Immobilization of Proteins into Microcapsules and Their Adsorption

Department of Chemical Science and Engineering, Miyakonojo National College of Technology,. 473-1 Yoshio-cho, Miyakonojo, Miyazaki 885-8567, Japan...
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Ind. Eng. Chem. Res. 2008, 47, 1527-1532

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Immobilization of Proteins into Microcapsules and Their Adsorption Properties with Respect to Precious-Metal Ions Shiro Kiyoyama* Department of Chemical Science and Engineering, Miyakonojo National College of Technology, 473-1 Yoshio-cho, Miyakonojo, Miyazaki 885-8567, Japan

Tatsuo Maruyama, Noriho Kamiya, and Masahiro Goto Department of Applied Chemistry, Graduate School of Engineering, Kyushu UniVersity, 744 Motooka, Fukuoka 819-0395, Japan

We report on protein immobilization in a microcapsule and its adsorption performance with respect to preciousmetal ions. Proteins including lysozyme, bovine serum albumin, chicken egg albumin, and soybean protein were immobilized by a microencapsulation method, and their adsorption properties with respect to ions of both precious metals (gold, platinum, and palladium) and base metals (zinc and copper) were investigated under various preparation conditions. Immobilized proteins could selectively adsorb precious-metal ions over base-metal ions. High immobilization efficiencies were obtained using lysozyme and soybean protein. The immobilization efficiency of the proteins was controlled by the surfactant concentration in the organic phase, the protein concentration, and the outer-aqueous-phase composition. We succeeded in immobilizing over 90% of the protein in the protein dose by optimizing the preparation conditions. Furthermore, the preciousmetal ions adsorbed by the immobilized proteins were completely recovered with thiourea. No leakage of proteins was observed after the adsorption and desorption experiments. These results indicate that the microcapsules immobilizing proteins prepared in the present study can be used as reusable precious-metalselective absorbents. Introduction Precious metals such as gold, platinum, and palladium are indispensable materials in the high-technology industry. In addition to the high demand for precious metals in a variety of fields, the supply of precious metals is not always stable, and this sometimes leads to price increases. To obtain a stable supply of precious metals and minimize their waste, an efficient technique for recovering precious metals needs to be developed. This is also important from the viewpoint of recycling of resources and environmental conservation. A solvent extraction method has been used for the recovery of precious metals. This method has a high selectivity and is easy to scale up, but it requires large amounts of organic solvents. For these reasons, solvent extraction processes are generally used for large-scale industrial operations. For a complex target, ion exchange is considered to be better than solvent extraction. In ion-exchange processes, an adsorbent plays an important role, and selection of the adsorbent often determines the success of the separation process. To date, a variety of adsorbents have been developed, and recently, immobilization1-4 and microencapsulation of an extractant have become candidate methods for creating novel adsorbents.5-8 Here, we focus on a protein as a separation tool, because proteins contain amino acid residues with a strong affinity for precious-metal ions. By using biological molecules as recovery tools for precious metals, considerable benefits such as reduction of environmental burdens, specificity that derives from the biological molecule’s structure, and cost reduction through use of biomass are expected. The recovery of precious metals using biological materials has been investigated.9-13 However, the biological materials used in these studies were * To whom correspondence should be addressed. Tel./Fax: +81(0)986-47-1224. E-mail: [email protected].

highly specialized materials, and the study on the selective recovery of precious metals was not sufficient. In a previous study, a protein was found to show a high affinity for precious metals.14 Thus, immobilization of proteins in a solid material could result in the construction of a novel absorbent for precious metals. In the present study, we attempted to immobilize various proteins in microporous microcapsules and investigated the effects of the preparation conditions on the immobilization and adsorption properties of various metal ions. Experimental Section Chemicals. The proteins used in this study were lysozyme from chicken egg white (hereafter called lysozyme; Sigma Chemical Co.), bovine serum albumin (BSA; Wako Pure Chemical), albumin from chicken egg (hereafter called ova; Wako Pure Chemical), and soybean protein (Fuji Oil Co., Ltd.). The precious metals used were gold, platinum, and palladium. The base metals used were zinc and copper. Each metal ion solution was prepared from a standard solution (1000 ppm for each metal ion) for atomic absorption analysis. An organic phase consisting of trimethylolpropane trimethacrylate (Trim; Wako Pure Chemical) as the frame material, hexaglycerin recinoleic acid (Sunsoft 818SX, hereafter called 818SX; Taiyo Kagaku) as the surfactant, and 2,2-azobis(4methoxy-2,4-dimethylvaleronitrile) (ADVN; Wako Pure Chemical) as the polymerization initiator was used. The outer aqueous phase consisted of distilled water, polyvinyl alcohol (PVA; Wako Pure Chemical) as a dispersion stabilizer, and decaglycerin monolauric acid (Sunsoft Q12S, hereafter called Q12S; Taiyo Kagaku) as the surfactant. According to need, 0.1 M hydrochloric acid solution (pH 1.1), 10 mM phosphate buffer (pH 2.5), 100 mM acetate buffer (pH 4.6), 100 mM boric acid

10.1021/ie0712636 CCC: $40.75 © 2008 American Chemical Society Published on Web 01/25/2008

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Figure 1. SEM images of various microcapsule-immobilized proteins: (a) lysozyme, (b) BSA, (c) ova, (d) soybean protein, (e) cross section of immobilized BSA, (f) cross section of immobilized soybean protein.

buffer (pH 9.6), or 0.1 M sodium hydroxide solution (pH 14.3) was used as the outer aqueous phase. Immobilization of Protein Using Microencapsulation Method. A prescribed amount of protein was dispersed in the organic phase using a vortex mixer to obtain solid-in-oil (S/O) emulsions. The prepared S/O emulsions were added to the outer aqueous phase, and then the mixture was heated at 323 K with stirring at a prescribed speed for 2 h. The immobilized proteins were collected by filtration, washed with distilled water, and finally freeze-dried. The immobilization efficiency of the proteins was estimated by measuring the loss of proteins to the outer aqueous phase by the BCA protein assay method. Adsorption Experiments. A sample of immobilized protein was immersed in aqueous HCl solution containing metal ions. After the solution had been shaken for 1 h, the immobilized protein was separated from the aqueous solution by filtration. The concentration of residual metal ions in the aqueous solution was determined by atomic absorption spectrophotometry (Shimadzu AA-6700). The adsorption ratio of metals ions was then calculated as the ratio of the concentrations before and after adsorption. Desorption Experiments. A sample of immobilized protein that had achieved adsorption equilibrium was immersed in a thiourea solution. After the solution had been shaken for 1 h, the immobilized protein was separated from the thiourea solution by filtration. The concentration of metal ions in the thiourea solution was measured by atomic absorption spectrophotometry. The desorption ratio of metal ions was then calculated as the ratio of the amount of desorbed metal ions to the amount of initially adsorbed metal ions.

Figure 2. Immobilization efficiency of each protein.

Results and Discussion Characteristics of Immobilized Protein Prepared by the Microcapsulation Method. SEM images of the microcapsuleimmobilized proteins are shown in Figure 1. Regardless of the kind of protein, the shape of the microcapsules was spherical, and there was considerable roughness on the surface of the microcapsules. Furthermore, large spherical hollows formed inside the microcapsules. The average diameter of the microcapsule-immobilized proteins was around 100 µm, and the standard deviation was ca. 30 µm. Immobilization Properties of Proteins and Their Adsorption Properties with Respect to Precious- and Base-Metal Ions. The immobilization efficiencies of the lysozyme, BSA, ova, and soybean proteins are summarized in Figure 2, where E represents the immobilization efficiency of protein per protein dosage. Each protein concentration was fixed at 2.5 wt %, and

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Figure 3. Adsorption properties of the different metal ions on the immobilized proteins. Figure 5. Effects of surfactant concentration in the organic phase on the immobilization efficiency of soybean protein and the adsorption properties of gold ions.

Figure 4. Adsorption properties of metal ions on the immobilized proteins calculated by taking account of immobilization efficiency and nonspecific adsorption.

the surfactant concentration was fixed at 5.0 wt % for the experiments reported in Figure 2. The pH in the outer aqueous phase was adjusted to be equal to the isoelectric point of each protein using a buffer solution. Of the proteins tested, lysozyme and soybean protein showed high immobilization yields, but hardly any BSA or ova was encapsulated. Figure 3 shows the adsorption properties of these immobilized proteins with respect to precious- and base-metal ions. Here, “MC only” means the microcapsule itself without proteins prepared at various outer-aqueous-phase pHs. Each immobilized protein selectively adsorbed precious-metal ions. In particular, microcapsule-immobilized lysozyme and soybean protein showed high adsorption levels. It is thought that the differences in adsorption properties among the proteins are caused by the immobilized quantity of protein. Unexpectedly, adsorption of precious-metal ions occurred on the microcapsules without proteins, whereas base-metal ions were hardly adsorbed. It was deduced that the precious-metal ions were adsorbed to the hydroxyl group of the surfactant.15-17 Here, adsorption of metal ions by protein only was calculated by taking account of immobilization efficiency and nonspecific adsorption; the results are presented in Figure 4. All of the proteins adsorbed preciousmetal ions selectively. According to these results, the immobilized proteins should be useful for the recovery of preciousmetal ions. Of the proteins studied, soybean protein can be obtained as biomass from the production of bean oil, and its price is very low compared to those of the other proteins. Therefore, we employed soybean protein as the immobilized

absorbent and focused on gold ions as the precious-metal ion in subsequent experiments. Effects of Surfactant Concentration in the Organic Phase on the Immobilization Properties of Soybean Protein and the Adsorption Properties of Gold Ions. Microcapsuleimmobilized soybean protein was prepared using various surfactant concentrations in the organic phase. Figure 5 shows the effects of the surfactant concentration in the organic phase on the immobilization properties of soybean protein and the adsorption behavior of gold ions. Here, LAds represents the leakage of soybean protein after the adsorption experiments. In the absence of the surfactant 818SX, no soybean protein was immobilized. With addition of the surfactant, soybean protein was immobilized in the microcapsules, but the immobilization efficiency was constant at 60%. We previously reported that the emulsion was destabilized by heating before the formation of the microcapsule membrane and that the leakage of core material occurred during the early stage of microcapsulation.18 From this result, soybean protein that was not immobilized in the microcapsules leaked out into the aqueous phase before the formation of the microcapsule membrane. On the other hand, the adsorption ratio increased with increasing surfactant concentration, despite the constant immobilization efficiency. The effect of the surfactant concentration on gold adsorption is illustrated in Figure 6. In the case of a low surfactant concentration in the organic phase, soybean protein was immobilized in an aggregated fashion, which resulted in low dispersion in the microcapsules. Along with the adsorption of gold ions, formation of a gold-soybean protein complex occurred near the surface of the microcapsules. The goldsoybean protein complex inhibited further adsorption of gold ions by the soybean protein present in the center of the microcapsules. On the other hand, in the case of a high surfactant concentration, smaller portions of soybean protein were immobilized, and the protein was highly dispersed in the microcapsules. Thus, if the gold-soybean protein complex formed, the soybean protein in the center of the microcapsule was used effectively for the adsorption of gold ion. In addition, no leakage of soybean protein was observed after the adsorption experiment had been performed. In industrial applications, leakage of absorbent would be a serious problem.19 The immobilized protein prepared in this study can completely solve this leakage problem. Effects of Soybean Protein Concentration on the Immobilization Properties of Soybean Protein and the Adsorp-

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Figure 6. Schematic illustration of gold-ion adsorption at various surfactant concentrations. Table 1. Composition of the Outer Aqueous Phase

Figure 7. Effects of soybean protein concentration on the immobilization efficiency of soybean protein and the adsorption properties of gold ions.

tion Properties of Gold Ions. To increase the amount of immobilized soybean protein, the effect of the soybean protein concentration was investigated (Figure 7). Here, the ratio of the soybean protein concentration to the 818SX concentration was fixed at 1:1, and E′ represents the amount of immobilized protein per unit weight of microcapsules. The immobilization efficiency of the soybean protein, E, decreased dramatically with increasing soybean protein concentration. The decrease in E appears to have been caused by the decrease in the frame material. In contrast, the adsorption ratio increased with increasing soybean protein concentration, even though the amount of immobilized protein per unit weight of microcapsules was constant. This was caused by the increase in the 818SX concentration and the high dispersion of the soybean protein. In addition, no leakage of protein was observed during the adsorption operations in these experiments. However, when the

sample

composition

a b c d e f g

CPVA ) 2 wt %, CQ12 ) 1 wt % (pH 5.5) CPVA)2 wt % (pH 5.5) 0.1 M HCl (pH 1.1) 100 mM phosphate buffer (pH 2.5) 100 mM acetate buffer (pH 4.6) 100 mM borate buffer (pH 9.6) 0.1 M NaOH (pH 14.1)

818SX concentration was 10 wt %, it was difficult to collect the immobilized protein, and loss of soybean protein was clearly observed. Effects of Outer-Aqueous-Phase Composition on the Immobilization Properties of Soybean Protein and the Adsorption Properties of Gold Ions. To improve the immobilization efficiency of soybean protein, we immobilized the protein with different outer-aqueous-phase compositions as reported in Table 1. The effects of the outer-aqueous-phase composition on the immobilization yield of soybean protein and the adsorption properties of gold ions are shown in Figure 8. The immobilization efficiency of soybean protein, E, was high when the outer-aqueous-phase composition labeled e was used. As the pH in the outer aqueous phase was equal to the isoelectric point of soybean protein, soybean protein was not dissolved in the outer aqueous phase. Thus, the soybean protein precipitated when it came into contact with the outer aqueous phase, and insolubilized soybean protein was immobilized in the microcapsule. According to these results, soybean protein was immobilized completely in the microcapsules. Desorption of Gold Ions Using Thiourea. The desorption of gold ions from immobilized soybean protein using different concentrations of thiourea solution as a stripping agent was investigated. The effect of the thiourea concentration on the desorption of gold ions from immobilized soybean protein is shown in Figure 9. The desorption ratio of gold ions from the immobilized soybean protein was constant at 60% regardless of the thiourea concentration. On the other hand, desorption of gold ions from microcapsules without soybean protein did not occur. The desorption ratio (60%) of gold ions means that the total quantity of gold ions absorbed by the soybean protein could be recovered. Thus, gold ions adsorbed by the immobilized

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(3) A concentration of less than 5% is desirable for soybean protein immobilized in microcapsules. (4) The immobilization efficiency of the proteins can be controlled by the pH in the outer aqueous phase. (5) The precious-metal ions adsorbed by the immobilized proteins can be completely recovered with thiourea. (6) No leakage of soybean protein was observed after the adsorption and desorption operations had been performed.

Literature Cited (1) Trochimczuk, A. W.; Kabay, N.; Arda, M. M.; Streat, M. Stabilization of Solvent Impregnated Resins (SIRs) by Coating with Water Soluble Polymers and Chemical Crosslinking. React. Funct. Polym. 2004, 59, 1-7. (2) Kabay, N.; Arda, M.; Trochimczuk, A.; Streat, M. Removal of Chromate by Solvent Impregnated Resins (SIRs) Stabilized by Coating and Chemical Crosslinking. I. Batch-Mode Sorption Studies. React. Funct. Polym. 2004, 59, 9-14. (3) Kabay, N.; Arda, M.; Trochimczuk, A.; Streat, M. Removal of Chromate by Solvent Impregnated Resins (SIRs) Stabilized by Coating and Chemical Crosslinking. II. Column-Mode Sorption/Elution Studies. React. Funct. Polym. 2004, 59, 15-22.

Figure 8. Effects of outer-aqueous-phase composition on the immobilization efficiency of soybean protein and the adsorption properties of gold ions.

(4) Kabay, N.; Solak, O.; Arda, M.; Topal, U.; Yu¨ksel, M.; Trochimczuk, A.; Streat, M. Packed Column Study of the Sorption of Hexavalent Chromium by Novel Solvent Impregnated Resins Containing Aliquat 336: Effect of Chloride and Sulfate Ions. React. Funct. Polym. 2005, 64, 7582. (5) Kamio, E.; Matsumoto, M.; Kondo, K. Extraction Mechanism of Rare Metals with Microcapsules Containing Organophosphorus Compounds. J. Chem. Eng. Jpn. 2002, 35, 178-185. (6) Shiomori, K.; Yoshizawa, H.; Fujikubo, K.; Kawano, Y.; Hatate, Y.; Kitamura, Y. Extraction Equilibrium of Precious Metals from Aqueous Acidic Solutions Using Divinylbenzene Homopolymeric Microcapsules Encapsulating Ternary Amine as a Core Material. Sep. Sci. Technol. 2003, 38, 4057-4077. (7) Shiomori, K.; Fujikubo, K.; Kawano, Y.; Hatate, Y.; Kitamura, Y.; Yoshizawa, H. Extraction and Separation of Precious Metals by a Column Packed with Divinylbenzene Homopolymeric Microcapsule Containing Trin-octylamine. Sep. Sci. Technol. 2004, 39, 1645-1662. (8) Yoshizawa, H.; Fujikubo, K.; Uemura, Y.; Kawano, Y.; Kondo, K.; Hatate, Y. Preparation of Divinylbenzene Homopolymeric Microcapsules with Highly Porous Membranes by in Situ Polymerization with Solvent Evaporation. J. Chem. Eng. Jpn. 1995, 28, 78-84.

Figure 9. Effect of thiourea concentration on the desorption of gold ions from immobilized soybean protein.

soybean protein could be recovered completely. In addition, no leakage of soybean protein was observed after the desorption operation. These results indicate that the immobilized protein prepared in this study can be reused as a precious-metal-selective adsorbent without leakage of absorbent. Conclusions Various proteins including lysozyme, BSA, ova, and soybean protein were immobilized by a microencapsulation method. The effects of the preparation conditions on the immobilization efficiency of protein, the adsorption properties of precious- and base-metal ions, and the desorption properties of gold ions were investigated. The following results were obtained: (1) Lysozyme and soybean protein can be effectively immobilized in microcapsules, and the immobilized protein selectively adsorbs precious-metal ions over base-metal ions. (2) In the presence of surfactant, soybean protein can be immobilized in the microcapsules.

(9) Antunes, A. P. M.; Watkins, G. M.; Duncan, J. R. Batch Studies on the Removal of Gold(III) from Aqueous Solution by Azolla filiculoides. Biotechnol. Lett. 2001, 23, 249-251. (10) Yong, P.; Rowson, N. A.; Farr, J. P. G.; Harris, I. R.; Macaskie, L. E. A Novel Electrobiotechnology for the Recovery of Precious Metals from Spent Automotive Catalysts. EnViron. Technol. 2003, 24, 289-298. (11) Yong, P.; Farr, J. P. G.; Harris, I. R.; Macaskie, L. E. Palladium Recovery by Immobilized Cells of DesulfoVibrio desulfuricans Using Hydrogen as the Electron Donor in a Novel Electrobioreactor. Biotechnol. Lett. 2002, 24, 205-212. (12) Yong, P.; Rowson, N. A.; Farr, J. P. G.; Harris, I. R.; Macaskie, L. E. Bioaccumulation of Palladium by DesulfoVibrio desulfuricans. J. Chem. Technol. Biotechnol. 2002, 77, 593-601. (13) Vargas, I. D.; Macaskie, L. E.; Guibal, E. Biosorption of Palladium and Platinum by Sulfate-Reducing Bacteria. J. Chem. Technol. Biotechnol. 2004, 79, 49-56. (14) Maruyama, T.; Sonokawa, S.; Kamiya, N.; Goto, M. Proteins and protein-rich biomass as environmental-friendly adsorbents selective for precious metal ions. EnViron. Sci. Technol. 2007, 41, 1359-1364. (15) He, J.; Ichinose, I.; Fujikawa, S.; Kunitake, T. Synthesis of Metal and Metal Oxide Nanoparticles in Nanospace of Ultrathin TiO2 Gel Films: Role of the Ion-exchange Site. Int. J. Nano Sci. 2003, 1, 507-513. (16) He, J.; Fujiwara, S.; Kuitake, T.; Nakao, A. Preparation of Porous and Nonporous Silica Nanofilms from Aqueous Sodium Silicate. Chem. Mater. 2003, 15, 3308-3313.

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(17) He, J.; Ichinose, I.; Kunitake, T.; Nakao, A.; Shiraishi, Y.; Toshima, N. Facile Fabrication of Ag-Pd Bimetallic Nanoparticles in Ultrathin TiO2 Gel Films: Nanoparticle Morphology and Catalytic Activity. J. Am. Chem. Soc. 2003, 125, 11034-11040. (18) Kiyoyama, S.; Shiomori, K.; Kawano, Y.; Hatate, Y. Entrapment of Water Soluble Material into Biodegradable Microcapsule Prepared by Solvent Evaporation. Kagaku Kogaku Ronbunshu 1998, 24, 791-796.

(19) Guan, Y.; Fei, Z.; Luo, M.; Jin, T.; Yao, S. Chromatographic Refolding of Recombinant Human Interferon Gamma by an Immobilized Sht GroEL191-345 Column. J. Chromatogr. A 2006, 1107, 192-197.

ReceiVed for reView September 19, 2007 ReVised manuscript receiVed November 27, 2007 Accepted November 30, 2007 IE0712636