Materials Science and Engineering C 33 (2013) 532–536
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis and characterization of amino acid containing Cu(II) chelated nanoparticles for lysozyme adsorption Gözde Baydemir a, Müge Andaç a, Ali Derazshamshir a, Deniz Aktaş Uygun b, Emir Özçalışkan c, Sinan Akgöl c,⁎, Adil Denizli a a b c
Hacettepe University, Department of Chemistry, Ankara, Turkey Adnan Menderes University, Department of Chemistry, Aydın, Turkey Ege University, Department of Biochemistry, İzmir, Turkey
a r t i c l e
i n f o
Article history: Received 9 August 2011 Received in revised form 14 August 2012 Accepted 28 September 2012 Available online 6 October 2012 Keywords: Lysozyme Nanoparticles IMAC Tryptophan
a b s t r a c t Immobilized metal ion affinity chromatography (IMAC) is a useful method for adsorption of proteins that have an affinity for transition metal ions. In this study, poly(hydroxyethyl methacrylate-methacryloyl-L-tryptophan) (PHEMATrp) nanoparticles were prepared by surfactant free emulsion polymerization. Then, Cu(II) ions were chelated on the PHEMATrp nanoparticles to be used in lysozyme adsorption studies in batch system. The maximum lysozyme adsorption capacity of the PHEMATrp nanoparticles was found to be 326.9 mg/g polymer at pH 7.0. The nonspecific lysozyme adsorption onto the PHEMA nanoparticles was negligible. In terms of protein desorption, it was observed that adsorbed lysozyme was readily desorbed in medium containing 1.0 M NaCl. The results showed that the metal-chelated PHEMATrp nanoparticles can be considered as a good adsorbent for lysozyme purification. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lysozyme (EC 3.2.1.17) is considered as a self-defense enzyme, which is produced in the serum, mucus and many organs of vertebrates [1]. Lysozyme is a small protein (14.4 kDa) which is commercially valuable enzyme and has widespread applications such as cell-disrupting reagent, antibacterial agent, and food additives [2,3]. Lysozyme is also an important index in the diagnosis of various diseases [4–6]. Commercial lysozyme can be obtained from chicken egg white in which the content is about 0.34%, using different non-chromatographic and chromatographic techniques of which only a few have been adopted to an industrial scale [7–12]. In the last decade, applications of nanomaterials have received increasingly great attention. Nanomaterials can provide the upper limit in the key factors that determine the efficiency of biocatalysts, including surface area/volume ratios, mass transfer resistance and biomolecule loading capacity [13], thus using nanomaterial could be an alternative method to obtain lysozyme with high purity, in large scale. Immobilized metal ion affinity chromatography (IMAC) is one of the most effective methods for the adsorption of proteins [14,15]. IMAC introduces an interesting approach for selectivity of materials on the basis of their affinities for chelated metal ions. The separation is based on the interaction of a Lewis acid (electron pair donor) with an electron acceptor group on the surface of the protein. Proteins are ⁎ Corresponding author. Tel.: +90 232 3115493; fax: +90 232 343 86 24. E-mail address: [email protected] (S. Akgöl). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.09.027
assumed to interact mainly through the imidazole group of histidine and, to a lesser extent, the indoyl group of tryptophan and the thiol group cysteine [16]. Protein retention on IMAC matrices is affected by a wide range of variables, such as the surrounding chemical environment, the nature of the chelating group and the metal ion specificity [17]. The lysozyme adsorption has been widely studied using different matrices to optimize the catalytic features [18–25]. In this study, N-methacryloyl-(L)-tryptophan (MATrp) was purposed as a metalchelating ligand for use in the IMAC of lysozyme. Poly(hydroxyethyl methacrylate-methacryloyl-L-tryptophan) (PHEMATrp) nanoparticles were obtained by surfactant free emulsion polymerization of MATrp and HEMA. Then, Cu(II) ions were chelated on the PHEMATrp nanoparticles. The Cu(II)-chelated PHEMATrp nanoparticles were used for lysozyme adsorption from aqueous solutions and egg white. The adsorption conditions (i.e., pH, enzyme concentration, ionic strength, temperature) and the reusability of the nanoparticles were also investigated. 2. Materials and methods 2.1. Materials Lysozyme (chicken egg white, EC 3.2.1.17), lyophilized Micrococcus lysodeikticus cells, L-tryptophan methyl ester, methacryloyl chloride, bovine serum albumin and immunoglobulin G were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2-Hydroxyethyl methacrylate
G. Baydemir et al. / Materials Science and Engineering C 33 (2013) 532–536
(HEMA) and ethylene glycol dimethacrylate (EGDMA) were purchased from Fluka AG (Switzerland). Poly(vinyl alcohol) (molecular weight: 100,000, 98% hydrolyzed) was obtained from Aldrich (USA). All other chemicals were guaranteed analytical grade reagents and used without further purification.
533
and stirred magnetically for 2 h. The concentration of Cu(II) ions in the resulting solution was determined with a graphite furnace atomic absorption spectrophotometer (AA800, Perkin-Elmer, Bodenseewerk, Germany). The amount of chelated Cu(II) ions was calculated by using the concentrations of the Cu(II) ions in the initial solution and in the equilibrium.
2.2. Synthesis of N-methacryloyl-(L)-tryptophan (MATrp) 2.5. Lysozyme adsorption from aqueous solutions The following experimental procedure was applied for the synthesis of N-methacryloyl-(L)-tryptophan (MATrp) [26]. L-Tryptophan methyl ester (5.0 g) and NaNO2 (0.2 g) were dissolved in 30 mL of K2CO3 aqueous solution (5%, w/v). This solution was cooled down to 0 °C. Methacryloyl chloride (4.0 mL) was slowly poured into this solution under nitrogen atmosphere and this solution was then stirred magnetically at room temperature for 2 h. At the end of this period, the pH of the solution was adjusted to 7.0 and subsequently the solution was extracted with ethyl acetate. The liquid phase was evaporated in a rotary evaporator. The residue (MATrp) was crystallized from ether and cyclohexane. The 1H NMR and 13C NMR spectra of MATrp were used to indicate the characteristic peaks from the groups in MATrp monomer using Bruker-400 MHz instrument (USA). Peaks in 1 H NMR were observed as CH3 1.80 ppm (s), CH2 3.42–3.36 ppm (q), CH 4.50–4.51 ppm (m), CH2 5.33 ppm (s), CH2 5.64 ppm (s), NH (amid) 7.15 ppm (d), 5H, indole 7.15–7.57 ppm (m), NH (indol) 8.25 ppm (d), OH (acid) 11.1 ppm (s). Peaks in 13C NMR spectrum were observed as CH3, 18.9 ppm; CH, 53.8 ppm; CH2, 65.4 ppm; C (vinyl), 110.7 ppm; CH (indole), 111.8 ppm; CH (benzene ring), 118.6 ppm; CH (benzene ring), 118.8 ppm; CH2 (vinyl), 120.0 ppm, CH (benzene ring), 121.37 ppm; CH (benzene ring), 124.0 ppm; C (indole), 127.6 ppm; C (benzene ring), 136.6 ppm; CH benzene ring, 139.9 ppm; C_O (amide), 167.9 ppm and C_O (acid), 173.9 ppm, respectively. 2.3. Synthesis and characterization of PHEMATrp nanoparticles The PHEMATrp nanoparticles were produced by surfactant free emulsion polymerization [27]. For the synthesis of PHEMATrp nanoparticles, the following experimental procedure was applied: 0.5 g of poly (vinyl alcohol) was dissolved in 45 mL of deionized water and added to the glass sealed polymerization reactor. Then, 0.6 mL of HEMA, 0.3 mL of EGDMA and 80 μL of MATrp comonomer were added into this solution and slowly shaken for 30 s. 0.0198 g of potassium peroxodisulfate (in 45 mL of water) was added in a reactor and conducted at 70 °C for 7 h. Polymerization was verified by observing the white color of the medium. After completion of the polymerization, the reactor content was cooled to room temperature. The PHEMATrp nanoparticles were incubated in ethanol solution (50%) for 1 h until all the unreacted monomers were removed. Then, PHEMATrp nanoparticles were further washed with deionized water. The PHEMA nanoparticles were prepared by using the same formulation without including MATrp. The characteristic functional groups of the PHEMATrp nanoparticles were analyzed by using a Fourier transform infrared (FTIR) spectrometer (Model FTS 7000; Varian Inc., Palo Alto, CA, USA). The average nanoparticle diameter, size distribution, and surface morphology of the nanoparticles were obtained by scanning electron microscopy (SEM) (Model XL-30S FEG, Philips, Eindhoven, The Netherlands). The amount of MATrp incorporation in the PHEMATrp nanoparticles was evaluated by elemental analysis from nitrogen stoichiometry using an elemental analyzer (Model CHNS-932, Leco Corp., St. Joseph, MI, USA). 2.4. Chelation of Cu(II) ions A 1000 ppm atomic absorption standard solution (containing 10% HNO3) was used as a source of Cu(II) ions. The PHEMATrp nanoparticles were mixed with Cu(II) solution (50 ppm, pH 5.0) at room temperature
Adsorption of lysozyme onto the PHEMATrp-Cu(II) nanoparticles was performed in a batch experimental set-up. Adsorption experiments were conducted at 25 °C for 120 min. The effects of pH, lysozyme concentration, salt dependence and temperature on adsorption were investigated. The lysozyme concentration was measured at 280 nm by using a double beam UV/Vis spectrophotometer (Model 1601, Shimadzu, Tokyo, Japan). The amount of adsorbed lysozyme on the PHEMATrp-Cu(II) nanoparticles was determined by measuring the initial and final concentrations of protein. Lysozyme desorption from the nanoparticles was performed with 1.0 M NaCl. In order to determine the reusability of PHEMATrp-Cu(II) nanoparticles, the lysozyme adsorption and desorption cycle was repeated five times using the same group of nanoparticles. 2.6. Lysozyme purification from chicken egg white and activity measurements Purification studies were carried out in a batch experimental set-up. Chicken egg white was separated from fresh eggs. The egg white was filtered through the cheese cloth in order to remove particulate matter (e.g., chalazae) and then it was diluted to 50% (v/v) with phosphate buffer (100 mM, pH 7.0). The diluted egg-white was homogenized in an ice bath and centrifuged at 4 °C, at 5000 rpm for 30 min. Diluted egg-white solution was transferred in a flask including the PHEMATrp nanoparticles. The mixture was stirred in a rotary mixer at room temperature for 2 h. Lysozyme adsorption was periodically checked by taking samples from the adsorption media. Thereafter, the PHEMATrp nanoparticles were removed from the adsorption media by centrifugation at 30,000 rpm and resuspended in washing buffer (acetate buffer, pH 4.0). Adsorbed lysozyme was eluted by using 1 M NaCl. All experiments were performed at least three times and the experimental error was less than 5%. The purity of the eluted fractions was analyzed with SDS-PAGE. Precast gels and electrophoresis reagents were from BioRad Laboratories (USA). Purified lysozyme activity was determined spectrophotometrically. For this purpose, 9.0 mg of M. lysodeikticus cells were suspended in 30 mL of carbonate buffer (0.1 M, pH 9.0). M. lysodeikticus cell suspension (2.9 mL) was mixed with 0.1 mL of appropriately diluted lysozyme solution. The lysis rate of M. lysodeikticus was recorded by the change in absorbance at 450 nm per minute. For the blank run, the same procedure was followed except that the enzyme solution was replaced with the buffer solution. One unit of activity was defined as the decrease in turbidity of 0.001 per minute at 450 nm at pH 9.0 and 25 °C under the specified conditions. 3. Results and discussion 3.1. Characterization of PHEMATrp nanoparticles In the present study, PHEMATrp nanoparticles containing hydrophobic amino acid based comonomer (i.e. tryptophan) were prepared via copolymerization of HEMA with MATrp comonomer. Details of the characterization of PHEMATrp nanoparticles were given in our previous study [27]. The FTIR spectra of both PHEMA and PHEMATrp nanoparticles have a characteristic stretching vibration band of hydrogen bonded alcohol, O\H at around 3440 cm−1. Additionally the characteristic stretching vibration band of carbonyl group 1700 cm−1, in
534
G. Baydemir et al. / Materials Science and Engineering C 33 (2013) 532–536
PHEMATrp nanoparticles was excessively sharp because of the extra carbonyl groups. The FTIR spectrum of PHEMATrp nanoparticles has the characteristic stretching vibration amide I and amide II absorption bands at 1650 and 1550 cm−1, respectively. The surface morphology of the PHEMATrp nanoparticles was investigated by SEM. As seen in Fig. 1, the PHEMATrp nanoparticles have a good spherical shape, and a uniform size and are non-porous. Particle size of PHEMATrp nanoparticles was found to be roughly 350 nm with an intensity of 93%. A major advantage of the non-porous adsorbents is that significant intraparticle diffusion resistance is absent; this is particularly useful for the rapid purification of lipase [27]. The incorporation of MATrp into the PHEMATrp nanoparticles was estimated by elemental analysis. The incorporation of MATrp was found to be 1.36 mmol/g of polymer using nitrogen stoichiometry. This nitrogen amount determined by elemental analysis comes from only the incorporated MATrp groups into the polymeric structure. It should be also noted that HEMA and other polymerization ingredients do not contain nitrogen. Additionally, the chelating capacity of nanoparticles for Cu(II) ions was investigated and the amount of adsorbed Cu(II) on the PHEMATrp nanoparticles was calculated as 1.02 mmol/g polymer. It was observed that there was no Cu(II) leakage from any of the Cu(II)-attached nanoparticles to adsorption–desorption media.
3.2. Adsorption of lysozyme from aqueous solutions 3.2.1. Effect of pH The amount of lysozyme adsorbed onto the PHEMATrp-Cu(II) nanoparticles as a function of pH was shown in Fig. 2. Lysozyme adsorption amount onto the PHEMATrp-Cu(II) nanoparticles shows a maximum value at pH 7.0 (326.9 mg/g polymer). Specific interactions such as electrostatic and coordination, between lysozyme and chelated Cu(II) ions at pH 7.0 may result both from the ionization states of several groups on amino acid side chains in lysozyme structure and from the conformational state of lysozyme molecules at this pH. As seen in Fig. 2, the adsorbed amount of lysozyme decreased at pH values lower and higher than pH 7.0. This could be generated by the ionization state of lysozyme and could be caused by repulsive electrostatic forces between adsorbed lysozyme molecules at the chelated Cu(II) ions via MATrp. Increase in conformational size and the lateral electrostatic repulsions between adjacent adsorbed lysozyme molecules may also cause a decrease in adsorption efficiency. Also, the
Fig. 2. Effect of pH on lysozyme adsorption; lysozyme concentration: 1.5 mg/mL; temperature: 25 °C, incubation time: 2 h.
change of coordination interaction at high and low pH should have a great influence [28]. 3.2.2. Effect of lysozyme concentration The adsorbed amount of lysozyme on the PHEMATrp-Cu(II) nanoparticles was dependent on the equilibrium concentration of lysozyme. The lysozyme adsorption amount increased with the increasing concentration of lysozyme, and a saturation value was reached at a concentration of 1.5 mg/mL, which shows saturation of the active binding sites of PHEMATrp-Cu(II) nanoparticles in Fig. 3. The steep slope of the initial part of adsorption curve represents a high affinity between lysozyme and Cu(II)-chelated groups. It becomes constant when the lysozyme concentration is greater than 1.5 mg/mL. A small amount of lysozyme molecules adsorbed onto the PHEMA nanoparticles which was about 2.21 mg/g polymer at pH 7.0. Cu(II) chelation significantly increased the lysozyme adsorption capacity of the nanoparticles up to 326.9 mg/g polymer at pH 7.0 in phosphate buffer. It is clear that this increase is due to the specific interaction between chelated Cu(II) ions and lysozyme molecules. Additionally, the adsorption study of PHEMATrp-Cu(II) nanoparticles was also implemented with other proteins such as bovine serum albumin (BSA) and immunoglobulin G (IgG). All the adsorption experiments were performed at the same conditions with lysozyme experiments and the adsorption studies indicated that the adsorption amounts of BSA and IgG were significantly low (1.2% which corresponds to 30 mg/g and 0.7% which corresponds to 17.5 mg/g, respectively) for the PHEMATrp-Cu(II) nanoparticles. 3.2.3. Evaluation of adsorption isotherm An adsorption isotherm is used to characterize the interaction of the each protein with the adsorbents. This provides a relationship between the concentration of protein in the solution and the amount of protein adsorbed onto solid phase when the two phases are at equilibrium. The results could be well fitted to either the Langmuir
Fig. 1. SEM micrograph of PHEMATrp-Cu(II) nanospheres.
Fig. 3. Effect of lysozyme concentration on lysozyme adsorption; pH: 7.0; temperature: 25 °C, incubation time: 2 h.
G. Baydemir et al. / Materials Science and Engineering C 33 (2013) 532–536
535
Table 1 Langmuir and Freundlich constants. Experimental Langmuir constants Qex (mg/g) PHEMATrp-Cu(II) 326.8
Freundlich constants
Qmax b R2 (mg/g) (mL/mg) 434.8
1.44
KF n (mg/g)
0.94 233.4
R2
2.04 0.82
(Ceq / q = 1 / qmax.b + Ceq / qmax) or Freundlich (q = Kf.Ce1/n) isotherms. Some model parameters were determined by nonlinear regression with commercially available software and are shown in Table 1. The Langmuir adsorption model can be applied in this affinity adsorbent system. It should also be noted that the maximum adsorption capacities (qmax) and the Langmuir constant were found to be 434.8 mg/g and 1.44 mL/mg for batch system. 3.2.4. Effects of salt concentration and temperature The effect of salt concentration on lysozyme adsorption is shown in Fig. 4. The adsorption capacity decreased with increasing (i.e., NaCl) salt concentration. The adsorption of lysozyme was decreased by about 68% as the NaCl concentration increased from 0 to 0.05 M. The decrease in the adsorption capacity as the NaCl concentration increases can be attributed to the repulsive electrostatic interactions between the PHEMATrp-Cu(II) nanoparticles and lysozyme molecules. Higher lysozyme adsorption capacities at low salt concentrations may be due to nonspecific adsorption and hence, signify the role of amino acid residues other than histidine, cysteine and tryptophan that are usually considered responsible for immobilized metal affinity phenomenon [29]. The effect of temperature on lysozyme adsorption was investigated in the range of 4 °C–45 °C (Fig. 5). The adsorption capacity of PHEMATrp-Cu(II) nanoparticles decreased with increasing temperature. The maximum adsorption capacity was obtained at 4 °C. The adsorption capacity of nanoparticles decreased about 37%, as the temperature increased from 4 °C to 45 °C. A possible explanation for this behavior is the exothermic nature of the adsorption process [30]. 3.3. Elution and repeated use
Fig. 5. Effect of temperature on lysozyme adsorption; lysozyme concentration: 1.5 mg/mL; pH: 7.0; incubation time: 2 h.
in enzyme activity. These results demonstrated the stability of present metal-chelated nanoparticles as an affinity adsorbent. 3.4. Lysozyme purification from chicken egg white and activity measurements Single step lysozyme purification from egg white was studied in a batch mode. The PHEMATrp nanoparticles provided an efficient single step method to purify lysozyme from diluted egg white, showing high adsorption capacity and high selectivity for lysozyme. The purity of the lysozyme desorbed from the metal chelated nanoparticles was determined by SDS-PAGE. The purity of the desorbed lysozyme was about 87% with an overall yield of about 78%, which is similar to 77% yield obtained using Streamline SP followed by dye affinity adsorption [31] and 80% yield using metal-affinity precipitation with Cu 2+ followed by gel filtration chromatography on Sephadex G75 [32]. Denizli et al. prepared lysozyme-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester) particles for the purification of lysozyme from egg-white [25]. They reported 89% purity with recovery of about 84%. Compared to the results in literature, the result by the PHEMATrp nanoparticles are better. The specific activity of the purified lysozyme with the PHEMATrp-Cu(II) nanoparticles was 27,500 U/mg. It should be noted that the specific activity of native lysozyme was 30,000 U/mg. As seen here there was no drastic decrease in specific activity during the purification studies. The PHEMATrp-Cu(II) nanoparticles provided an efficient method to purify lysozyme from diluted egg white, showing high adsorption capacity and high selectivity for lysozyme.
In the affinity separation processes, an advantage of a support material is its reusability. The lysozyme adsorbed onto the PHEMATrp-Cu(II) nanoparticles was desorbed with 1.0 M NaCl solution and desorption ratio was calculated as 90% at the end of 2 h. During the desorption of lysozyme, no leakage of Cu(II) ions was observed from the Cu(II) chelated nanoparticles. The adsorption–desorption cycle was repeated 10 times using the same nanoparticles in order to show the reusability of the nanoparticles (Fig. 6). It was observed that the PHEMATrp-Cu(II) nanoparticles can be used many times without decreasing their adsorption capacities significantly. In addition, at the end of ten adsorption and desorption cycles, there was no remarkable reduction
IMAC is a significant bioseparation technique for biomolecules [32–35]. In addition to its affinity-like selectivity, IMAC exhibits the advantages of a higher recovery yield with the use of mild, non-denaturing elution conditions, compared to biospecific chromatographic methods [13]. In this study, nanoparticles were used
Fig. 4. Effect of ionic strength on lysozyme adsorption; lysozyme concentration: 1.5 mg/mL; pH: 7.0; temperature: 25 °C, incubation time: 2 h.
Fig. 6. Reusability of PHEMATrp-Cu(II) nanospheres; lysozyme concentration: 1.5 mg/mL; pH: 7.0; temperature: 25 °C, incubation time: 2 h.
4. Conclusion
536
G. Baydemir et al. / Materials Science and Engineering C 33 (2013) 532–536
as metal-chelated affinity matrix. Nanoparticles provide an ideal remedy to the conflicting issues usually encountered in the optimization of adsorbed enzymes; minimum diffusional limitation, maximum surface area per unit mass and high enzyme loading [36]. The maximum lysozyme adsorption capacity by using metal chelated nanoparticles was found to be 326.9 mg/g polymer. Adsorbed lysozyme was desorbed up to 90% by using 1.0 M NaCl as the elution agent. The metal-chelated nanoparticles can be used as affinity adsorbents with their high reusability capacity. References [1] T. Jing, H. Du, Q. Dai, H. Xia, J. Niu, Q. Hao, S. Mei, Y. Zhou, Biosens. Bioelectron. 26 (2010) 301–306. [2] D.S. Freitas, J. Abrahano-Neto, Int. J. Pharm. 392 (2010) 111–117. [3] G. Zhang, Q. Cao, N. Li, K. Li, F. Liu, Talanta 83 (2011) 1515–1520. [4] K.A. Near, M.J. Lefford, J. Clin. Microbiol. 30 (1992) 1105–1110. [5] B. Porstmann, K. Jung, H. Scmechta, U. Evers, M. Pergande, T. Porstman, H.-J. Kramn, H. Krause, Clin. Biochem. 22 (1989) 349–355. [6] S. Yu, A. Luo, D. Biswal, J.Z. Hilt, D.A. Puleo, Talanta 83 (2010) 156–161. [7] T.D. Durance, in: J.S. Sim, S. Nakai (Eds.), Egg Uses and Processing Technologies. New DevelopmentsInternational CAB, Wallingford, 1994, pp. 77–93. [8] R. Ghosh, Z.F. Cui, Biotechnol. Bioeng. 68 (2000) 191–203. [9] E. Li-Chan, S. Nakai, J. Sim, D.B. Bragg, K.V. Lo, Food Sci. 51 (1986) 1032–1036. [10] R. Dembczynski, W. Bialas, K. Regulski, T. Jankowski, Process. Biochem. 45 (2010) 369–374. [11] S. Öncel, L. Uzun, B. Garipcan, A. Denizli, Ind. Eng. Chem. Res. 44 (2005) 7049–7056. [12] E.B. Altıntaş, A. Denizli, Int. J. Biol. Macromol. 38 (2006) 99–106. [13] L. Wang, R. Xu, Y. Chen, R. Jiang, J. Mol. Catal. B Enzym. 69 (2011) 120–126.
[14] J. Porath, J. Carlson, I. Olsson, G. Belfrage, Nature 258 (1975) 598–599. [15] S. Akgöl, D. Türkmen, A. Denizli, J. Appl. Polym. Sci. 93 (2004) 2669–2677. [16] A. Denizli, M. Alkan, B. Garipcan, S. Özkara, E. Pişkin, J. Chromatogr. B 795 (2003) 93–103. [17] M.B. Riberio, M. Vijayalakshmi, D. Todorova-Balvay, S.M.A. Bueno, J. Chromatogr. B 861 (2008) 64–73. [18] A. Denizli, S. Şenel, M.Y. Arıca, Colloids Surf. B: Biointerfaces 11 (1998) 113–122. [19] M.-H. Liao, D.-H. Chen, Biotechnol. Lett. 24 (2002) 1913–1917. [20] N. Basar, L. Uzun, A. Guner, A. Denizli, Int. J. Biol. Macromol. 41 (2007) 234–242. [21] E.B. Altıntaş, N. Tuzmen, N. Candan, A. Denizli, J. Chromatogr. B 853 (2007) 105–113. [22] C. Garcia-Diego, J. Cuellar, Chem. Eng. J. 143 (2008) 337–348. [23] D. Shao, K. Xu, X. Song, J. Hu, W. Yang, C. Wang, J. Colloid Interface Sci. 336 (2009) 526–532. [24] S. Akgol, N. Ozturk, A. Denizli, J. Appl. Polym. Sci. 115 (2010) 1608–1615. [25] M. Odabasi, R. Say, A. Denizli, Mater. Sci. Eng. C 27 (2007) 90–99. [26] E.B. Altıntaş, A. Denizli, Mater. Sci. Eng. C 29 (2009) 1627–1634. [27] D.A. Uygun, M.E. Çorman, N. Ozturk, S. Akgol, A. Denizli, Mater. Sci. Eng. C 30 (2010) 1285–1290. [28] H. Yavuz, M. Odabaşı, S. Akgöl, A. Denizli, J. Biomater. Sci. Polym. Ed. 16 (2005) 673–684. [29] S. Sharma, G.P. Agorwal, J. Colloid Interf. Sci. 243 (2001) 61–72. [30] M. Karatas, S. Akgol, H. Yavuz, R. Say, A. Denizli, Int. J. Biol. Macromol. 40 (2007) 254–260. [31] I. Roy, M.V. Rao, M.N. Gupta, Appl. Biochem. Biotechnol. 111 (2003) 55. [32] I. Roy, M.V. Rao, M.N. Gupta, Biotechnol. Appl. Biochem. 37 (2003) 9. [33] E.B. Altintas, H. Yavuz, R. Say, A. Denizli, J. Biomater. Sci. Polym. Ed. 17 (2006) 213–226. [34] D. Türkmen, H. Yavuz, A. Denizli, Int. J. Biol. Macromol. 38 (2006) 126–133. [35] N. Tüzmen, T. Kalburcu, A. Denizli, Process. Biochem. 47 (2012) 26–33. [36] H. Koo, M.S. Huh, J.H. Ryu, D.-E. Lee, I.-C. Sun, K. Choi, K. Kim, I.C. Kwon, Nano Today 6 (2011) 204–220.
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis and characterization of amino acid containing Cu(II) chelated nanoparticles for lysozyme adsorption Gözde Baydemir a, Müge Andaç a, Ali Derazshamshir a, Deniz Aktaş Uygun b, Emir Özçalışkan c, Sinan Akgöl c,⁎, Adil Denizli a a b c
Hacettepe University, Department of Chemistry, Ankara, Turkey Adnan Menderes University, Department of Chemistry, Aydın, Turkey Ege University, Department of Biochemistry, İzmir, Turkey
a r t i c l e
i n f o
Article history: Received 9 August 2011 Received in revised form 14 August 2012 Accepted 28 September 2012 Available online 6 October 2012 Keywords: Lysozyme Nanoparticles IMAC Tryptophan
a b s t r a c t Immobilized metal ion affinity chromatography (IMAC) is a useful method for adsorption of proteins that have an affinity for transition metal ions. In this study, poly(hydroxyethyl methacrylate-methacryloyl-L-tryptophan) (PHEMATrp) nanoparticles were prepared by surfactant free emulsion polymerization. Then, Cu(II) ions were chelated on the PHEMATrp nanoparticles to be used in lysozyme adsorption studies in batch system. The maximum lysozyme adsorption capacity of the PHEMATrp nanoparticles was found to be 326.9 mg/g polymer at pH 7.0. The nonspecific lysozyme adsorption onto the PHEMA nanoparticles was negligible. In terms of protein desorption, it was observed that adsorbed lysozyme was readily desorbed in medium containing 1.0 M NaCl. The results showed that the metal-chelated PHEMATrp nanoparticles can be considered as a good adsorbent for lysozyme purification. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lysozyme (EC 3.2.1.17) is considered as a self-defense enzyme, which is produced in the serum, mucus and many organs of vertebrates [1]. Lysozyme is a small protein (14.4 kDa) which is commercially valuable enzyme and has widespread applications such as cell-disrupting reagent, antibacterial agent, and food additives [2,3]. Lysozyme is also an important index in the diagnosis of various diseases [4–6]. Commercial lysozyme can be obtained from chicken egg white in which the content is about 0.34%, using different non-chromatographic and chromatographic techniques of which only a few have been adopted to an industrial scale [7–12]. In the last decade, applications of nanomaterials have received increasingly great attention. Nanomaterials can provide the upper limit in the key factors that determine the efficiency of biocatalysts, including surface area/volume ratios, mass transfer resistance and biomolecule loading capacity [13], thus using nanomaterial could be an alternative method to obtain lysozyme with high purity, in large scale. Immobilized metal ion affinity chromatography (IMAC) is one of the most effective methods for the adsorption of proteins [14,15]. IMAC introduces an interesting approach for selectivity of materials on the basis of their affinities for chelated metal ions. The separation is based on the interaction of a Lewis acid (electron pair donor) with an electron acceptor group on the surface of the protein. Proteins are ⁎ Corresponding author. Tel.: +90 232 3115493; fax: +90 232 343 86 24. E-mail address: [email protected] (S. Akgöl). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.09.027
assumed to interact mainly through the imidazole group of histidine and, to a lesser extent, the indoyl group of tryptophan and the thiol group cysteine [16]. Protein retention on IMAC matrices is affected by a wide range of variables, such as the surrounding chemical environment, the nature of the chelating group and the metal ion specificity [17]. The lysozyme adsorption has been widely studied using different matrices to optimize the catalytic features [18–25]. In this study, N-methacryloyl-(L)-tryptophan (MATrp) was purposed as a metalchelating ligand for use in the IMAC of lysozyme. Poly(hydroxyethyl methacrylate-methacryloyl-L-tryptophan) (PHEMATrp) nanoparticles were obtained by surfactant free emulsion polymerization of MATrp and HEMA. Then, Cu(II) ions were chelated on the PHEMATrp nanoparticles. The Cu(II)-chelated PHEMATrp nanoparticles were used for lysozyme adsorption from aqueous solutions and egg white. The adsorption conditions (i.e., pH, enzyme concentration, ionic strength, temperature) and the reusability of the nanoparticles were also investigated. 2. Materials and methods 2.1. Materials Lysozyme (chicken egg white, EC 3.2.1.17), lyophilized Micrococcus lysodeikticus cells, L-tryptophan methyl ester, methacryloyl chloride, bovine serum albumin and immunoglobulin G were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2-Hydroxyethyl methacrylate
G. Baydemir et al. / Materials Science and Engineering C 33 (2013) 532–536
(HEMA) and ethylene glycol dimethacrylate (EGDMA) were purchased from Fluka AG (Switzerland). Poly(vinyl alcohol) (molecular weight: 100,000, 98% hydrolyzed) was obtained from Aldrich (USA). All other chemicals were guaranteed analytical grade reagents and used without further purification.
533
and stirred magnetically for 2 h. The concentration of Cu(II) ions in the resulting solution was determined with a graphite furnace atomic absorption spectrophotometer (AA800, Perkin-Elmer, Bodenseewerk, Germany). The amount of chelated Cu(II) ions was calculated by using the concentrations of the Cu(II) ions in the initial solution and in the equilibrium.
2.2. Synthesis of N-methacryloyl-(L)-tryptophan (MATrp) 2.5. Lysozyme adsorption from aqueous solutions The following experimental procedure was applied for the synthesis of N-methacryloyl-(L)-tryptophan (MATrp) [26]. L-Tryptophan methyl ester (5.0 g) and NaNO2 (0.2 g) were dissolved in 30 mL of K2CO3 aqueous solution (5%, w/v). This solution was cooled down to 0 °C. Methacryloyl chloride (4.0 mL) was slowly poured into this solution under nitrogen atmosphere and this solution was then stirred magnetically at room temperature for 2 h. At the end of this period, the pH of the solution was adjusted to 7.0 and subsequently the solution was extracted with ethyl acetate. The liquid phase was evaporated in a rotary evaporator. The residue (MATrp) was crystallized from ether and cyclohexane. The 1H NMR and 13C NMR spectra of MATrp were used to indicate the characteristic peaks from the groups in MATrp monomer using Bruker-400 MHz instrument (USA). Peaks in 1 H NMR were observed as CH3 1.80 ppm (s), CH2 3.42–3.36 ppm (q), CH 4.50–4.51 ppm (m), CH2 5.33 ppm (s), CH2 5.64 ppm (s), NH (amid) 7.15 ppm (d), 5H, indole 7.15–7.57 ppm (m), NH (indol) 8.25 ppm (d), OH (acid) 11.1 ppm (s). Peaks in 13C NMR spectrum were observed as CH3, 18.9 ppm; CH, 53.8 ppm; CH2, 65.4 ppm; C (vinyl), 110.7 ppm; CH (indole), 111.8 ppm; CH (benzene ring), 118.6 ppm; CH (benzene ring), 118.8 ppm; CH2 (vinyl), 120.0 ppm, CH (benzene ring), 121.37 ppm; CH (benzene ring), 124.0 ppm; C (indole), 127.6 ppm; C (benzene ring), 136.6 ppm; CH benzene ring, 139.9 ppm; C_O (amide), 167.9 ppm and C_O (acid), 173.9 ppm, respectively. 2.3. Synthesis and characterization of PHEMATrp nanoparticles The PHEMATrp nanoparticles were produced by surfactant free emulsion polymerization [27]. For the synthesis of PHEMATrp nanoparticles, the following experimental procedure was applied: 0.5 g of poly (vinyl alcohol) was dissolved in 45 mL of deionized water and added to the glass sealed polymerization reactor. Then, 0.6 mL of HEMA, 0.3 mL of EGDMA and 80 μL of MATrp comonomer were added into this solution and slowly shaken for 30 s. 0.0198 g of potassium peroxodisulfate (in 45 mL of water) was added in a reactor and conducted at 70 °C for 7 h. Polymerization was verified by observing the white color of the medium. After completion of the polymerization, the reactor content was cooled to room temperature. The PHEMATrp nanoparticles were incubated in ethanol solution (50%) for 1 h until all the unreacted monomers were removed. Then, PHEMATrp nanoparticles were further washed with deionized water. The PHEMA nanoparticles were prepared by using the same formulation without including MATrp. The characteristic functional groups of the PHEMATrp nanoparticles were analyzed by using a Fourier transform infrared (FTIR) spectrometer (Model FTS 7000; Varian Inc., Palo Alto, CA, USA). The average nanoparticle diameter, size distribution, and surface morphology of the nanoparticles were obtained by scanning electron microscopy (SEM) (Model XL-30S FEG, Philips, Eindhoven, The Netherlands). The amount of MATrp incorporation in the PHEMATrp nanoparticles was evaluated by elemental analysis from nitrogen stoichiometry using an elemental analyzer (Model CHNS-932, Leco Corp., St. Joseph, MI, USA). 2.4. Chelation of Cu(II) ions A 1000 ppm atomic absorption standard solution (containing 10% HNO3) was used as a source of Cu(II) ions. The PHEMATrp nanoparticles were mixed with Cu(II) solution (50 ppm, pH 5.0) at room temperature
Adsorption of lysozyme onto the PHEMATrp-Cu(II) nanoparticles was performed in a batch experimental set-up. Adsorption experiments were conducted at 25 °C for 120 min. The effects of pH, lysozyme concentration, salt dependence and temperature on adsorption were investigated. The lysozyme concentration was measured at 280 nm by using a double beam UV/Vis spectrophotometer (Model 1601, Shimadzu, Tokyo, Japan). The amount of adsorbed lysozyme on the PHEMATrp-Cu(II) nanoparticles was determined by measuring the initial and final concentrations of protein. Lysozyme desorption from the nanoparticles was performed with 1.0 M NaCl. In order to determine the reusability of PHEMATrp-Cu(II) nanoparticles, the lysozyme adsorption and desorption cycle was repeated five times using the same group of nanoparticles. 2.6. Lysozyme purification from chicken egg white and activity measurements Purification studies were carried out in a batch experimental set-up. Chicken egg white was separated from fresh eggs. The egg white was filtered through the cheese cloth in order to remove particulate matter (e.g., chalazae) and then it was diluted to 50% (v/v) with phosphate buffer (100 mM, pH 7.0). The diluted egg-white was homogenized in an ice bath and centrifuged at 4 °C, at 5000 rpm for 30 min. Diluted egg-white solution was transferred in a flask including the PHEMATrp nanoparticles. The mixture was stirred in a rotary mixer at room temperature for 2 h. Lysozyme adsorption was periodically checked by taking samples from the adsorption media. Thereafter, the PHEMATrp nanoparticles were removed from the adsorption media by centrifugation at 30,000 rpm and resuspended in washing buffer (acetate buffer, pH 4.0). Adsorbed lysozyme was eluted by using 1 M NaCl. All experiments were performed at least three times and the experimental error was less than 5%. The purity of the eluted fractions was analyzed with SDS-PAGE. Precast gels and electrophoresis reagents were from BioRad Laboratories (USA). Purified lysozyme activity was determined spectrophotometrically. For this purpose, 9.0 mg of M. lysodeikticus cells were suspended in 30 mL of carbonate buffer (0.1 M, pH 9.0). M. lysodeikticus cell suspension (2.9 mL) was mixed with 0.1 mL of appropriately diluted lysozyme solution. The lysis rate of M. lysodeikticus was recorded by the change in absorbance at 450 nm per minute. For the blank run, the same procedure was followed except that the enzyme solution was replaced with the buffer solution. One unit of activity was defined as the decrease in turbidity of 0.001 per minute at 450 nm at pH 9.0 and 25 °C under the specified conditions. 3. Results and discussion 3.1. Characterization of PHEMATrp nanoparticles In the present study, PHEMATrp nanoparticles containing hydrophobic amino acid based comonomer (i.e. tryptophan) were prepared via copolymerization of HEMA with MATrp comonomer. Details of the characterization of PHEMATrp nanoparticles were given in our previous study [27]. The FTIR spectra of both PHEMA and PHEMATrp nanoparticles have a characteristic stretching vibration band of hydrogen bonded alcohol, O\H at around 3440 cm−1. Additionally the characteristic stretching vibration band of carbonyl group 1700 cm−1, in
534
G. Baydemir et al. / Materials Science and Engineering C 33 (2013) 532–536
PHEMATrp nanoparticles was excessively sharp because of the extra carbonyl groups. The FTIR spectrum of PHEMATrp nanoparticles has the characteristic stretching vibration amide I and amide II absorption bands at 1650 and 1550 cm−1, respectively. The surface morphology of the PHEMATrp nanoparticles was investigated by SEM. As seen in Fig. 1, the PHEMATrp nanoparticles have a good spherical shape, and a uniform size and are non-porous. Particle size of PHEMATrp nanoparticles was found to be roughly 350 nm with an intensity of 93%. A major advantage of the non-porous adsorbents is that significant intraparticle diffusion resistance is absent; this is particularly useful for the rapid purification of lipase [27]. The incorporation of MATrp into the PHEMATrp nanoparticles was estimated by elemental analysis. The incorporation of MATrp was found to be 1.36 mmol/g of polymer using nitrogen stoichiometry. This nitrogen amount determined by elemental analysis comes from only the incorporated MATrp groups into the polymeric structure. It should be also noted that HEMA and other polymerization ingredients do not contain nitrogen. Additionally, the chelating capacity of nanoparticles for Cu(II) ions was investigated and the amount of adsorbed Cu(II) on the PHEMATrp nanoparticles was calculated as 1.02 mmol/g polymer. It was observed that there was no Cu(II) leakage from any of the Cu(II)-attached nanoparticles to adsorption–desorption media.
3.2. Adsorption of lysozyme from aqueous solutions 3.2.1. Effect of pH The amount of lysozyme adsorbed onto the PHEMATrp-Cu(II) nanoparticles as a function of pH was shown in Fig. 2. Lysozyme adsorption amount onto the PHEMATrp-Cu(II) nanoparticles shows a maximum value at pH 7.0 (326.9 mg/g polymer). Specific interactions such as electrostatic and coordination, between lysozyme and chelated Cu(II) ions at pH 7.0 may result both from the ionization states of several groups on amino acid side chains in lysozyme structure and from the conformational state of lysozyme molecules at this pH. As seen in Fig. 2, the adsorbed amount of lysozyme decreased at pH values lower and higher than pH 7.0. This could be generated by the ionization state of lysozyme and could be caused by repulsive electrostatic forces between adsorbed lysozyme molecules at the chelated Cu(II) ions via MATrp. Increase in conformational size and the lateral electrostatic repulsions between adjacent adsorbed lysozyme molecules may also cause a decrease in adsorption efficiency. Also, the
Fig. 2. Effect of pH on lysozyme adsorption; lysozyme concentration: 1.5 mg/mL; temperature: 25 °C, incubation time: 2 h.
change of coordination interaction at high and low pH should have a great influence [28]. 3.2.2. Effect of lysozyme concentration The adsorbed amount of lysozyme on the PHEMATrp-Cu(II) nanoparticles was dependent on the equilibrium concentration of lysozyme. The lysozyme adsorption amount increased with the increasing concentration of lysozyme, and a saturation value was reached at a concentration of 1.5 mg/mL, which shows saturation of the active binding sites of PHEMATrp-Cu(II) nanoparticles in Fig. 3. The steep slope of the initial part of adsorption curve represents a high affinity between lysozyme and Cu(II)-chelated groups. It becomes constant when the lysozyme concentration is greater than 1.5 mg/mL. A small amount of lysozyme molecules adsorbed onto the PHEMA nanoparticles which was about 2.21 mg/g polymer at pH 7.0. Cu(II) chelation significantly increased the lysozyme adsorption capacity of the nanoparticles up to 326.9 mg/g polymer at pH 7.0 in phosphate buffer. It is clear that this increase is due to the specific interaction between chelated Cu(II) ions and lysozyme molecules. Additionally, the adsorption study of PHEMATrp-Cu(II) nanoparticles was also implemented with other proteins such as bovine serum albumin (BSA) and immunoglobulin G (IgG). All the adsorption experiments were performed at the same conditions with lysozyme experiments and the adsorption studies indicated that the adsorption amounts of BSA and IgG were significantly low (1.2% which corresponds to 30 mg/g and 0.7% which corresponds to 17.5 mg/g, respectively) for the PHEMATrp-Cu(II) nanoparticles. 3.2.3. Evaluation of adsorption isotherm An adsorption isotherm is used to characterize the interaction of the each protein with the adsorbents. This provides a relationship between the concentration of protein in the solution and the amount of protein adsorbed onto solid phase when the two phases are at equilibrium. The results could be well fitted to either the Langmuir
Fig. 1. SEM micrograph of PHEMATrp-Cu(II) nanospheres.
Fig. 3. Effect of lysozyme concentration on lysozyme adsorption; pH: 7.0; temperature: 25 °C, incubation time: 2 h.
G. Baydemir et al. / Materials Science and Engineering C 33 (2013) 532–536
535
Table 1 Langmuir and Freundlich constants. Experimental Langmuir constants Qex (mg/g) PHEMATrp-Cu(II) 326.8
Freundlich constants
Qmax b R2 (mg/g) (mL/mg) 434.8
1.44
KF n (mg/g)
0.94 233.4
R2
2.04 0.82
(Ceq / q = 1 / qmax.b + Ceq / qmax) or Freundlich (q = Kf.Ce1/n) isotherms. Some model parameters were determined by nonlinear regression with commercially available software and are shown in Table 1. The Langmuir adsorption model can be applied in this affinity adsorbent system. It should also be noted that the maximum adsorption capacities (qmax) and the Langmuir constant were found to be 434.8 mg/g and 1.44 mL/mg for batch system. 3.2.4. Effects of salt concentration and temperature The effect of salt concentration on lysozyme adsorption is shown in Fig. 4. The adsorption capacity decreased with increasing (i.e., NaCl) salt concentration. The adsorption of lysozyme was decreased by about 68% as the NaCl concentration increased from 0 to 0.05 M. The decrease in the adsorption capacity as the NaCl concentration increases can be attributed to the repulsive electrostatic interactions between the PHEMATrp-Cu(II) nanoparticles and lysozyme molecules. Higher lysozyme adsorption capacities at low salt concentrations may be due to nonspecific adsorption and hence, signify the role of amino acid residues other than histidine, cysteine and tryptophan that are usually considered responsible for immobilized metal affinity phenomenon [29]. The effect of temperature on lysozyme adsorption was investigated in the range of 4 °C–45 °C (Fig. 5). The adsorption capacity of PHEMATrp-Cu(II) nanoparticles decreased with increasing temperature. The maximum adsorption capacity was obtained at 4 °C. The adsorption capacity of nanoparticles decreased about 37%, as the temperature increased from 4 °C to 45 °C. A possible explanation for this behavior is the exothermic nature of the adsorption process [30]. 3.3. Elution and repeated use
Fig. 5. Effect of temperature on lysozyme adsorption; lysozyme concentration: 1.5 mg/mL; pH: 7.0; incubation time: 2 h.
in enzyme activity. These results demonstrated the stability of present metal-chelated nanoparticles as an affinity adsorbent. 3.4. Lysozyme purification from chicken egg white and activity measurements Single step lysozyme purification from egg white was studied in a batch mode. The PHEMATrp nanoparticles provided an efficient single step method to purify lysozyme from diluted egg white, showing high adsorption capacity and high selectivity for lysozyme. The purity of the lysozyme desorbed from the metal chelated nanoparticles was determined by SDS-PAGE. The purity of the desorbed lysozyme was about 87% with an overall yield of about 78%, which is similar to 77% yield obtained using Streamline SP followed by dye affinity adsorption [31] and 80% yield using metal-affinity precipitation with Cu 2+ followed by gel filtration chromatography on Sephadex G75 [32]. Denizli et al. prepared lysozyme-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester) particles for the purification of lysozyme from egg-white [25]. They reported 89% purity with recovery of about 84%. Compared to the results in literature, the result by the PHEMATrp nanoparticles are better. The specific activity of the purified lysozyme with the PHEMATrp-Cu(II) nanoparticles was 27,500 U/mg. It should be noted that the specific activity of native lysozyme was 30,000 U/mg. As seen here there was no drastic decrease in specific activity during the purification studies. The PHEMATrp-Cu(II) nanoparticles provided an efficient method to purify lysozyme from diluted egg white, showing high adsorption capacity and high selectivity for lysozyme.
In the affinity separation processes, an advantage of a support material is its reusability. The lysozyme adsorbed onto the PHEMATrp-Cu(II) nanoparticles was desorbed with 1.0 M NaCl solution and desorption ratio was calculated as 90% at the end of 2 h. During the desorption of lysozyme, no leakage of Cu(II) ions was observed from the Cu(II) chelated nanoparticles. The adsorption–desorption cycle was repeated 10 times using the same nanoparticles in order to show the reusability of the nanoparticles (Fig. 6). It was observed that the PHEMATrp-Cu(II) nanoparticles can be used many times without decreasing their adsorption capacities significantly. In addition, at the end of ten adsorption and desorption cycles, there was no remarkable reduction
IMAC is a significant bioseparation technique for biomolecules [32–35]. In addition to its affinity-like selectivity, IMAC exhibits the advantages of a higher recovery yield with the use of mild, non-denaturing elution conditions, compared to biospecific chromatographic methods [13]. In this study, nanoparticles were used
Fig. 4. Effect of ionic strength on lysozyme adsorption; lysozyme concentration: 1.5 mg/mL; pH: 7.0; temperature: 25 °C, incubation time: 2 h.
Fig. 6. Reusability of PHEMATrp-Cu(II) nanospheres; lysozyme concentration: 1.5 mg/mL; pH: 7.0; temperature: 25 °C, incubation time: 2 h.
4. Conclusion
536
G. Baydemir et al. / Materials Science and Engineering C 33 (2013) 532–536
as metal-chelated affinity matrix. Nanoparticles provide an ideal remedy to the conflicting issues usually encountered in the optimization of adsorbed enzymes; minimum diffusional limitation, maximum surface area per unit mass and high enzyme loading [36]. The maximum lysozyme adsorption capacity by using metal chelated nanoparticles was found to be 326.9 mg/g polymer. Adsorbed lysozyme was desorbed up to 90% by using 1.0 M NaCl as the elution agent. The metal-chelated nanoparticles can be used as affinity adsorbents with their high reusability capacity. References [1] T. Jing, H. Du, Q. Dai, H. Xia, J. Niu, Q. Hao, S. Mei, Y. Zhou, Biosens. Bioelectron. 26 (2010) 301–306. [2] D.S. Freitas, J. Abrahano-Neto, Int. J. Pharm. 392 (2010) 111–117. [3] G. Zhang, Q. Cao, N. Li, K. Li, F. Liu, Talanta 83 (2011) 1515–1520. [4] K.A. Near, M.J. Lefford, J. Clin. Microbiol. 30 (1992) 1105–1110. [5] B. Porstmann, K. Jung, H. Scmechta, U. Evers, M. Pergande, T. Porstman, H.-J. Kramn, H. Krause, Clin. Biochem. 22 (1989) 349–355. [6] S. Yu, A. Luo, D. Biswal, J.Z. Hilt, D.A. Puleo, Talanta 83 (2010) 156–161. [7] T.D. Durance, in: J.S. Sim, S. Nakai (Eds.), Egg Uses and Processing Technologies. New DevelopmentsInternational CAB, Wallingford, 1994, pp. 77–93. [8] R. Ghosh, Z.F. Cui, Biotechnol. Bioeng. 68 (2000) 191–203. [9] E. Li-Chan, S. Nakai, J. Sim, D.B. Bragg, K.V. Lo, Food Sci. 51 (1986) 1032–1036. [10] R. Dembczynski, W. Bialas, K. Regulski, T. Jankowski, Process. Biochem. 45 (2010) 369–374. [11] S. Öncel, L. Uzun, B. Garipcan, A. Denizli, Ind. Eng. Chem. Res. 44 (2005) 7049–7056. [12] E.B. Altıntaş, A. Denizli, Int. J. Biol. Macromol. 38 (2006) 99–106. [13] L. Wang, R. Xu, Y. Chen, R. Jiang, J. Mol. Catal. B Enzym. 69 (2011) 120–126.
[14] J. Porath, J. Carlson, I. Olsson, G. Belfrage, Nature 258 (1975) 598–599. [15] S. Akgöl, D. Türkmen, A. Denizli, J. Appl. Polym. Sci. 93 (2004) 2669–2677. [16] A. Denizli, M. Alkan, B. Garipcan, S. Özkara, E. Pişkin, J. Chromatogr. B 795 (2003) 93–103. [17] M.B. Riberio, M. Vijayalakshmi, D. Todorova-Balvay, S.M.A. Bueno, J. Chromatogr. B 861 (2008) 64–73. [18] A. Denizli, S. Şenel, M.Y. Arıca, Colloids Surf. B: Biointerfaces 11 (1998) 113–122. [19] M.-H. Liao, D.-H. Chen, Biotechnol. Lett. 24 (2002) 1913–1917. [20] N. Basar, L. Uzun, A. Guner, A. Denizli, Int. J. Biol. Macromol. 41 (2007) 234–242. [21] E.B. Altıntaş, N. Tuzmen, N. Candan, A. Denizli, J. Chromatogr. B 853 (2007) 105–113. [22] C. Garcia-Diego, J. Cuellar, Chem. Eng. J. 143 (2008) 337–348. [23] D. Shao, K. Xu, X. Song, J. Hu, W. Yang, C. Wang, J. Colloid Interface Sci. 336 (2009) 526–532. [24] S. Akgol, N. Ozturk, A. Denizli, J. Appl. Polym. Sci. 115 (2010) 1608–1615. [25] M. Odabasi, R. Say, A. Denizli, Mater. Sci. Eng. C 27 (2007) 90–99. [26] E.B. Altıntaş, A. Denizli, Mater. Sci. Eng. C 29 (2009) 1627–1634. [27] D.A. Uygun, M.E. Çorman, N. Ozturk, S. Akgol, A. Denizli, Mater. Sci. Eng. C 30 (2010) 1285–1290. [28] H. Yavuz, M. Odabaşı, S. Akgöl, A. Denizli, J. Biomater. Sci. Polym. Ed. 16 (2005) 673–684. [29] S. Sharma, G.P. Agorwal, J. Colloid Interf. Sci. 243 (2001) 61–72. [30] M. Karatas, S. Akgol, H. Yavuz, R. Say, A. Denizli, Int. J. Biol. Macromol. 40 (2007) 254–260. [31] I. Roy, M.V. Rao, M.N. Gupta, Appl. Biochem. Biotechnol. 111 (2003) 55. [32] I. Roy, M.V. Rao, M.N. Gupta, Biotechnol. Appl. Biochem. 37 (2003) 9. [33] E.B. Altintas, H. Yavuz, R. Say, A. Denizli, J. Biomater. Sci. Polym. Ed. 17 (2006) 213–226. [34] D. Türkmen, H. Yavuz, A. Denizli, Int. J. Biol. Macromol. 38 (2006) 126–133. [35] N. Tüzmen, T. Kalburcu, A. Denizli, Process. Biochem. 47 (2012) 26–33. [36] H. Koo, M.S. Huh, J.H. Ryu, D.-E. Lee, I.-C. Sun, K. Choi, K. Kim, I.C. Kwon, Nano Today 6 (2011) 204–220.
