Preparation of Fe(III)-immobilized collagen fiber for lysozyme adsorption

Colloids and Surfaces A: Physicochem. Eng. Aspects 301 (2007) 85–93

Preparation of Fe(III)-immobilized collagen fiber for lysozyme adsorption Ai Xia Lu, Xue Pin Liao, Rong Qing Zhou, Bi Shi ∗ The Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu 610065, China Received 7 September 2006; received in revised form 23 November 2006; accepted 6 December 2006 Available online 10 December 2006

Abstract A novel adsorbent was prepared by immobilizing Fe(III) onto collagen fiber matrix, and its adsorption to lysozyme was studied. Fe(III) was uniformly dispersed in collagen fibers mainly through chemical reaction and could endure the extraction of water. It was found that the Fe(III)immobilized collagen fiber (FICF) exhibited high adsorption capacity to lysozyme. The adsorption capacity was 395 mg/g at 303 K when initial concentration of lysozyme was 2.5 mg/mL. The adsorption capacity was significantly influenced by pH, and it reached a maximum value around pH 8.0. The adsorption capacity increased with the increase of temperature. The adsorption capacity of lysozyme remarkably decreased when the concentration of NaCl was increased from 0 to 0.25 mol/L. However, the adsorption capacity increased slightly as the concentration of NaCl was further increased. The adsorption isotherms can be described by the Langmuir equation. Further analysis indicated that the adsorption kinetic data can be well fitted by the pseudo-second-order rate model, and the adsorption capacities calculated by the model were consistent with those of the actual measurements. In addition, Fe(III)-immobilized collagen fiber presented excellent column adsorption kinetic properties and high binding capacity. The adsorption behavior of the column was almost unchanged in adsorption–desorption cycles. The purification of lysozyme from chicken egg white powder by using FICF was investigated. The purity of lysozyme obtained was 100%, and recovery extent of lysozyme was 70.5%. © 2006 Elsevier B.V. All rights reserved. Keywords: Collagen fiber; Fe(III); Immobilization; Adsorption; Lysozyme

1. Introduction The efficient separation and purification of proteins has become one of important prerequisites in modern biomedical and pharmaceutical industries. Although the separation processes concern numerous proteins with diverse characteristics, the strategies of bioseparation for these targets consist of four similar sequential steps: removal of insolubles by filtration or centrifugation; isolation of proteins by solvent extraction or adsorption; purification by chromatography or electrophoresis; polishing by freeze drying or crystallization [1]. The method of adsorption has been widely used for separation of proteins because of the selectivity and specificity in protein binding based on electrostatic, ion-exchange, hydrophobic and affinity interaction between the adsorbent surface and the protein surface [2]. Since immobilized metal affinity (IMA) was introduced in 1975 [3], the adsorptive separation of proteins by using immo-

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bilized metal affinity chromatography (IMAC) has become a well-developed technique. Silica, copolymer microparticles and chemical modified agarose had been used as loading matrices for preparing IMAC, which showed satisfactory property in purification of proteins [4–8]. In terms of economy, ease of preparation, stability and adsorption capacity, immobilized metal affinity adsorbents offer several advantages over other specific adsorbents, such as ion-exchange cellulose, chitosan and DEAE sephadex [9–12]. It was reported that metal-chelated polyamide hollow fiber showed a promising increase of the adsorption capacity to lysozyme. The non-specific adsorption of lysozyme on the polyamide hollow fiber was only 1.8 mg/g, while the maximum capacities of Zn(II), Cu(II) and Ni(II)chelated hollow fibers reached 144.2, 75.2 and 68.6 mg/g, respectively [13]. Lysozyme occurs naturally in chicken or goose egg white, animal tissues, human urine, etc. Attempts have been made to separate and purify lysozyme. For example, Klint and Eriksson have reported the purification of lysozyme from chicken egg white by using ultrastable zeotite Y with 56% yield [14]. Ghosh et al. have described the purification of lysozyme by polysulphone hollow-fiber ultrafiltration, and the purity of

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lysozyme obtained was 80–90% [15]. Carboxymethyl [16], poly(vinyl alcohol)-perfluoropolymer [17] and polyvinylidine fluoride [18] ion-exchangers were also used to purify lysozyme from chicken egg white or rabbit kidney. The lysozyme yield by using ion-exchanger chromatography was between 65 and 98%. The affinity chromatography by immobilizing dye-ligands, such as Cibacron Blue (F)3GA [19,20], Procion Red HE7B [21], Procion Brown MX-5BR and Procion Green H-4G [22], onto polymers has been used to separate lysozyme from chicken egg white. The lysozyme yield by using dye-affinity chromatography was between 63 and 87.6%, but the purity of the lysozyme obtained was unsatisfactory. Lysozyme was usually selected as a model protein to investigate adsorption capacity of metal ions-affinity adsorbent to proteins [7,13], but no report about purification of lysozyme by IMAC is found. Collagen fiber, an abundant natural biomass, comes from the skin of animals and is traditionally used as raw material of leather manufacturing. Collagen molecule is composed of three polypeptide chains with triple helical structure and they are aggregated through hydrogen bonds to form collagen fiber [23]. Collagen fiber is water insoluble, but it is a hydrophilic material. Based on the principles of leather manufacturing, collagen fiber which has abundant functional groups is capable of chemically reacting with many kinds of metal ions, such as Cr(III), Fe(III), Zr(IV), etc. [24]. Therefore, collagen fiber is ready to be used as a carrier of metal ions. In the present study, a novel adsorbent was prepared by immobilizing Fe(III) onto collagen fiber. Considering the fact that lysozyme is usually selected as a model of proteins in investigating separation processes of proteins [25–27], the adsorption behaviors of lysozyme on the adsorbent were studied. 2. Materials and methods 2.1. Materials and reagents 2.1.1. Protein and protein reagent Crystalline lysozyme (EC 3.2.1.7) was purchased from Seikagaku Kogyo Company, Ltd. (Tokyo, Japan). Crude dried egg white power (A-5253) was purchased from Sigma. Micrococcus lysodeikticus, used for the analysis of lytic activity, was purchased from Institute of Microbiology, Chinese Academy of Sciences. Coomassie assay reagent, used as protein dye, was purchased from Pierce (Rockford, IL, USA). 2.1.2. Preparation of collagen fiber Collagen fiber was prepared according to the procedures in our previous work [28]. Briefly, bovine skin was cleaned, limed, split and delimed according to the procedures of leather processing in order to remove the non-collagen components. Then the skin was treated with aqueous solution of acetic acid (conc. 16.0 g/L) for three times to remove mineral substances. After the pH of skin was adjusted to 4.8–5.0 by using acetic acid-sodium acetate buffer solution, the skin was dehydrated by absolute ethyl alcohol, dried in vacuum to moisture content ≤10.0%, ground and sieved. As a result, the collagen fiber was obtained with par-

ticle size of 0.1–0.25 mm, moisture ≤12.0%, ash content ≤0.3% and pH 5.0–5.5. 2.1.3. Immobilization of Fe(III) on collagen fiber 15.0 g collagen fiber was soaked in 400 mL deionized water at room temperature for 24 h. The pH of the deionized water was pre-adjusted to 1.7–2.0 by HCOOH and H2 SO4 . Then, 50.6 mmol Fe2 (SO4 )3 was added and reacted at 30 ◦ C with constant stirring for 4 h. A proper amount of NaHCO3 solution (15%, w/w) was gradually added within 2 h in order to increase the pH of the solution to 4.0–4.5 and then continuously reacted at 40 ◦ C for another 4 h. When the reaction was completed, the product was collected by filtration, washed with deionized water and dried in vacuum at 50 ◦ C for 12 h, and then the adsorbent of the Fe(III)-immobilized collagen fiber (FICF) was obtained. 2.2. Measurement methods Lysozyme concentration was assayed by Bradford’s method using a lysozyme calibration curve [29]. Enzyme activity of lysozyme was determined according to the method described by Shugar [30]. Fe(III) ions in residual solution after immobilization reaction, adsorption or elution were determined by means of ICP-AES (ICP, Perkin-Elmer Optima 2100DV, German). The BET surface areas of collagen fiber and FICF were measured by Surface Area and Porosity Analyzer (TriStar3000, Micrometitics, America). Thermal stability of both collagen fiber and FICF was measured by Differential Scanning Calorimetry (2000PC, NETZSCH, Germany). 2.3. Adsorption behaviors of lysozyme on FICF 2.3.1. Effect of pH on adsorption capacity A series of 0.5 mg/mL lysozyme solutions were prepared by using 0.01 M acetate buffer solution (pH 4.0), 0.01 M phosphate buffer solution (pH 6.0, 7.0 and 8.0), and 0.01 M carbonate buffer solution (pH 9.0 and 10.0), respectively. 0.100 g dry FICF adsorbent was suspended in 25 mL of the sample solutions individually and shaken at 278 K for 60 min. After the adsorption, the residual solutions were filtered and FICF was washed with 10 mL of the same buffer solution. The amount of lysozyme adsorbed on FICF was calculated from the difference between the amount of lysozyme in the initial solution and that in the filtrate and washing solution. The adsorption capacity was calculated by mass balance before and after adsorption, and expressed as mg/g (mg lysozyme per g dry adsorbent). The adsorption capacity of collagen fiber without immobilizing reaction was also determined as control. In addition, Fe(III) ions in the solutions after adsorption were determined by means of ICP-AES. 2.3.2. Effect of salt concentration on adsorption capacity 0.100 g dry FICF adsorbent was suspended in 25 mL phosphate buffer solution with pH 8.0, in which the concentration of lysozyme was 0.5 mg/mL, and the concentrations of NaCl were 0, 0.1, 0.25, 0.5 and 1.0 M, respectively. The adsorption procedures were as the same as Section 2.3.1.

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2.3.3. Adsorption isotherms 0.100 g dry FICF adsorbent was suspended in 25 mL phosphate buffer solution with pH 8.0, in which the concentrations of lysozyme were 0.5, 1.0, 1.5, 2.0 and 2.5 mg/mL, respectively. The adsorption was carried out with shaking for 24 h at 278, 288 and 303 K, respectively. The determination procedures for adsorption capacity were as the same as Section 2.3.1.

Every batch adsorption experiments above were conducted twice, and it was found that the results were reproducible with an error of less than 10%. The adsorption isotherms were presented with error bars, and others were presented with the average values.

2.3.4. Adsorption kinetics 0.100 g dry FICF adsorbent was suspended in 25 mL phosphate buffer solution with pH 8.0, in which the concentrations of lysozyme were 0.5 and 1.0 mg/mL, respectively. The adsorption was carried out with shaking at 303 K. The adsorption capacity of lysozyme during adsorption process was analyzed at a regular interval. The determination procedures for adsorption capacity were as the same as Section 2.3.1.

3.0 g dry FICF adsorbent was soaked in deionized water for 24 h and then filled into a column with diameter 11 mm. The column was equilibrated by 0.01 M sodium phosphate buffer (pH 8.0), and then 2 L of 1.0 mg/mL chicken egg white power solution prepared by the equilibration buffer was pumped into the column with flow-rate of 1.1 mL/min. After washing the column with 0.01 M sodium phosphate to remove unbound proteins, the lysozyme adsorbed on column was eluted by using a mixture solution of 0.3 M imidazole and 0.25 M NaCl in 0.01 M phosphate buffer with pH 6.0. Fractions were collected throughout the experiment and total amount of proteins was assayed. The purity of eluted lysozyme was measured by HPLC with ZOBAX 300 filtration column. A 20 ␮L sample loop was employed for sample injection and 6 M urea in 0.2 M phosphate buffer (pH 7.5) was used as mobile phase. The flow-rate of mobile phase was 0.8 mL/min.

2.3.5. Batch desorption studies 0.100 g dry FICF adsorbent was suspended in 25 mL of phosphate buffer solution with pH 8.0, in which the concentration of lysozyme was 0.5 mg/mL, and the adsorption was conducted by constant shaking at 278 K for 60 min. Then the solution was filtered and the adsorbent was transferred into 10 mL of different desorption solutions. Desorption studies were conducted by constant shaking at 278 K for 20 min. The lysozyme amount desorbed and enzyme activity were determined. Recovery extent of lysozyme was calculated from the amount of lysozyme adsorbed on FICF and the amount of lysozyme desorbed from FICF. Remained lytic activity of desorbed lysozyme was calculated from enzyme activity of lysozyme adsorbed and desorbed. In addition, Fe(III) ions in the eluted solution were also determined by means of ICP-AES. 2.3.6. Column adsorption kinetics 1.5 g dry FICF adsorbent was soaked in deionized water for 24 h and then filled into a column of diameter 11 mm and height 185 mm. The bed volume (BV) of the column was 15.9 mL. The column was equilibrated with 300 mL phosphate buffer solution (pH 8.0). Then 0.5 mg/mL lysozyme solution prepared with the same buffer solution was bumped into the column at a constant volume velocity of 0.81 BV/h. When the adsorption was saturated, the column was washed with 50 mL of the same buffer solution to remove un-adsorbed lysozyme, and then the column was regenerated by a mixture solution (0.3 M imidazole and 0.25 M NaCl in 0.01 M phosphate buffer solution with pH 6.0) at a volume velocity of 1.93 BV/h. Effluent was collected by an automatic collector and the concentration of lysozyme in the effluent was analyzed. After desorption, the column was reused for adsorption–desorption cycles.

2.4. Purification of lysozyme from chicken egg white power

3. Results and discussion 3.1. The features of Fe(III)-immobilized collagen fiber Fig. 1 is the Scanning Electronic Microscope (SEM) photos of collagen fiber and FICF. It is obvious that the collagen fiber without loading of Fe(III) is compact and stark, while the FICF is well dispersed because of so-called tanning effect. Fe(III) was uniformly impregnated into collagen fibers and no evident precipitate stack of Fe(III) was found. In addition, no Fe(III) was detected in the residual solution of Fe(III) impregnation reaction. Therefore, the amount of Fe(III) loaded on collagen fiber was approximately 6.75 mmol Fe(III)/g. The general properties of collagen fiber and FICF are summarized in Table 1. The BET surface area of FICF is larger than that of collagen fiber, which is consistent with the observation of SEM. As it can be seen in Table 1, the immobilizing reaction of Fe(III) on collagen fibers can efficiently improve the denaturation temperature of collagen fiber, so that the FICF adsorbent has higher thermal stability that favors practical uses. The adsorption capacity of FICF to lysozyme was 94.7 mg/g when the initial concentration of lysozyme was 0.5 mg/mL, about 6 times higher than that of collagen fiber. Therefore, Fe(III) immobilized on collagen fiber plays an important role for increase of adsorption

Table 1 The general properties of collagen fiber and FICF Sample

Temperature of denaturation (◦ C)

BET surface area (m2 /g)

Adsorption capacity to lysozyme (mg/g)a

Collagen fiber FICF

60–65 82–87

1.96 5.47–6.23

16.5 94.7

a

Initial concentration: 0.5 mg/mL; media volume: 25 mL; pH 8.0; 278 K; 60 min; 0.100 g dry adsorbent.

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Fig. 2. Effect of pH on adsorption capacity of lysozyme.

[6]. The adsorption of lysozyme on metal-immobilized materials is mainly through chelating bonding between metal ion and histidine residue of lysozyme, and the pKa values of histidine residue in most of proteins are in the range of 5.5–8.5 [33,34]. Therefore, the optimal pH for lysozyme adsorption on FICF is around 8.0. It is similar to the results of lysozyme adsorption on metalchelated polyamide hollow membrane [13]. Furthermore, it was found that no Fe(III) was released from collagen fiber during the adsorption process in the pH range of 4.0–10.0, suggesting the suitability of FICF for practical application. In addition, the control experiment showed that the adsorption capacity of FICF was much higher than that of collagen fiber in the pH range of 6.0–10. 3.3. Effect of salt concentration on adsorption capacity Fig. 1. SEM photographs of collagen fiber and FICF (20000×).

capacity. Our experiments indicated that the adsorption capacity of FICF to lysozyme was increased as the rise of temperature. These facts suggest that chemical adsorption mechanism might be involved in the adsorption process. However, the adsorption of lysozyme on collagen fiber without Fe(III) might be due to hydrogen and/or hydrophobic interactions. The adsorption capacity of lysozyme on FICF is higher than other comparative adsorbents reported, such as ultrastable zeolite Y (26.7 mg/g) [31] and Fe(III) complexed microparticles (67.7 mg/g) [32].

The influence of NaCl concentration on adsorption capacity is presented in Fig. 3. The adsorption capacity of lysozyme remarkably decreased when the concentration of NaCl was increased from 0 to 0.25 mol/L, which suggests that electrolyte greatly

3.2. Effect of pH on adsorption capacity The adsorption capacity of lysozyme on FICF is significantly influenced by pH, as shown in Fig. 2. It is evident that the adsorption capacity increases with the increase of pH and it reaches a maximum value around pH 8.0. The adsorption of proteins on IMAC is mainly through chelating bonding between metal ions and amino acid residues. The ionization of amino acid residues at a weakly alkaline pH favors the reaction and therefore induces protein adsorption on the metal-immobilized matrixes

Fig. 3. Effect of ionic strength on adsorption capacity.

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affects the chelating bonding of lysozyme on FICF. This might be due to the fact that the addition of electrolyte has changed the pKa of coordinate groups on lysozyme and thus weakens the chelating reaction. As further increase of NaCl concentration, the adsorption capacity increased slightly, which might be due to the enhancement of hydrophobic interaction [6] and/or the effect of salting out. In fact, hydrophobic and electrostatic interactions between lysozyme and FICF may take place in the adsorption process, and can not be neglected under certain circumstances [6,35,36]. Similar phenomena was also observed for the adsorption of proteins on Fe(III)-IDA [37]. It was suggested that the proteins were bound by the adsorbent via metal chelating at lower salt concentration, while the adsorption mechanism was hydrophobic interaction at relative higher salt concentration [37]. Thus, FICF can acts as metal chelating adsorbent at low salt concentration, and as hydrophobic interaction adsorbent at high salt concentration. The specific area determination of FICF (BET approach) indicates that the specific area of FICF is around 6.0 m2 /g, which is much smaller than that of porous adsorbents. However, the adsorption capacity of FICF to lysozyme is significant and the adsorption rate is also satisfactory. In addition, FICF is in fibrous structure. Therefore, it can be suggested that the adsorption process is taking place on the outer surface of the fibrously structured FICF and thus the intraparticle diffusion resistance can be neglected. 3.4. Adsorption isotherms The adsorption isotherms of lysozyme on FICF are illustrated in Fig. 4. In order to further understand the adsorption mechanism of lysozyme on FICF, thermodynamic analysis was carried out. The maximum adsorption (qmax ) of lysozyme at each temperature and the enthalpy change (H) of adsorption process were obtained according to the Langmuir equation (Eq. (1)) and Van Hoff equation (Eq. (2)), respectively. qe =

qmax bce (1 + bce )

Fig. 4. Adsorption isotherms of lysozyme on FICF.

(1)

89

Table 2 Langmuir parameters of lysozyme on FICF Temperature (K)

qmax (mg/g)a

b × 102

R2

H (kJ/mol)

278 288 303

275 349 427

0.79 0.88 1.36

0.991 0.990 0.992

19.5

a Initial concentration: 2.5 mg/mL; media volume: 25 mL; pH 8.0; 24 h; 0.100 g dry adsorbent.

log b =

−H + constant (2.303RT )

(2)

where qe and Ce are the amount of lysozyme adsorbed (mg/g) and the bulk concentration (mg/L) at equilibrium, respectively; qmax is the maximum lysozyme adsorption (mg/g); b is the Langmuir coefficient relating to the strength of adsorption; H is the change of enthalpy (kJ/mol); R is the gas universal constant (8.314 J/mol); and T is temperature (K). It can be found from Fig. 4 that the Langmuir equation gives perfect fittings to the adsorption isotherms. Similarly, it was reported that the adsorption isotherms of catalase on Fe(III)-derived poly(hydroxyethyl methacrylate) membranes could be also fitted with Langmuir model [38]. The results listed in Table 2 indicate that the enthalpy change of adsorption process is smaller than typical chelating bonding and ionic bonding, but higher than electrostatic and Van der Waal forces. In addition, the qmax and qe increased with the rise of temperature, implying the existence of chemical adsorption. Therefore, it can be concluded that multi-interactions are included in the adsorption process of lysozyme on FICF, although the chelating bonding of proteins on metal-immobilized materials has been confirmed by researchers [35]. 3.5. Adsorption kinetics Fig. 5 shows the adsorption kinetics of lysozyme on FICF. It can be seen that the adsorption rate at early stage was nearly the same even the initial concentration of lysozyme was different. However, with the increase of initial concentration of lysozyme,

Fig. 5. Adsorption kinetics of lysozyme on FICF.

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Table 3 Comparison of the first-order and second-order rate constants Initial concentration (mg/mL)

qe.exp. a (mg/g)

First-order rate kinetic model k1

0.5 1.0 a

103 136

× 102

(min−1 )

3.87 2.66

Second-order rate kinetic model

qe.cal. (mg/g)

R2

k2 × 102 (g/mg min)

qe.cal. (mg/g)

R2

31.6 83.1

0.969 0.967

5.34 1.88

99.5 130

0.999 0.996

Media volume: 25 mL; 278 K; pH 8.0; 0.100 g dry adsorbent.

the equilibrium adsorption capacity increased and the time for attaining equilibrium was lagged. In order to analyze the adsorption kinetics of lysozyme, the pseudo-first-order rate and the pseudo-second-order rate kinetic models were applied to fit the experimental data. The pseudofirst-order-rate equation of Lagergren is most widely used for describing the adsorption of solute from a liquid solution [39]. It can be given as: log(qe − qt ) =

log qe − k1 t 2.303

(3)

where qe and qt are the amount of lysozyme adsorbed on adsorbent (mg/g) at equilibrium and at time t (min), respectively, and k1 is the rate constant of pseudo-first-order adsorption (min−1 ). The pseudo-second-order rate model was proposed by Ritchie for describing the adsorption kinetics of gases on solids [40], and it was also applied to the adsorption of solutes on the adsorbents [41]. The pseudo-second-order rate equation can be expressed as: t 1 t = + 2 qt k2 q e qe

(4)

where k2 is the constant of pseudo-second-order rate (mg/g min). Based on the experimental data of qt , the equilibrium adsorption capacity qe and the k2 can be determined from the slope and intercept of a plot of t/qt against t. Table 3 lists the fitting results of the rate constants by using the pseudo-first-order and pseudo-second-order models. The correlation coefficient R2 of the pseudo-second-order adsorption model has extremely high value (>0.996), and the adsorption capacities calculated by the model are close to those determined by experiments. However, the correlation coefficient R2 of the pseudo-first-order adsorption model is not satisfactory.

Therefore, these facts suggested that the pseudo-second-order adsorption model could be used to describe the adsorption kinetics of lysozyme on FICF, and the adsorption should be rate-controlling step [41]. 3.6. Batch desorption studies A series of solutions were used to test their ability to recover the lysozyme adsorbed on FICF. The results are summarized in Table 4. In general, the lysozyme adsorbed on FICF can not be desorbed by water, and the desorption extent is only 11.6% by using 0.25 mol/L NaCl even though the salt can remarkably decrease the adsorption capacity of lysozyme on FICF, as shown in Fig. 3. Desorbed by using 0.3 mol/L imidazole, 92.9% enzymatic activity of the recovered lysozyme is preserved, but the extent of recovery of lysozyme is only 43.4%. The mixture solution, 0.25 mol/L NaCl and 0.3 mol/L imidazole in 0.01 mol/L phosphate buffer solution with pH 6.0, can effectively recover lysozyme from FICF. The extent of recovery is 96.7%, and 94.1% enzymic activity is remained. Other mixture solutions, such as 0.25 M NaCl and 0.3 M imidazole, cannot effectively desorb lysozyme. These facts imply that the synergistic effect of the components in desorption solution might take place and the multiple mechanisms might be involved in the interaction between lysozyme and FICF. In view of the proposed mechanism [6,35], the formation of coordinated compound between protein and immobilized metal ion is considered to be the major binding mode. In addition, the electrostatic and hydrophobic interactions may be also involved in the adsorption of lysozyme on FICF. It was noticed that no Fe(III) was detected in desorption solutions. This is due to the fact that Fe(III) is chemically bonded with collagen fiber [24].

Table 4 Desorption of lysozyme from FICFa Desorbents

Recovery extent of lysozyme (%)

Remained lytic activity of desorbed lysozyme (%)

Deionized water 0.25 M NaCl 0.3 M imidazole 0.01 M acetate buffer (pH 4.0) 0.01 M phosphate buffer (pH 6.0) 0.25 M NaCl and 0.3 M imidazole 0.25 M NaCl in 0.01 M acetate buffer (pH 4.0) 0.25 M NaCl in 0.01 M phosphate buffer (pH 6.0) 0.25 M NaCl and 0.3 M imidazole in 0.01 M acetate buffer (pH 4.0) 0.25 M NaCl and 0.3 M imidazole in 0.01 M phosphate buffer (pH 6.0)

0 11.6 43.4 23.9 30.3 42.2 44.1 59.4 72.6 96.7

– 78.4 92.9 68.4 75.3 88.2 72.8 85.7 76.5 94.1

a

Media volume: 10 mL; 278 K; 20 min.

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adsorption time for determination of batch adsorption capacity is 24 h where the adsorption equilibration had been attained. However, the average retention time of lysozyme in column was only 1.2 h where the adsorption equilibration was not attained. Therefore, the continuous adsorption capacity is lower than batch adsorption capacity. It has been found that a simple two parameters model can be used to simulate the typical S-shaped breakthrough curves [1,43]. The model suggested by Belter et al. can be expressed in following form [44]. √ {1 + erf[(N − N0 )/( 2σN0 )]} C = (8) C0 2

Fig. 6. Breakthrough curves of lysozyme on FICF column.

3.7. Column adsorption kinetics Breakthrough curves of lysozyme in FICF column are shown in Fig. 6. The breakthrough point was about 50 BV under experimental conditions. The shape of breakthrough curve is determined by the shape of equilibrium isotherm and is influenced by the individual transport processes in column and in adsorbent [42]. In general, a sharp breakthrough curve can be obtained if the adsorption isotherm is a favorable isotherm, such as Langmuir, as shown in Fig. 4. The sharp breakthrough curve implies efficient adsorption performance and higher availability of column. Furthermore, the adsorption capacity of the regenerated column was almost unchanged compared with the new one. The dynamic adsorption capacity (DAC) in column was calculated from lysozyme breakthrough curves. When the concentration of lysozyme in effluent was 5% of feeding concentration (named breakthrough point), the total amount of lysozyme adsorbed was calculated by using the method of Griffith et al. [43]. It is given as:   t(C/C0 =0.05)  1−C Q5% = u × C0 dt (5) C0 0 where Q5% is the total amount of lysozyme adsorbed at 5% breakthrough point (mg/g), u is feeding rate (BV/h), t is the time of adsorption (h), C0 and C are the concentrations of lysozyme in feeding solution and in effluent at the moment of t, respectively (mg/mL). DAC is calculated as: Q5% DAC = (6) m where m is the mass of adsorbent (g). Average retention time (τ) is calculated as: BV (7) u where BV is bed volume of column (15.9 mL). The continuous adsorption capacity in column was 268 mg/g, which was lower than batch adsorption capacity (349 mg/g). The τ=

where C and C0 are the effluent and influent concentration, respectively; erf[x] is the error function of x; N is the bed volume number of effluent; N0 is the bed volume number when the effluent concentration is half of the influent concentration; and σ represents the standard deviation which is a measure of the slope of the breakthrough curve. The likely mechanism influencing the shape of the breakthrough curve may be deduced from the relationships between σ 2 and the superficial velocity and column length. This model gives satisfactory fitting to the experimental data, as shown in Fig. 6. The parameters of N0 and σ are 64.5 and 0.0812, respectively for the first adsorption, and they are 63.2 and 0.0530, respectively for the second adsorption. But the relationship of N0 and σ with the parameters, such as feeding velocity and column length need to be further investigated. Our studies indicated that FICF is not porous material, so the adsorption of lysozyme should take place at the surface of the adsorbent. Hence, it is relatively easy to regenerate adsorbent after saturation adsorption has been attained. Fig. 7 shows the elution curves of FICF after first and second adsorption. FICF adsorbent can be easily regenerated by using only 60 mL mixture solution of 0.25 mol/L NaCl and 0.3 mol/L imidazole in 0.01 mol/L phosphate buffer solution with pH 6.0. The maximum concentration of lysozyme in the eluate was 14.4 and 14.1 mg/mL for the first and second regeneration, respectively, much greater than the feed concentration of lysozyme solution, which implies that lysozyme in solution can be concentrated by FICF column. As indicated in Table 1, the specific area of FICF is much smaller than other adsorbents, such as silicon and polyvinylidine fluoride. Therefore, the adsorption and desorption process should mainly take place at the outer surface of the adsorbent, and the diffusion mass transfer can be ignored. As a result, the adsorption and desorption rate is faster compared with common porous adsorbents, which makes it possible to completely recover its adsorption ability after regeneration. 3.8. Separation of lysozyme from chicken egg white powder The separation of lysozyme from chicken egg white powder by using FICF column was undertaken. The purity of lysozyme desorbed from the column was found to be 100% determined by HPLC, as shown in Fig. 8, whilst the recovery extent of lysozyme was 70.5%. The purity of lysozyme obtained by adsorption

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separation of FICF is remarkably higher than that of other approaches, such as by using RB-10 (76%) and RG-5 (92%) membranes [22]. 4. Conclusion A new metal-immobilized affinity adsorbent can be prepared by immobilizing Fe(III) onto collagen fiber. Experiment results indicated that this novel adsorbent can effectively adsorb lysozyme from aqueous solution, and the excellent adsorption– desorption behaviors of the adsorbent promise it to be useful in practical applications. The recovery extent of lysozyme by using FICF column was 70.5%, and the purity of separated lysozyme measured by HPLC was 100%. Compared with raw collagen fiber, the adsorption capacity of FICF is greatly increased. It can be concluded that Fe(III) plays a key role for the adsorption of lysozyme on FICF. Therefore, the formation of coordinated compound between protein and Fe(III) should be considered to be the major binding mode. However, electrostatic and hydrophobic interactions may be also involved in the adsorption of lysozyme on FICF. Furthermore, the adsorption of FICF to other proteins should be investigated to understand the relation between the protein structure and adsorption behaviors. Acknowledgments The research was financially supported by National Major Research Plan (pilot) of China (2004CCA06100), National Natural Science Foundation of China (20476062), The Key Program of Natural Science Found of China (20536030) and National Science Fund for Distinguished Young Scholar (20325619). References

Fig. 7. Elution curves of lysozyme adsorbed on FICF.

Fig. 8. HPLC of egg white (A), commercial lysozyme (B) and elute (C).

[1] P.A. Belter, E.L. Cussler, W.S. Hu, Bioseparation: Downstream Processing for Biotechnology, Wiley Press, New York, 1988. [2] T. Kawai, K. Saito, W. Lee, Protein binding to polymer brush, based on ion-exchange, hydrophobic and affinity interactions, J. Chromatogr. B 790 (2003) 131–142. [3] J. Porath, J. Carlsson, I. Olsson, G. Belfrage, Metal chelate affinity chromatography, a new approach to protein fractionation, Nature 258 (1975) 598–599. [4] L.F. Ho, S.Y. Li, S.C. Lin, W.H. Hsu, Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents, Process Biochem. 39 (2004) 1573–1581. [5] C.M. Zhanq, S.A. Reslewic, C.E. Glata, Suitability of immobilized metal affinity chromatography for protein purification from canola, Biotechnol. Bioeng. 68 (2000) 52–58. [6] J.W. Wong, R.L. Albriqht, N-H.L. Wanq, Immobilized metal ion affinity chromatography (IMAC) chemistry and bioseparation applications, Sep. Purif. Method 20 (1991) 49–106. [7] W.Y. Chen, C.F. Wu, C.C. Liu, Interaction of imidazole and protein with immobilized Cu(II) ions: effects of structure, salt concentration, and pH in affinity and binding capacity, J. Colloid Interface Sci. 180 (1996) 135–143. [8] E.K.M. Ueda, P.W. Gout, L. Morganti, Current and prospective application of metal ion-protein binding, J. Chromatogr. A 988 (2003) 1–23. [9] A. Denizli, H. Yavuz, Y. Arica, Monosize and non-porous p(HEMA-coMMA) microparticles designed as dye- and metal-chelate affinity sorbents, Colloid Surf. A 174 (2000) 307–317.

A.X. Lu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 301 (2007) 85–93 [10] M.A. Vijayalakshmi, Pseudobiospecific ligand affinity chromatography, Trends Biotechnol. 7 (1989) 71–76. [11] J. Benesch, P. Tengvall, Blood protein adsorption onto chitosan, Biomaterials 23 (2002) 2561–2568. [12] B.C.C. Pessela, R. Torres, M. Fuentes, C. Mateo, R. Munilla, A. Vian, A.V. Carrascosa, J.L. Garcia, J.M. Guisan, L.R. Fernandes, Selective and mild adsorption of large proteins on lowly activated immobilized metal ion affinity chromatography matrices: purification of multimeric thermophilic enzymes overexpressed in Escherichia coli, J. Chromatogr. A 1055 (2004) 93–98. [13] S. Senel, A. Kassab, Y. Arica, R. Say, A. Denizli, Comparison of adsorption performances of metal-chelated polyamide hollow fibre membranes in lysozyme separation, Colloid Surf. B 24 (2002) 265–275. [14] D. Klint, H. Eriksson, Condition for the adsorption of proteins on ultrastable zeotite Y and its use in protein purification, Protein Expr. Purif. 10 (1997) 247–255. [15] R. Ghosh, S. Silva, Z.F. Cui, Lysozyme separation by hollow-fibre ultrafiltration, Biochem. Eng. J. 6 (2000) 19–24. [16] M. Fang, H. John, A. Fernando, H. John, Study on separation of conalbumin and lysozyme from high concentration fresh egg white at high flow rates by a novel ion-exchanger, Biotechnol. Bioeng. 42 (1993) 1086–1090. [17] G.E. McCreath, R.O. Owen, D.C. Nash, H.A. Chase, Preparation and use of ion-exchange chromatographic supports based on perfluoropolymers, J. Chromatogr. A 773 (1997) 73–83. [18] R. Ghosh, Purification of lysozyme by microporous PVDF membranebased chromatographic process, Biochem. Eng. J. 14 (2003) 109–116. [19] X.D. Tong, X.Y. Dong, Y. Sun, Lysozyme adsorption and purification by expanded bed chromatography with a small-sized dense adsorbent, Biochem. Eng. J. 12 (2002) 117–124. [20] E.B. Altintas, A. Denizli, Monosize poly(glycidyl methacrylate) beads for dye-affinity of lysozyme, Int. J. Biol. Macromol. 38 (2006) 99–106. [21] R.O. Owen, H.A. Chase, Direct purification of lysozyme using continuous counter-current expended bed adsorption, J. Chromatogr. B 757 (1997) 41–49. [22] M.Y. Arica, M. Yilmaz, E. Yalcin, G. Bayramoglu, Affinity membrane chromatography: relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification, J. Chromatogr. B 805 (2004) 315–323. [23] W. Friess, Collagen-biomaterial for drug delivery, Eur. J. Pharm. Biopharm. 45 (1998) 113–136. [24] N.A. Evans, B. Milligan, K.C. Montgomery, Collagen crosslinking: new binding sites for mineral tannage, J. Am. Leather Chem. As. 82 (1987) 86–95. [25] Y. Yokoyama, R. Ishiguro, H. Maeda, M. Mukaiyama, K. Kameyama, K. Hiramatsu, Quantitative analysis of protein adsorption on a planar surface by Fourier transform infrared spectroscopy: lysozyme adsorbed on hydrophobic silicon-containing polymer, J. Colloid Interface Sci. 268 (2003) 23–32. [26] Z.G. Peng, K. Hidajat, M.S. Uddin, Adsorption and desorption of lysozyme on nano-sized magnetic particles and its conformational changes, Colloid Surf. B 35 (2004) 169–174.

93

[27] N. Mcrianne, A. Pierre, L. Susana, K. Maaret, M. Sari, Fractionation of model proteins using their physiochemical properties, Colloid Surf. A 138 (1998) 185–205. [28] X.P. Liao, M.N. Zhang, B. Shi, Collagen fibre immobilized tannins and their adsorption of Au(III), Ind. Chem. Eng. Res. 43 (2004) 2222– 2227. [29] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the pronciple of protein-dye binging, Anal. Biochem. 72 (1976) 248–254. [30] D. Shugar, The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme, Biochem. Biophys. Acta 8 (1952) 302– 309. [31] D. Kint, H. Eriksson, Condition for the adsorption of proteins on ultrastable zeolite Y and it usein protein purification, Protein Expr. Purif. 10 (1997) 247–255. [32] A. Denizli, H. Yavuz, Y. Arica, Momosize and non-porous p(HEMA-coMMA) microparticles designed as dye- and metal-chelate affinity sorbents, Colloid Surf. A 174 (2000) 307–317. [33] G.D. Fasman, Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton FL, 1977. [34] K.D. Jurgens, R. Baumann, Ultrasonic absorption studies of protein-buffer interactions, Eur. Biophys. J. 12 (1985) 217–222. [35] E.S. Hemdan, J. Porath, Development of immobilized metal affinity chromatography. II. Interaction of amino acids with immobilized nickel iminodiacetate, J. Chromatogr. 323 (1985) 255–264. [36] E.S. Hemdan, J. Porath, Development of immobilized metal affinity chromatography. III. Interaction of oligopeptides with immobilized nickel iminodiacetate, J. Chromatogr. 323 (1985) 265–272. [37] Z.E.L. Rass, C. Horvath, Metal chelate-interaction chromatography of proteins with iminodiacetic acid-bonded stationary phases on silica support, J. Chromatogr. 359 (1986) 241–253. [38] M.Y. Arica, A. Denizli, B. Salih, E. Piskin, V. Hasirci, Catalase adsorption onto Cibacron blue F3GA and Fe(III)-derivatized poly(hydroxyethyl methacrylate) membranes and application to a continuous system, J. Membr. Sci. 129 (1997) 65–76. [39] C.W. Cheung, J.F. Porter, G. Mckay, Sorption kinetic analysis for the removal of cadmium ions from effluents using bone char, Water Res. 35 (2001) 605–612. [40] A.G. Ritchie, J. Chem, Alternative to the Elovich equation for kinetics of adsorption of gases on solids, J. Chem. Soc. Faraday Trans. 73 (1977) 1650–1653. [41] G. McKay, Y.S. Ho, Pseudo-second order model for sorption processes, Process Biochem. 34 (1999) 451–465. [42] K.H. Chu, Improved fixed bed models for metal biosorption, Chem. Eng. J. 97 (2004) 233–239. [43] C.M. Griffith, J. Morris, M. Robichaud, M.J. Annen, A.V. McCormick, M.C. Flickinger, Fluidization characteristics of and protein adsorption by porous zirconium oxide particles in a fluidized-bed, J. Chromatogr. A 776 (1997) 179–195. [44] P.A. Belter, E.L. Cussler, W.S. Hu, Bioseparations: Downstream Processing for Biotechnonogy, Wiley, New York, 1988.