Alginate Hybrid Beads for Efficient Enzyme

Sep 26, 2013 - ABSTRACT: A novel kind of matrix for enzyme immobilization, the boehmite/alginate hybrid beads, was fabricated under mild conditions in...
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Fabrication of Boehmite/Alginate Hybrid Beads for Efficient Enzyme Immobilization Qinghong Ai,† Dong Yang,†,§ Yuanyuan Zhu,† and Zhongyi Jiang*,‡,§ †

Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 30072, China ‡ Key Laboratory for Green Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China § Synergetic Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China ABSTRACT: A novel kind of matrix for enzyme immobilization, the boehmite/alginate hybrid beads, was fabricated under mild conditions in this study. The boehmite particles were first prepared via a mild sol−gel technology and were then entrapped into the inherent network of calcium alginate. The characterization results suggest that the hybridization between alginate and boehmite belongs to a physical blending, in which the electrostatic interaction acts an important role. Yeast alcohol dehydrogenase (YADH), which is of high importance for the transformation of CO2, was encapsulated in the boehmite/alginate hybrid beads. The enzyme was loaded as high as 99.4% and uniformly distributed in these hybrid beads according to the fluorescence experiment. It is worthy of noting that the encapsulated YADH can almost reach “zero leaching” after incubation for 67 h and preserve 86.6% activity after 12-recycle using. This kind of hybrid materials may become a promising matrix for the immobilization of industrial enzymes.

1. INTRODUCTION Much effort has been focused on the immobilized enzyme in recent years for its expected superiorities over free enzyme, such as the easy separation from the product and the enzyme reuse.1−9 Besides the immobilized protocol, the matrix is another important factor affecting the immobilized enzyme performance. Over the past decade, organic/inorganic hybrid materials have attracted much interest among various immobilized matrices, since they combine the flexibility, lightweight, good processing ability of organic moiety and high mechanical strength, good chemical resistance, and thermal stability of inorganic moiety.10−14 Till now, a number of organic/inorganic hybrid matrices have been extensively developed in the field of enzyme immobilization.15−21 For example, Macario et al.15 entrapped the lipase in the surfactant micelles that was self-assembled with silica to prepare active heterogeneous biocatalyst under the conditions free of organic solvent. Allouche et al.19 fabricated biomimetic core−shell gelatin/silica nanoparticles through a nanoemulsion route. Zhang et al.20 encapsulated yeast alcohol dehydrogenase (YADH) in a kind of biomimetic alginate/silica hybrid microcapsule with an egg-like hierarchical structure via a one-pot method. The silica-based materials have been the most commonly used and well-studied inorganic moiety in the organic/inorganic hybrid matrices to date.17,18,21 The type of inorganic moiety need be extended to wider inorganic materials. Boehmite (γ-AlOOH), the oxide−hydroxide phase of aluminum, is a kind of traditional inorganic material with high thermal, chemical, and mechanical stability as well as its good biocompatibility, which has been applied broadly as the absorbents,22 catalysts,23 and inorganic fillers in the membrane24,25 etc. In our previous study,26 boehmite was used as a carrier to encapsulate enzyme for the first time, and the © 2013 American Chemical Society

encapsulated enzyme showed excellent immobilization efficiency, enhanced pH, thermal and storage stability compared to its free counterpart. Nevertheless, the serious leakage of boehmite particles results in their poor recycling stability, which hinders their practical application for the immobilized carrier. Herein, boehmite/alginate hybrid beads are designed and fabricated by a facile method in order to solve the leakage problem of boehmite via the organic/inorganic hybridization idea. In recent years, alginate has become one of the most widely used polymers as the carrier of enzymes and drugs, due to its low cost, natural biocompatibility, excellent gelation ability, and mild processing characteristics. Moreover, much effort has been devoted to the hybridization of alginate to improve its performance. 27,28 Formate dehydrogenase (FateDH), formaldehyde dehydrogenase (FaldDH), and alcohol dehydrogenase (ADH) are three enzymes in the consecutive reduction of CO2 to methanol described as FateDH

FaldDH

ADH

NADH

NADH

NADH

CO2 ⎯⎯⎯⎯⎯⎯→ HCOOH ⎯⎯⎯⎯⎯⎯→ HCHO ⎯⎯⎯⎯⎯⎯→ CH3OH

The coimmobilization of this enzyme cascade system has been investigated in our previous study.29 To deeply understand the whole process and acquire the optimal operating conditions, it is necessary to study three reactions separately.30 In this study, we concentrated on the third reaction, and encapsulated yeast alcohol dehydrogenase (YADH) in these hybrid beads to evaluate the catalytic performance of immobilized enzyme. Received: Revised: Accepted: Published: 14898

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2.4. Characterization. The structure morphology of the samples was characterized by a scanning electron microscope (SEM, XL30, Philips) with an accelerating voltage of 20 kV. The cross-section EDS mapping of boehmite/alginate hybrid beads was carried out by an energy dispersive X-ray spectroscope (EDX), which was directly connected to the SEM. Transmission electron microscopy (TEM) observation was performed on a JEM-100CX II instrument. Fluorescence microscope photos were taken by using an Olympus BX51 microscope with a 100 oil immersion objective lens (Olympus, Tokyo, Japan) to demonstrate the dispersion of YADH in the boehmite/alginate hybrid beads. The infrared (IR) spectra of the samples were obtained using a Nicolet-6700 Fourier transform infrared (FTIR) spectrometer, and thirty-two scans were accumulated with a resolution of 4 cm−1 for each spectrum. Differential scanning calorimetry (DSC, NETZSCH DSC 200F3 A) was used to determine the glass transition temperature of freeze-dried alginate and boehmite/alginate hybrid beads under constant N2 purging with a 20 mL·min−1 rate. 2.5. Catalytic Activity of YADH. The catalytic activity of YADH was measured by the concentration change of coenzyme NADH in the reversible transformation of HCHO to CH3OH catalyzed by YADH. HCHO and NADH were dissolved in a Tris-HCl buffer (0.05 mol·L−1, pH 7.0) to prepare the substrate solution, in which their concentrations were 0.01 mol·L−1 and 133 μmol·L−1, respectively. Subsequently, 0.1 mg of free or encapsulated YADH was added into a 9 mL substrate solution under continuous magnetic stirring. The absorbance decrease at 340 nm caused by NADH consumption was recorded after the enzyme was mixed in the above solution for 10 min. One unit of catalytic activity is defined as the enzyme amount producing 1 μmol of CH3OH per minute under the assay conditions (25 °C, pH 7.0). The YADH concentration was determined by the Brandford method, and the catalytic activity of free and encapsulated YADH was given as U·mg of protein−1. The optimum pH and temperature were determined by measuring the catalytic activity of encapsulated YADH in a 0.05 mol·L−1 Tris-HCl buffer with different pH values (6.0−9.0) and at different temperatures (20−60 °C), respectively. The activity of encapsulated YADH at optimum pH or temperature is defined as 100%, while the activities at other pH values or temperatures are the relative values with regard to it. The stability experiments of encapsulated YADH include four parts, i.e. the pH, thermal, storage, and recycling stability. Briefly, the pH stability was investigated by measuring the residual activity of encapsulated YADH after having been incubated in a 0.05 mol·L−1 Tris-HCl buffer with different pH values (6.0−9.0) at 25 °C for 3 h. Similarly, the thermal stability was investigated by measuring the residual activity of encapsulated YADH after having been incubated in a 0.05 mol· L−1 Tris-HCl buffer (pH 7.0) with different temperatures (20− 60 °C) for 3 h. The storage stability was determined through selectively measuring the residual activity of encapsulated YADH after having been stored for 1 d, 8 d, 15 d, 22 d, and 29 d at 4 °C, respectively. The recycling stability was determined by measuring the residual activity of encapsulated YADH after each reaction cycle at room temperature and neutral pH. Simply, 1.5 g of immobilized YADH were dispersed in a 20 mL substrate solution at 25 °C and pH 7.0, and their activity was tested as described above. Subsequently, the hybrid beads containing YADH were collected after reacting for 30 min, thoroughly washed with Tris-HCl buffer, and then reused in

2. EXPERIMENTAL SECTION 2.1. Materials. Yeast alcohol dehydrogenase (YADH, EC1.1.1.1, MW = 141 kDa, from Saccharomyces cerevisiae, 445 U·mg of protein−1), reduced form of nicotinamide adenine dinucleotide (NADH, grade I, 98%), and tris(hydroxymethyl) aminomethane (Tris) were purchased from Sigma-Aldrich Chemical Co. Ltd. Sodium aluminate was purchased from Guangfu Technology Development Co. Ltd. (Tianjin, China). Sodium alginate was obtained from Jiangtian Chemicals Co. Ltd. (Tianjin, China). All other chemical reagents were commercially analytical grade and used without further purification. Deionized water was used throughout the study. 2.2. Fabrication of Boehmite/Alginate Hybrid Beads. Boehmite particles were synthesized at first by a sol−gel method according to our previous literature.26 Briefly, a sodium aluminate solution containing 0.7 mol·L−1 Al3+ was prepared by completely dissolving 5.74 g of sodium aluminate in 100 mL of water, and its pH value was adjusted to be 8.0 by adding concentrated hydrochloric acid (36.5 wt./%). This solution was incubated at 80 °C for 8 h in a water bath under continuous magnetic agitation, and then the precipitate was collected by centrifugation and washed with water for 5 times to remove residual ions. A boehmite sol was obtained by mixing 1 mL of deionized water with 1.5 g of precipitates thoroughly. Finally, the boehmite sol was separated by centrifugation after aging for 30 min at room temperature in order to get the wet boehmite gel. The boehmite/alginate hybrid beads were prepared by a onepot method. First, sodium alginate was dissolved in deionized water to prepare an alginate solution at a final concentration of 2.5% (w/v), and 1 mL Tris-HCl buffer (pH = 7.0, 0.05 mol· L−1) was added into 1.5 g of wet boehmite gel to prepare a boehmite suspension. Then, the prepared sodium alginate gel and boehmite suspension were blended at a volume ratio of 2:1 completely. After incubation for 10 min, 2 mL of the mixture was added dropwise into 20 mL of CaCl2 solution (0.2 mol· L−1) by a syringe with an inner diameter of 0.5 mm. At last, boehmite/alginate hybrid beads were obtained after being filtered and rinsed twice with excess deionized water. The preparation of pure alginate beads is the same as that of boehmite/alginate hybrid beads except that the sodium alginate gel replaces the mixture of sodium alginate and boehmite. 2.3. YADH Immobilized in Boehmite/Alginate Hybrid Beads. YADH was immobilized in the boehmite/alginate hybrid beads by the same method as mentioned above. Simply, 1 mL of free YADH solution (1 mg·mL−1, 0.05 mol·L−1 TrisHCl buffer at pH 7.0) was added into 1.5 mg of wet boehmite sol. After the mixture was incubated for 30 min under continuously stirring, the boehmite gel containing YADH was collected by centrifugation and rinsed twice with deionized water to get rid of enzyme adsorbed on the surface. This gel was mixed with a 2.5% (w/v) alginate solution thoroughly at a volume ratio of 1: 2 and then added dropwise into 20 mL of CaCl2 solution (0.2 mol·L−1) by a syringe. After the suspension was incubated for 30 min at room temperature, YADH immobilized in the boehmite/alginate hybrid beads was obtained by filtration and rinsing twice with deionized water. YADH immobilized in pure alginate beads was also prepared by the same method as the control. The amount of immobilized enzyme was confirmed indirectly by determining the quantities of protein in supernatant and free enzyme solution through the Bradford method. 14899

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Figure 1. Optical images of alginate (left) and boehmite/alginate hybrid beads (right).

Figure 2. Surface SEM images of boehmite/alginate hybrid beads (a), alginate beads (b) and boehmite particles (c) after freeze-drying. The inset in (a) and (b) is an intact bead, and the inset in (c) is the TEM image of boehmite.

fresh reaction medium for the next reaction cycle. In all the stability experiments, the initial activity of encapsulated YADH is assumed as 100%, while other activities are the relative values through comparison with the initial activity. Each result was obtained by averaging three individual experiments. 2.6. Immobilization Capability of Boehmite/alginate Hybrid Beads. The immobilization capability of boehmite/ alginate hybrid beads was evaluated by measuring their swelling ratio, encapsulation efficiency, and leakage rate for YADH. The swelling experiment was carried out by incubating a certain amount of boehmite/alginate hybrid beads in the buffer with different pH values (pH = 6, 7, 9) at 20 or 60 °C for a period of time. Then, they were dried by filter papers and weighted. The swelling ratio is calculated based on the following equation swelling ratio (%) =

WS × 100 WI

encapsulation efficiency (%) C V + CCVC = 100 − S S × 100 CIVI

(2)

where CI and VI represent the initial concentration and volume of free YADH solution, respectively; CS and VS represent the residual YADH concentration and volume of the supernatant after encapsulation in boehmite, respectively; and CC and VC represent the residual YADH concentration and volume of the supernatant after encapsulation in boehmite/alginate hybrid beads, respectively. The YADH release experiment was carried out by soaking 1.5 g boehmite/alginate hybrid beads containing YADH in a 10 mL Tris-HCl buffer solution (0.05 mol·L−1, pH = 7.0) under magnetic stirring, and the time-dependent concentration of YADH released into the buffer solution was determined by Bradford method. The initial weight of YADH encapsulated in the boehmite/alginate hybrid beads is defined as 100%. The leakage ratio is calculated as the following equation

(1)

where WI and WS represent the initial weight of hybrid beads and the weight of hybrid beads after incubated for a period of time, respectively. The encapsulation efficiency of YADH in the boehmite/ alginate hybrid beads was calculated indirectly based on the following equation

leakage ratio (%) =

CiVi × 100 MT

(3)

where Ci, Vi, and MT represent the YADH concentration released in the buffer solution, the buffer solution volume, and 14900

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Figure 3. The cross-section SEM image (right) and EDX element mapping (Al and Ca, left) of a boehmite/alginate hybrid bead.

the initial amount of YADH encapsulated in the boehmite/ alginate hybrid beads, respectively.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Boehmite/ Alginate Hybrid Beads. The boehmite/alginate hybrid beads were fabricated by dropwise adding the boehmite/ sodium alginate suspension into a Ca2+ solution. The optical images in Figure 1 demonstrate that both fresh alginate and boehmite/alginate hybrid beads have a regular spherical shape about 2 mm in diameter. The alginate beads are translucent, while the hybrid beads are milk white, which can be as an indication of the formation of boehmite/alginate hybrid. The microstructure of boehmite/alginate hybrid beads after freezedrying is observed by SEM. As shown in Figure 2a, a boehmite/ alginate hybrid bead remains the spherical structure with the diameter about 700 μm. Some regions are sunken on the surface, which may be caused by the different tensions due to the heterogeneous structure of boehmite/alginate hybrid beads. Moreover, the hybrid bead exhibits a compact and smooth surface, while the alginate bead (Figure 2b) has a porous and wrinkled surface. This result hints that this kind of hybrid bead may have a lower leakage rate of enzyme molecules than pure alginate matrix. As for boehmite particles in Figure 2c, they are composed of a lot of needle-like nanoparticles, in accordance with the previous study.26 The cross-section image of a hybrid bead is presented in Figure 3, which is also homogeneous and smooth. The corresponding EDS map for the Al and Ca element demonstrates that the boehmite particles are homogeneously embedded in the calcium alginate network.31,32 In order to identify the molecule structure of boehmite/ alginate hybrid beads, FTIR spectra of boehmite, alginate, and boehmite/alginate hybrid beads were conducted (Figure 4). The characteristic absorption bands at 1632 and 1070 cm−1 in the IR spectrum of boehmite (Figure 4a) are assigned to adsorbed H2O and the Al−O bond with tetrahedral symmetry, respectively.26 There are also two characteristic absorption bands at 1597 and 1420 cm−1 in the IR spectrum of alginate (Figure 4b), which can be attributed to the symmetric and asymmetrical vibration of COO−, respectively.33,34 Compared to the IR spectra of boehmite and alginate, a new band is not present in the IR spectrum of boehmite/alginate hybrid beads. The alginate band at 1597 cm−1 disappears, which is overlapped

Figure 4. FTIR spectra of boehmite (a), alginate (b), and boehmite/ alginate hybrid beads (c).

by the band of adsorbed H2O at 1632 cm−1. Therefore, the hybridization between alginate and boehmite belongs to a physical blending, in which the static electricity effect acts an important role. In addition, the loose network of the boehmite gel is also helpful for the incorporation of alginate molecules to form the hybrid structure. The glass transition temperature of alginate/boehmite hybrid beads was determined via the differential scanning calorimetry in order to evaluate their thermal stability. As shown in Figure 5, the alginate/boehmite hybrid beads after freeze-drying have a higher glass transition temperature (Tg, 104.4 °C) than pure alginate beads (79.7 °C). This result indicates that the hybrid beads possess the better thermal stability than pure alginate beads, which can be attributed to the restricted flexibility of alginate chains arisen from the formation of hybrid structure. The swelling property is directly related to the stability of immobilized enzyme during the long-period reaction, since it can result in the change of the matrix structure and thus, causing the enzyme leakage. As demonstrated in Figure 6, the pH value and temperature can influence obviously the swelling ratio of YADH encapsulated in the alginate and boehmite/ alginate hybrid beads. The boehmite/alginate hybrid beads exhibit a lower swelling ratio than pure alginate beads at pH 7− 9 and 20−60 °C, while a comparative swelling ratio at pH 6 and 20 °C. Moreover, the swelling ratio of both alginate and boehmite/alginate hybrid beads increases gradually with the pH 14901

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particles during the self-assembly of boehmite nanoneedles on account of the electrostatic interaction between negatively charged YADH molecules and positively charged boehmite nanoneedles.26 Next, these boehmite particles containing YADH mix with alginate solution under vigorous agitation. When the mixture is added dropwise into the CaCl2 solution, calcium alginate forms the reticular structure due to the crosslinking interaction between alginate and Ca2+ ions. At the same time, the boehmite particles are entrapped in the reticular structure, and the hybrid layer forms on the interface between calcium alginate and boehmite particles. Finally, YADH molecules are effectively immobilized in the boehmite/alginate hybrid beads. The fluorescence image of encapsulated fluorescein-5-isothiocyanate (FITC)-labeled YADH in the boehmite/alginate hybrid beads is demonstrated in the inserted picture in Scheme 1. It can be observed that the fluorescence light disperses homogeneously in the entire bead, revealing no apparent aggregation of enzyme molecules. 3.2. Performances of Encapsulated YADH in Boehmite/Alginate Hybrid Beads. A series of performances of encapsulated YADH in the boehmite/alginate hybrid beads are conducted, such as encapsulation efficiency, leakage rate, optimal temperature and pH, and stability. The encapsulation efficiency of YADH in the boehmite/alginate hybrid beads is calculated to be 99.4% according to eq 2, i.e. all enzyme molecules are almost encapsulated in the hybrid beads. This high encapsulation efficiency can be attributed to the strong electrostatic interaction between YADH and boehmite nanoneedles and the formation of the boehmite/alginate hybrid. The encapsulated amount and specific activity of YADH in the boehmite/alginate hybrid beads are 560 U·g of hybrid beads−1 and 29.9 U·mg of protein−1, respectively. Compare to that of free YADH (445 U·mg of protein−1), and YADH encapsulated in the boehmite (92 U·mg of protein−1) and alginate (35.6 U· mg of protein−1) beads, the lower specific activity may be caused by the more compact structure as well as the higher substrate diffusion resistance.35−37 Generally, the activity of immobilized enzymes is affected by the bead diameter; therefore, the hybrid beads with different diameters were prepared, and the effect of the bead diameter on the enzyme activity was studied. The calculated specific activities of YADH encapsulated in the boehmite/alginate hybrid beads with average diameters of 2, 2.6, 3.2, and 3.7 mm are 29.9, 27.3, 20.6, and 19.6 U·mg of protein−1 after reacting for 1 h, respectively. With the increase of the bead size, the

Figure 5. DSC thermograms of freeze-dried alginate/boehmite hybrid beads (a) and alginate beads (b).

Figure 6. Swelling ratio of encapsulated YADH in the alginate (b, c, f, h) and boehmite/alginate hybrid beads (a, d, e, g) incubated in the Tris-HCl buffer with different pH values and at 20 or 60 °C. (a, b) pH = 7, 60 °C; (c, d) pH = 6, 20 °C; (e, f) pH = 7, 20 °C; (g, h) pH = 9, 20 °C.

increase at 20 °C, indicating that these beads are stable at the neutral or closely neutral conditions. The alkaline environment can destroy partly the ion or coordination bonds between Ca2+ and alginate and, thus, leading to the alginate network become loose. Since the boehmite possesses the high resistance to temperature and pH conditions, its incorporation can suppress the alginate swelling. A schematic representation that YADH is encapsulated into the boehmite/alginate hybrid beads is shown in Scheme 1. At first, YADH molecules are encapsulated in the boehmite

Scheme 1. Illustration of YADH Encapsulated in Boehmite/Alginate Hybrid Beads

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activity of encapsulated YADH declines gradually, in accordance with the literature’s report.38 This result is mainly caused by the substrate diffusion limitation, i.e. the large bead has the longer substrate diffusion distance, which leads to the activity decreasing of encapsulated YADH. The leakage rate of encapsulated YADH was assessed by measuring the ratio of YADH leaked from the carriers after immersed in the buffer solution for a period of time. Figure 7

Figure 7. The leakage rate of YADH immobilized in boehmite, alginate, and boehmite/alginate particles.

shows the leakage rate of YADH encapsulated in the boehmite, alginate, and boehmite/alginate particles. It can be observed that the leakage rate of YADH encapsulated in the boehmite/ alginate hybrid beads is almost zero after 67 h incubation, while encapsulated YADH in the boehmite and alginate particles leaks seriously after 40 h incubation. It is well-known that the alginate gel possesses an incompact three-dimensional network and easily swells in the aqueous solution, resulting in the enzyme leakage.39,40 Boehmite is a kind of hydrophilic material and also swells in the aqueous solution, causing the enzyme leakage.26 The formation of boehmite/alginate hybrids improves the swelling resistance of alginate and boehmite, thus cutting down their leakage drawback. Enzyme, as a kind of sensitive catalyst, demonstrates the maximum activity at specific temperature and pH value and loses most of its activity when the surrounding deviates slightly. Figure 8 illustrates the effect of temperature and pH on the activity of YADH immobilized in the boehmite, alginate, and boehmite/alginate hybrid beads. It can be seen from Figure 8a that the highest relative activity of YADH encapsulated in the alginate and boehmite/alginate hybrid beads is at 40 °C, which is consistent with free YADH. YADH immobilized in the boehmite particles has an optimum temperature at 50 °C, indicating that the boehmite possesses a higher thermal resistance. These three samples have the same optimal pH value of 7.0 (Figure 8b), in accordance with free enzyme. High operational stability is essential for immobilized enzymes in order to achieve the low cost benefit. The stability of the boehmite/alginate hybrid beads, including operational (thermal and pH), storage, and recycling stability, was evaluated through investigating the activities of immobilized YADH under different conditions. At the same time, YADH encapsulated in the boehmite and alginate beads was also investigated as the control. As shown in Figure 9, YADH encapsulated in the boehmite/alginate beads demonstrates a good pH and thermal stability, which is similar to YADH encapsulated in the boehmite beads. This phenomenon can be

Figure 8. The effect of temperature (a) and pH (b) on the specific activity of immobilized YADH in boehmite, alginate, and boehmite/ alginate particles.

Figure 9. The thermal (a) and pH (b) stabilities of immobilized YADH in boehmite, alginate, and boehmite/alginate particles.

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YADH encapsulated in the boehmite/alginate hybrid beads preserves 98.9% of the initial activity after reused for 5 times. It is interesting that the catalytic activities in the sixth and seventh cycles are higher than that in the first cycle, which is caused by the fact that a slightly swelling of hybrid beads promotes the catalytic activity through decreasing mass transfer resistance.41 In addition, further research of the continued recycle reactions demonstrates that 86.6% initial activity retains after 12 cycles. Just as described above, YADH encapsulated in the boehmite/alginate hybrid beads has a higher stability than that encapsulated in the boehmite or alginate beads solely. It is a common phenomenon in the field of the enzyme immobilization that the organic/inorganic hybrid materials exhibit the improved performance as the carrier of enzyme immobilization compared with the pure organic or inorganic materials.27,30 In this kind of hybrid bead, boehmite endows the bead with resistance to the temperature and pH condition, while alginate endows the bead with good gelation ability. In addition, both alginate and boehmite offer the suitable microenvironment for the encapsulated enzyme. Most importantly, boehmite can form the compact hybrid structure with alginate, which can inhibit the leakage of the encapsulated enzyme. Therefore, the stability of the enzyme encapsulated in the boehmite/alginate hybrid beads is greatly enhanced.

ascribed by the fact that the immobilized YADH molecules have the similar microenvironment in these two kinds of matrices. Comparatively, the relative activity of YADH encapsulated in the alginate beads decreases more, indicating that its operational stability is not very good. The storage stability of the boehmite/alginate hybrid beads was evaluated by measuring the remaining activities of immobilized YADH after long-term storage at 4 °C. As demonstrated in Figure 10a, YADH encapsulated in the

4. CONCLUSIONS A novel kind of matrix, the boehmite/alginate hybrid beads, has been fabricated, and their structure has been characterized. YADH was encapsulated in the boehmite/alginate hybrid beads in order to investigate its potential as the enzyme immobilization matrix. The immobilized YADH demonstrates low leakage and high loading and stabilities, which can be assigned to the compact structure of the forming boehmite/ alginate hybrid. Since the boehmite/alginate hybrid beads can successfully solve the high-leakage problem of boehmite particles and well retain their intrinsic merits including high encapsulation efficiency and stability, they may become a novel kind of matrix for the efficient immobilization of a variety of industrial biocatalysts.

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Figure 10. The storage (a) and recycling (b) stabilities of immobilized YADH in boehmite, alginate, and boehmite/alginate particles.

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 22 2350 0086. Fax: +86 22 2350 0086. E-mail: [email protected].

boehmite/alginate hybrid beads keep about 90% relative activity after 30 d, which is only a little lower than YADH encapsulated in the boehmite beads. However, the relative activity of YADH encapsulated in the alginate beads declines sharply to 62.6% under the same conditions. This result indicates that the formation of the boehmite/alginate hybrid hardly affects the good storage stability of the boehmite matrix. The recycling stability is a significant character of immobilized enzymes, which can be used to assess its potential reuse or use in a long-term enzymatic reaction. In this study, the recycling experiments were carried out through monitoring the residual activity of encapsulated YADH after every successive enzymatic reaction, and the results are shown in Figure 10b. The catalytic activity of YADH encapsulated in the alginate beads declines gradually after each cycle, and only 40% of the initial activity remains after seven cycles. The activity of YADH encapsulated in the boehmite beads decreases directly to 62.7% after one cycle and then remains almost unchanged after seven cycles. This poor recycling stability can be ascribed to easy dispersion of boehmite aggregates. Comparatively,

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by National Science Fund for Distinguished Young Scholars (21125627), the National Basic Research Program of China (2009CB724705), and the Program of Introducing Talents of Discipline to Universities (No. B06006).

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REFERENCES

(1) Zhou, Z.; Hartmann, M. Progress in enzyme immobilization in ordered mesoporous materials and related applications. Chem. Soc. Rev. 2013, 42, 3894−3912. (2) Tran, D. N.; Balkus, K. J. Perspective of recent progress in immobilization of enzymes. ACS Catal. 2011, 1, 956−968. (3) Brady, D.; Jordaan, J. Advances in enzyme immobilisation. Biotechnol. Lett. 2009, 31, 1639−1650.

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preparation of antifouling mixed matrix membranes. J. Membr. Sci. 2012, 401−402, 132−402. (25) Song, K. H.; Kim, H. Y.; Kim, B. M.; Lee, K. R. Boehmite solcoated membrane preparation for the separation of aqueous alcohol. Desalination 2008, 234, 244−251. (26) Zhu, Y. Y.; Jiang, Z. Y.; Zhang, L.; Shi, J. F.; Yang, D. Sol-gel derived boehmite as an efficient and robust carrier for enzyme encapsulation. Ind. Eng. Chem. Res. 2012, 51, 255−261. (27) Taqieddin, E.; Amiji, M. Enzyme immobilization in novel alginate-chitosan core-shell microcapsules. Biomaterials. 2004, 25, 1937−1945. (28) Kurayama, F.; Suzuki, S.; Bahadur, N. M.; Furusawa, T.; Ota, H.; Sato, M.; Suzuki, N. Preparation of aminosilane-alginate hybrid microcapsules and their use for enzyme encapsulation. J. Mater. Chem. 2012, 22, 15405−15411. (29) Hong, W.; Huang, S. F.; Jiang, Z. Y. Effects of modification of silica gel and ADH on enzyme activity for enzymatic conversion of CO2 to methanol. Catal. Today 2004, 98, 545−552. (30) Xu, S. W.; Lu, Y.; Jiang, Z. Y.; Wu, H. Silica nanotubes-doped alginate gel for yeast alcohol dehydrogenase immobilization. J. Mol. Catal. B: Enzym. 2006, 43, 68−73. (31) Yang, Y.; Lan, J.; Li, X. C. Study on bulk aluminum matrix nanocomposite fabricated by ultrasonic dispersion of nano-sized SiC particles in molten aluminum alloy. Mater. Sci. Eng. A 2004, 380, 378− 383. (32) Wang, C. C.; Yan, Y. J.; Zhang, L. W.; Cui, M. Y.; Xie, G. L.; Cao, B. S. Maxwell-Wagner relaxation in CaCu3Ti4O12/Ag composites. Scr. Mater. 2006, 54, 1501−1504. (33) Broderick, E.; Lyons, H.; Pembroke, T.; Byrne, H.; Murray, B.; Hall, M. The characterisation of a novel, covalently modified, amphiphilic alginate derivative, which retains gelling and non-toxic properties. J. Colloid Interface Sci. 2006, 298, 154−161. (34) Sarmento, B.; Ferreira, D.; Veiga, F.; Ribeiro, A. Characterization of insulin-loaded alginate nanoparticles produced by ionotropic pre-gelation through DSC and FTIR studies. Carbohydr. Polym. 2006, 66, 1−7. (35) Elnashar, M. M. M.; Danial, E. N.; Awad, G. E. A. Novel carrier of grafted alginate for covalent immobilization of inulinase. Ind. Eng. Chem. Res. 2009, 48, 9781−9785. (36) Wang, J. Y.; Yu, H. R.; Xie, R.; Ju, X. J.; Yu, Y. L.; Chu, L. Y.; Zhang, Z. Alginate/protamine/silica hybrid capsules with ultrathin membranes for laccase immobilization. AIChE J. 2013, 59, 380−389. (37) Baeumler, H.; Georgieva, R. Coupled enzyme reactions in multicompartment microparticles. Biomacromolecules 2010, 11, 1480− 1487. (38) Kurayama, F.; Suzuki, S.; Bahadur, N. M.; Furusawa, T.; Ota, H.; Sato, M.; Suzuki, N. Preparation of aminosilane-alginate hybrid microcapsules and their use for enzyme encapsulation. J. Mater. Chem. 2012, 22, 15405−15411. (39) Blandino, A.; Macias, M.; Cantero, D. Immobilization of glucose oxidase within calcium alginate gel capsules. Process Biochem. 2001, 36, 601−606. (40) Blandino, A.; Macias, M.; Cantero, D. Glucose oxidase release from calcium alginate gel capsules. Enzyme Microb. Technol. 2000, 27, 319−324. (41) Shen, Q. Y.; Yang, R. J.; Hua, X.; Ye, F. Y.; Zhang, W. B.; Zhao, W. Gelatin-templated biomimetic calcification for beta-galactosidase immobilization. Process Biochem. (Oxford, U. K.) 2011, 46, 1565−1571.

(4) Sheldon, R. A. Enzyme immobilization: The quest for optimum performance. Adv. Synth. Catal. 2007, 349, 1289−1307. (5) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 2007, 40, 1451−1463. (6) Garcia-Galan, C.; Berenguer-Murcia, A.; Fernandez-Lafuente, R.; Rodrigues, R. C. Potential of different enzyme immobilization strategies to improve enzyme performance. Adv. Synth. Catal. 2011, 353, 2885−2904. (7) Lei, C. H.; Shin, Y. S.; Liu, J.; Ackerman, E. J. Entrapping enzyme in a functionalized nanoporous support. J. Am. Chem. Soc. 2002, 124, 11242−11243. (8) Kim, J.; Grate, J. W.; Wang, P. Nanostructures for enzyme stabilization. Chem. Eng. Sci. 2006, 61, 1017−1026. (9) Mayoral, A.; Arenal, R.; Gascon, V.; Marquez-Alvarez, C.; Blanco, R. M.; Diaz, I. Designing functionalized mesoporous materials for enzyme immobilization: Locating enzymes by using advanced TEM techniques. ChemCatChem 2013, 5, 903−909. (10) Andrade, G.; Barbosa-Stancioli, E. F.; Mansur, A. A. P.; Vasconcelos, W. L.; Mansur, H. S. Design of novel hybrid organicinorganic nanostructured biomaterials for immunoassay applications. Biomed. Mater. 2006, 1, 221−234. (11) Hwang, E. T.; Lee, H.; Kim, J. H.; Tatavarty, R.; Gu, M. B. Highly-stable magnetically-separable organic-inorganic hybrid microspheres for enzyme entrapment. J. Mater. Chem. 2011, 21, 6491−6493. (12) Sanchez, C.; Julian, B.; Belleville, P.; Popall, M. Applications of hybrid organic-inorganic nanocomposites. J. Mater. Chem. 2005, 15, 3559−3592. (13) Sanchez, C.; Belleville, P.; Popall, M.; Nicole, L. Applications of advanced hybrid organic-inorganic nanomaterials: from laboratory to market. Chem. Soc. Rev. 2011, 40, 696−753. (14) Sanchez, C.; Rozes, L.; Ribot, F.; Laberty-Robert, C.; Grosso, D.; Sassoye, C.; Boissiere, C.; Nicole, L. ″Chimie douce″: A land of opportunities for the designed construction of functional inorganic and hybrid organic-inorganic nanomaterials. C. R. Chim. 2010, 13, 3−39. (15) Macario, A.; Moliner, M.; Corma, A.; Giordano, G. Increasing stability and productivity of lipase enzyme by encapsulation in a porous organic-inorganic system. Microporous Mesoporous Mater. 2009, 118, 334−340. (16) Avnir, D.; Coradin, T.; Lev, O.; Livage, J. Recent bioapplications of sol-gel materials. J. Mater. Chem. 2006, 16, 1013−1030. (17) Hoffmann, F.; Froeba, M. Vitalising porous inorganic silica networks with organic functions-PMOs and related hybrid materials. Chem. Soc. Rev. 2011, 40, 608−620. (18) Li, J.; Wu, H.; Liang, Y. P.; Jiang, Z. Y.; Jiang, Y. J.; Zhang, L. Facile fabrication of organic-inorganic hybrid beads by aminated alginate enabled gelation and biomimetic mineralization. J. Biomater. Sci., Polym. Ed. 2013, 24, 119−134. (19) Allouche, J.; Boissiere, M.; Helary, C.; Livage, J.; Coradin, T. Biomimetic core-shell gelatine/silica nanoparticles: a new example of biopolymer-based nanocomposites. J. Mater. Chem. 2006, 16, 3120− 3125. (20) Zhang, L.; Jiang, Y. J.; Shi, J. F.; Sun, X. H.; Li, J.; Jiang, Z. Y. Biomimetic polymer-inorganic hybrid microcapsules for yeast alcohol dehydrogenase encapsulation. React. Funct. Polym. 2008, 68, 1507− 1515. (21) Shao, J.; Huang, L. L.; Yang, Y. M. Immobilization of polyphenol oxidase on alginate-SiO2 hybrid gel: stability and preliminary applications in the removal of aqueous phenol. J. Chem. Technol. Biotechnol. 2009, 84, 633−635. (22) Nédez, C.; Boitiaux, J. P.; Cameron, C. J.; Didillon, B. Optimization of the textural characteristics of an alumina to capture contaminants in natural gas. Langmuir 1996, 12, 3927−3931. (23) Liu, H.; Deng, J.; Li, W. Synthesis of nickel nanoparticles supported on boehmite for selective hydrogenation of p-nitrophenol and p-chloronitrobenzene. Catal. Lett. 2010, 137, 261−266. (24) Vatanpour, V.; Madaeni, S. S.; Rajabi, L.; Zinadini, S.; Derakhshan, A. A. Boehmite nanoparticles as a new nanofiller for 14905

dx.doi.org/10.1021/ie4021649 | Ind. Eng. Chem. Res. 2013, 52, 14898−14905