elimination of pollutants such as phenol and aniline in wastewater treatments. In these reactions, H2 O2 , which can inhibit the activity of enzyme, is usually utilized as a substrate. Thus, the stability of HRP in H2 O2 solution was investigated. As shown in Fig. 4, about 93.91% of enzymatic activity for encapsulated HRP was retained after incubating 30 min in H2 O2 solution (8.0 M), while for free HRP, only 53.18% of initial activity was retained. These results indicated that immobilization can protect HRP from inactivation induced by hydrogen peroxide. 3.5. Kinetic parameters of free and encapsulated HRP Fig. 4. Tolerance of HRP against inactivating agents. Initial activity of HRP was 1.106 U, maximum activity of the free and encapsulated HRP were 1.057 U and 1.100 U after incubation 30 min.
3.4. Tolerance of HRP against inactivating agents The tolerance of free and encapsulated HRP against different inactivating chemicals was tested to foresee their potential effect in applications where such conditions may be present. As shown in Fig. 4, the residual activities of free and encapsulated HRP were measured after incubating 30 min with several chemical agents (BaCl2 , CuCl2 , CaCl2 , MnCl2 , H2 O2 , methanol, and acetone) at 25 ◦ C [47]. Compared to free HRP, less decrease in the activities was detected for the encapsulated HRP, which presented a residual activity approximately 34–44% higher than free HRP against CaCl2 , MnCl2 , methanol and acetone, 26–29% higher than free HRP against BaCl2 and CuCl2 . These results indicate that the HRP encapsulated in biomimetic titania exhibited a remarkably higher tolerance against inactivating agents.
The kinetics of the free and encapsulated HRP were investigated follows the Michaelis–Menten equation. The increase in Km value and the decrease in Vmax value indicated the reduction in the affinity of HRP for binding substrate after HRP immobilization. As seen in the Table 1, these results were similar with other investigations reporting significant affinity decreases for the immobilized biocatalyst [48]. Taken together, all these data indicated that the encapsulated HRP behaved as a lower affinity for the substrate than free HRP, which may be caused by the steric hindrance of the active site by the support, or the loss of enzyme flexibility necessary for substrate binding [47,49]. 3.6. Reusability of encapsulated HRP on phenolic compounds and dye removal The main purpose of enzyme immobilization is to increase the frequency of use in industrial applications, thus the cost associated could be largely reduced. The reusability of encapsulated HRP to treat phenolic compounds and Direct Black-38 was investigated in
Fig. 5. Reusability of encapsulated HRP on phenolic compounds and dye removal. Conditions: HRP content 130 U, H2 O2 8 mM 50 mL, (a) Phenol 8 mM 50 mL, (b) 2-chlorophenol 60 mM 50 mL, (c) Direct Black-38 120 mg/L 50 mL.
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the present study. To distinguish between enzymatic degradation and physical adsorption by the support, a control experiment was carried out by using the titania particles without enzyme loading. As shown in Fig. 5a, the removal efficiency of phenol by free and encapsulated HRP was about 70.17% and 92.99%, respectively, in the first treatment cycle. After six consecutive operations, the phenol removal efficiency for encapsulated HRP was more than 50%. After eight repeated tests, the removal efficiency of 2-chlorophenol by encapsulated HRP decreased from 87.97% to 53.62% (Fig. 5b). The formation and accumulation of dark precipitates on the encapsulated HRP can be observed. As shown in Fig. 5c, the removal of Direct Black-38 by HRP was investigated. In the first treatment cycle, the decolorization efficiency of the free and encapsulated HRP was 46.82% and 79.72%, respectively. After 5 consecutive operations, each of 10 h duration, the decolorization efficiency was decreased to 51.58% for the encapsulated HRP. It must be pointed out that, the removal efficiency of titaina particles by physical adsorption was below 20% for all the tests, which indicated that the encapsulated HRP performed good activity and reusability. The loss of catalytic activity that occurred during the repetitive use can be attributed to enzyme inactivation as a result of radical attack [49]. Additionally, removal efficiency declined in subsequent cycles can be attributed to the mass transfer limitation or blockage of enzyme active sites due to the accumulating of polymerization products in the interior environment of the immobilized HRP particles [50]. 4. Conclusions In the current work, HRP was encapsulated in phospholipidtemplated titania particles through a biomimetic process and the properties of the immobilized HRP were investigated. The encapsulated HRP showed improved pH and thermal stabilities. The tolerance capacity against inactivating agents was also enhanced. The encapsulated HRP presented high efficiency in the repeated elimination of the phenolics and dye. Overall, the results of this study presented the phospholipid-templated titania encapsulation as a promising method for enzyme immobilization and the immobilized HRP can be easily used in industry applications. Acknowledgments This work was supported by the National Nature Science Foundation of China (Nos. 21006020, 21276060, and 21276062), the Application Basic Research Plan Key Basic Research Project of Hebei Province (11965150D) and the Natural Science Foundation of Tianjin (13JCYBJC18500). References [1] Sassolas A, Blum LJ, Leca-Bouvier BD. Immobilization strategies to develop enzymatic biosensors. Biotechnol Adv 2012;30:489–511. [2] Brady D, Jordaan J. Advances in enzyme immobilisation. Biotechnol Lett 2009;31:1639–50. [3] Hanefeld U, Gardossi L, Magner E. Understanding enzyme immobilization. Chem Soc Rev 2009;38:453–68. [4] Sheldon RA, van Pelt S. Enzyme immobilisation in biocatalysis: why, what and how. Chem Soc Rev 2013;42:6223–35. [5] Betancor L, Luckarift HR. Bioinspired enzyme encapsulation for biocatalysis. Trends Biotechnol 2008;26:566–72. [6] Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Technol 2007;40:1451–63. [7] Pramparo L, Stüber F, Font J, Fortuny A, Fabregat A, Bengoa C. Immobilisation of horseradish peroxidase on Eupergit® C for the enzymatic elimination of phenol. J Hazard Mater 2010;177:990–1000. [8] Yu JH, Ju HX. Preparation of porous titania sol–gel matrix for immobilization of horseradish peroxidase by a vapor deposition method. Anal Chem 2002;74:3579–83. [9] Bayramoglu G, Altintas B, Arica MY. Cross-linking of horseradish peroxidase adsorbed on polycationic films: utilization for direct dye degradation. Bioprocess Biosyst Eng 2012;35:1355–65.
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