Magnetic Polymeric Beads Functionalized with Different Mixed-Mode

Dec 11, 2013 - Veli Cengiz Ozalp,. § and M. Yakup Arica*. ,†. † ... School of Medicine, Istanbul Kemerburgaz University, 34217 Istanbul, Turkey. ...
1 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/IECR

Magnetic Polymeric Beads Functionalized with Different MixedMode Ligands for Reversible Immobilization of Trypsin Gulay Bayramoglu,†,‡ Veli Cengiz Ozalp,§ and M. Yakup Arica*,† †

Biochemical Processing and Biomaterial Research Laboratory, Gazi University, 06500 Teknikokullar-Ankara, Turkey Department of Chemistry, Faculty of Sciences, Gazi University, 06500 Teknikokullar-Ankara, Turkey § School of Medicine, Istanbul Kemerburgaz University, 34217 Istanbul, Turkey ‡

S Supporting Information *

ABSTRACT: In this study, we describe a preparation of magnetic affinity support carrying different ligands for immobilization of trypsin via adsorption. The magnetic support was synthesized in the bead form using glycidylmethacrylate (GMA) and methylmethacrylate (MMA) monomers. Three different ligands (i.e., p-aminobenzoic acid, L-phenylalanine and paminobenzamidine,) were attached on the aminated magnetic beads surface via glutaraldhyde coupling. Specific surface area of the mp(GMA/MMA) beads was found to be 21.4 m2/g. The maximum trypsin adsorption was observed at pH 7.0 for paminobenzoic acid and L-phenylalanine and at pH 8.0 for p-aminobenzamidine carrying ligand. The maximum amounts of the enzyme adsorbed on the p-aminobenzoic acid-, L-phenylalanine-, and p-aminobenzamidine-attached magnetic beads reached 99.6, 84.2, and 75.9 mg/g with an enzyme activity recovery of 69.4, 73.2, and 22.9%, respectively. The L-phenylalanine ligandattached support displayed a higher activity recovery than those of the p-aminobenzoic acid- and the p-aminobenzamidineattached magnetic beads. This carrier showed also very good storage and operational stability. Trypsin immobilized on paminobenzoic acid showed significant activity toward casein. Trypsin could be repeatedly adsorbed and desorbed with all of the ligand-attached beads without a noticeable loss in the adsorption capacity.

■

INTRODUCTION

resistant to microbial degradation and several chemicals. In addition, magnetic separation technique using magnetic polymeric beads is a quick and easy method for sensitive and reliable capture of inorganic or organic pollutants.21 This method is also nonlaborious, cheap, and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization.22 The magnetic character implies that they respond to a magnet, making sampling and collection easier and faster, but their magnetization disappears once the magnetic field is removed. Magnetic separation is relatively rapid and easy, requiring a simple apparatus, compared to centrifugal separation. Recently, there has been increased interest in the use of magnetic carriers in protein purification. Trypsin (EC 3.4.21.4) is a serine protease found in the digestive system, where it breaks down proteins. Trypsin specifically hydrolyzes peptide bonds at the carboxyl side of lysine and arginine residues. It is used for numerous biotechnological processes, such as protein primary structure analysis, to breakdown casein in milk for baby food and to resuspend cells adherent to the cell culture dish wall during the process of harvesting cells.23,24 In this protocol, the preparation and modification of magnetic p(GMA/MMA) beads were described. Three different affinity ligands (i.e., p-aminobenzoic acid, phenylalanine, and p-aminobenzamidine) attached to magnetic beads’ surfaces

For adsorption of proteins, pseudospecific small ligands, dyes, amino acids, and chelated metal ions have been used instead of biospecific ligands.1−4 Small ligand molecules (such as paminobenzamidine, L-histidine, L-phenylalanine, L-glutamic acid, and various dye molecules) have been reported to be fairly selective and efficient ligands for the adsorption of various proteins.5,6 There are many reports that adsorption of proteins on the support surface quantitatively changed, depending on the type of ligands attached.7−10 The adsorption of proteins is a very complex process, which can be determined by several factors. Among these, the chemical structure, surface roughness, the degree of hydrophilicity of surface, electrostatic interactions of the protein molecules with each other and with surface, and the structural stability of protein molecules are the most important.11−13 Enzyme adsorption on the functionalized support has been a popular strategy for most large scale applications because of the easy regeneration of support and no waste after enzyme inactivation.14,15 Poor biocatalytic efficiency of adsorbed enzymes, however, often limits the development of large-scale enzymatic reaction to compete with traditional chemical processes.15−17 Improvements of immobilized enzyme efficiency can be achieved by manipulating the structure of carrier materials for enzyme adsorption. The most important advantages of this method are stability of the activity of adsorbed enzyme and reuse of the enzyme and support material for different purposes because of reversibility of the method.18−20 The acrylic-based support materials in the bead form is almost ideal to perform the adsorption of proteins because it is very stable in a range of buffers from pH 1.0 to 11.0 and © 2013 American Chemical Society

Received: Revised: Accepted: Published: 132

August 13, 2013 November 19, 2013 December 11, 2013 December 11, 2013 dx.doi.org/10.1021/ie402656p | Ind. Eng. Chem. Res. 2014, 53, 132−140

Industrial & Engineering Chemistry Research

Article

6 h. After the reaction period, the beads were filtered and the final HCl concentration in the solution was assayed by a potentiometric titration with 0.05 mol/L NaOH solution. The average size and size distribution of the magnetic beads were determined by screen analysis using molecular sieves. The specific surface area of the beads was measured by a surface area apparatus (Quantachrome Nova 2200 E, U. S. A.) and calculated using the BET (Brunauer, Emmett, and Teller) method. The FTIR spectra of the mp(GMA/MMA)-A, mp(GMA/MMA)-ABAc, mp(GMA/MMA)-PhA, and mp(GMA/MMA)-ABAm were obtained using an FTIR spectrophotometer (Shimadzu, FTIR 8000 Series, Japan). The magnetization curves of the mp(GMA/MMA) and mp(GMA/ MMA)-ABAm beads were determined with a vibrating sample magnetometer (VSM, model 155, Digital Measurement System, Inc., Westwood, MA, U. S. A.). The water content of the magnetic beads was determined at 25 °C using a gravimetric method as described, previously. The amounts of immobilized ligands on the p(GMA/MMA)-GA beads were also determined by measuring the absorbance of the reaction solution before and after coupling reaction of the used ligands at 280 by using a double beam UV/vis spectrophotometer (PG Instrument Ltd., Model T80+; PRC). A calibration curve constructed with each ligand solution of known concentration was used in the calculation of ligand in the solutions (see Figure 1).

were tested for immobilization of trypsin via adsorption. The adsorption conditions (i.e., medium pH and concentration of trypsin) were varied to evaluate their effects on the adsorption capacities of the functionalized magnetic beads. After immobilization of trypsin, the immobilized enzyme activities were determined under various experimental conditions using BApNA and casein as substrates. Additionally, the reusability of each affinity support was studied for trypsin adsorption.

■

MATERIALS AND METHODS Materials. Trypsin (EC 3.4.21.4; from bovine pancreas Type XI; about 7500 U/mg solids), methylmethacrylate (MMA), glycidyl methacrylate (GMA), and ethylene glycol dimethacrylate (EGDMA) were supplied by Sigma-Aldrich Chemical Co. (St. Louis, MO, U. S. A.). Inhibitor from monomers (i.e., MMA, GMA, and EGDMA) was removed using aluminum oxide fine coarse powder (Sigma-Aldrich, Type CG-20) in a column (diameter 2.5 cm; height 20 cm). Ethylene diamine (EDA), glutaraldehyde (GA), p-aminobenzoic acid, Lphenylalanine, and p-aminobenzamidine were supplied from Sigma-Aldrich (Germany). All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany). All solutions were prepared using ultrapure water. Preparation of Magnetic Affinity Beads. The magnetic p(GMA/MMA) beads were prepared in two sequential steps as described previously.25 Briefly, in the first step, ferric-p(GMA/ MMA) beads were prepared via suspension polymerization. In the second step, magnetization of the p(GMA/MMA) beads was performed by a conventional coprecipitation reaction of iron oxide in the beads. During this magnetization reaction, the aminolysis reaction was also taken place by splitting of epoxy groups in to amine and hydroxyl groups. Finally, the aminated and magnetic p(GMA/MMA)-A beads were separated from the reaction medium, washed first in ethanol solution (70%, 250 mL) for 30 min, and then purified water. The beads were finally dried in a vacuum oven at 50 °C and stored at room temperature. The aminated magnetic beads (5.0 g) (i.e., mp(GMA/ MMA)-A) were equilibrated in phosphate buffer (20 mL, 50 mM, pH 8.0) for 6 h and transferred to the same fresh medium containing glutaraldehyde (50 mL, 0.5% (v/v)). The activation reaction was carried out at 25 °C for 6 h while continuously stirring the medium. The excess glutaraldehyde was removed and washed with distilled water, acetic acid (0.1 M, 200 mL), and phosphate buffer solution (0.1 M, pH 7.0). The resulting mp(GMA/MMA)-GA were used for attachment of 4-aminobenzoic acid, L-phenylalanine, and p-aminobenzamidine ligands. The mp(GMA/MMA)-GA beads were transferred in carbonate buffer (0.1 M, pH 10) for 4 h, containing each ligand (2.0 mg/ mL ligand, in 25 mL). The coupling reactions of the ligands on the glutaraldehyde activated magnetic beads were carried out at 22 °C in a shaking water bath for 6 h. Different ligands attached magnetic beads were identified as mp(GMA/MMA)-ABAc, mp(GMA/MMA)-ABAm, and mp(GMA/MMA)-PhA, respectively. Characterization of Magnetic Beads. The available epoxy group content of the mp(GMA/MMA) beads was determined by the pyridine−HCl method as described previously.26 The amount of free amino groups of the mp(GMA/MMA) beads was determined by potentiometric titration by allowing the beads (0.1 g) to soak in water (10 mL) for 24 h. Then, HCl solution (0.1 mol/L, 20 mL) was added to the mixture and incubated in a shaking water bath at 35 °C for

Figure 1. Schematic representation of the preparations of magnetic beads.

Immobilization of Trypsin via Adsorption. The adsorption of trypsin on the mp(GMA/MMA)-ABAc, mp(GMA/MMA)-L-PhA, and mp(GMA/MMA)-ABAm beads was studied in a batch system. The effect of pH on the adsorption capacity of the functionalized magnetic beads was investigated over the pH range 4.0−9.0 at 25 °C. To determine the effect of initial trypsin concentration on the adsorption rate and capacity, the initial concentration of trypsin was varied between 0.2−2.0 mg/L at pH 7.5. Solutions of the trypsin, 133

dx.doi.org/10.1021/ie402656p | Ind. Eng. Chem. Res. 2014, 53, 132−140

Industrial & Engineering Chemistry Research

Article

presented as a percentage of the activity of free enzyme of same quantity. The effects of pH and temperature on the free and immobilized trypsin preparations were studied in the pH range 4.0−9.0 and temperature range 20−60 °C, respectively. The results of dependence on pH and temperature are presented in a normalized form with the highest value of each set being assigned the value of 100% activity. Determination of the Kinetic Constants. The relationship between the initial rate (V) of the enzymatic reaction and BApNA concentration (S) (between 5 and 50 mM) in phosphate buffer (50 mM, pH 7.5) at 25 °C was measured, from which the Michaelis constants Km and Vmax of the free and immobilized trypsin preparations determined. The Km and Vmax values for the free and immobilized trypsin preparations were calculated from Lineweaver−Burk plots using the initial rate of the enzymatic reaction

containing 0.2−2.0 mg/mL, were prepared in phosphate buffer solution. Adsorption experiments were performed by mechanically agitating at 150 rpm, at 25 ± 2 °C for 2.0 h. The adsorption volume was 10 mL, and magnetic affinity beads (50 mg) were used in each test. After magnetic separation, the amount of unadsorbed trypsin in supernatant solutions was analyzed for trypsin content using a double beam UV−vis spectrophotometer (PG Instrument Ltd., Model T80+, PRC) at 280 nm. For each adsorption experiment, the average of three replicates was reported. For each set of data present, standard statistical methods were used to determine the mean values and standard deviations. Confidence intervals of 95% were calculated for each set of samples in order to determine the margin of error. Adsorption Isotherms Studies. Two theoretical isotherm models (Langmuir and Freundlich) were used to analyze the experimental data. The former model is based on the assumption of surface homogeneity such as equally available adsorption sites, monolayer surface coverage, and no interaction between adsorbed species and described by the following equation:

q = qmCeq /Kd + Ceq

1/V = {(K m/Vmax ) ·[1/S] + (1/Vmax )

where [S] is the concentration of substrate, V and Vmax represent the initial and maximum rate of reactions, respectively. Km was the Michaels constant. Casein Hydrolysis Studies. The free and immobilized trypsin on the mp(GMA/MMA)-ABAc and mp(GMA/MMA)L-PhA preparations were incubated with casein solution (1.0 mg/mL, 3.0 mL) prepared in Tris−HCl buffer (pH 7.5, 50 mM) containing 10 mM CaCl2. Samples were preincubated at 25 °C for 5 min before addition of the free and immobilized trypsin preparations. At a predetermined time interval, the reaction was stopped by addition of trichloroacetic acid solution (1.0 mL, 0.1%, v/v) to precipitate the unhydrolyzed casein. The suspension was centrifuged at 15 000 rpm for 10 min. The soluble aromatic aminoacids in the supernatants were then measured at 280 nm. Thermal Stabilities of the Free and Immobilized Trypsin Preparations. The thermal stabilities of free and immobilized trypsin on the p-aminobenzoic acid and phenylalanine functionalized magnetic beads were determined by measuring the residual enzymatic activity of two different temperatures (i.e., 60 and 70 °C) in a phosphate buffer (0.1 M, pH 7.5) for 2 h. After every 15 min time interval, a sample was removed and assayed for enzymatic activity as described above. The results were given as percent activity. Reusability of the Differently Functionalized Magnetic Beads. In order to determine the reusability of the differently functionalized magnetic beads, trypsin adsorption and desorption cycle was repeated six times using the same ligand attached magnetic beads. Trysin desorption from the magnetic beads was carried out with glycine−HCl buffer (0.1 M, pH 2.3) containing 0.5 M NaCl. Desorption medium was stirred magnetically at 150 rpm at 25 °C for 120 min. The equilibrium desorption time was found to be 60 min. The amount of protein in desorption medium was determined as described above. The desorption ratio of trypsin was calculated by using the following expression:

(1)

where q is the adsorbed trypsin concentration (mg/g), Ceq the equilibrium trypsin concentration in solution (mg/mL), qm the maximum amount of adsorbed trypsin per gram of differently functionalized magnetic beads (mg/g), and Kd is the Langmuir equilibrium constant (mL/mg). The Freundlich adsorption isotherm is frequently used to describe the adsorption. It relates to adsorbed concentration as the power function of solute concentration. This empirical equation takes the form: q = KF(C)1/ n

(3)

(2)

where KF and n are the Freundlich constants characteristic of the system. KF and n are indicators of the adsorption capacity and adsorption intensity, respectively. The slope and the intercept of the linear Freundlich equation are equal to 1/n and ln KF, respectively. Activity Assay of Free and Immobilized Trypsin Preparations. Trypsin will hydrolyze ester and amide linkages of several synthetic substrates. Amidase activity of the trypsin was measured by using artificial substrate (N-benzoyl-D-Larginine-p-nitroanilide) BApNA. In the determination of activity of the free enzyme, the reaction medium consisted of Tris−HCl buffer (pH 7.5, 50 mM, 2.5 mL) containing 10 mM CaCl2 and 0.1 mL of BApNA (0.5 M BApNA in DMSO). The reaction mixtures were preincubated in water bath at 25 °C for 5 min, and the assay was started by the addition of the enzyme solution (0.1 mL) into the assay medium. After 5 min, the reaction was stopped by addition of 0.3 mL of 30% acetic acid solution. After enzymatic hydrolysis of BApNA p-nitroanilide is formed. The amount of p-nitroanilide released was monitored via the increase in absorbance at 410 nm, and the rate of formation of p-nitroanilide is proportional to the trypsin activity. So, the activity of enzyme can be calculated using the extinction coefficient of p-nitroanilide (ε = 8270 M−1/cm−1). For the determination of immobilized trypsin activity, 10 mg of trypsin immobilized magnetic beads was introduced to the enzymatic reaction medium instead of free enzyme solution as described above. The activity of the immobilized trypsin was

Desorption ratio (%) = (enzyme released/enzyme adsorbed on the support) × 100 (4) 134

dx.doi.org/10.1021/ie402656p | Ind. Eng. Chem. Res. 2014, 53, 132−140

Industrial & Engineering Chemistry Research

■

Article

RESULTS AND DISCUSSION

Table 1. Some Properties of the Magnetic p(GMA/MMA) Beads

Properties of the Magnetic Beads. The ligand molecules have functional groups such as −SO3H, −NH2, −COOH, −OH, and aromatic rings for ionic, polar, and hydrophobic interactions. Small ligand molecules offer several advantages over other ligands in terms of economy, stability, and ease of immobilization.6 In this work, three different ligands molecules were incorporated on the magnetic beads, viz. p-aminobenzamidine, p-aminobenzoic acid, and phenylalanine. The former ligand has an amidine moiety and is a reversible competitive inhibitor of trypsin, trypsin-like enzymes, and serine proteases. On the other hand, the later two ligands containing free −COOH groups were incorporated on the magnetic beads for ion-exchange interactions with the basic protein trypsin. The poly(methylmetacrylate-co-glycidyl methacrylate) “mp(GMA/MMA)” beads were produced by suspension polymerization in the presence of EGDMA as cross-linker. The preparation route of magnetic beads is presented in Figure 1. After thermal magnetization and amination reaction, the beads were functionalized with three different mixed mode ligands (i.e., p-aminobenzoic acid, L-phenylalanine, and p-aminobenzamidine) for immobilization of trypsin via adsorption (Figure 2). Some properties of the magnetic p(GMA/MMA) beads are summarized in Table 1. The surface morphology of the p(HPMA/EGDMA)-g-p(GMA) beads was investigated by SEM (Figure 3). As seen in this figure, the porous surface

bead size available epoxy group content free amine groups content free aldehyde groups content specific surface area swelling ratio density p-aminobenzoic acid content L-phenylalanin content p-aminobenzamidine content weight fraction of polymer weight fraction of Fe3O4

75−150 μm 1.67 mmol/g 0.87 mmol/g 0.49 mmol/g 21.4 m2/g 38% 1.123 g/cm3 4.78 μmol/g 6.47 μmol/g 7.63 μmol/g 89.2% 10.8%

Figure 3. SEM micrograph of magnetic p(GMA/MMA) beads.

properties of the magnetic beads would favor higher immobilization capacity for the mixed-mode ligands and adsorption of trypsin due to increase in the surface area. A vibrating sample magnetometer was employed to measure the magnetic properties of the functionalized beads. A typical room temperature magnetization curve of mp(GMA/MMA)-A was obtained (data not shown), and the saturation magnetization of the magnetic beads was found to be 28.6 emu/g. Fast separation of dispersed magnetic beads from the solution in the presence of an external magnetic field was also easily visible (Figure 4). Therefore, the presented magnetic beads can be easily separated from reaction medium within a few seconds by a conventional permanent magnet. When the applied magnetic force is removed, the magnetic beads can easily be dispersed by simple shaking.

Figure 2. Schematic representation of ligands attachments and enzyme immobilization.

Figure 4. Separation of dispersed magnetic beads from the medium with an external magnet. 135

dx.doi.org/10.1021/ie402656p | Ind. Eng. Chem. Res. 2014, 53, 132−140

Industrial & Engineering Chemistry Research

Article

Effect of pH on Trypsin Adsorption Efficiency. The pH value of the solution affects both external charge distribution of trypsin molecules and the functional groups on the differently functionalized magnetic beads. In order to investigate the effects of pH on the trypsin adsorption efficiency and capacity of mp(GMA/MMA)-ABAc, mp(GMA/MMA)-PhA, and mp(GMA/MMA)-ABAm beads, the medium pH was changed between pH 4.0 and 9.0. The maximum trypsin adsorption for mp(GMA/MMA)-ABAc and mp(GMA/MMA)-PhA beads was observed at around pH 7.5. The maximum adsorption pH value for mp(GMA/MMA)-ABAm beads was around 8.0 (Figure 5). As seen in this figure, the electrostatic interaction

Figure 6. Effect of initial trypsin concentration on the immobilization efficiency on the different ligands functionalized magnetic beads.

benzamidine ligand attached magnetic beads. The reason for this may probably be due to the interactions of trypsin through the active side with on the p-aminobenzamidine ligand, which blocked the substrate from reaching the active sites of the enzyme. It should be note that p-aminobenzamidine is considered as the prototype of trypsin-like serine protease inhibitors.27 Adsorption Isotherms. The adsorption isotherm was obtained from a batch experiment at 25 °C, and the results are presented in Table 2. The corresponding semireciprocal plots and Scatchard plots gave linear plots for the mp(GMA/ MMA)-ABAc, mp(GMA/MMA)-PhA, and mp(GMA/MMA)ABAm beads, and the correlation coefficients of semireciprocal plots (R2) was greater than 0.970 for all the tested adsorbents, indicating that the Langmuir model could be applied in these systems (Table 2). The calculated qmax values were very close to the experimental qexp values (Table 2). Therefore, the adsorption of trypsin onto all the tested adsorbents could be described in terms of the Langmuir model. The apparent dissociation constant (Kd) estimated from the intercept is a measure of stability of the complex formed between a protein and a ligand under specified experimental conditions. For example, a large Kd value indicates that the protein has a low binding affinity for the adsorbent. The Kd values were the same order of magnitude (around 1.0 × 10−6 M) for the mp(GMA/ MMA)-ABAc, mp(GMA/MMA)-PhA, and mp(GMA/MMA)ABAm beads. The Freundlich plots for trypsin adsorption on the mp(GMA/MMA)-ABAc, mp(GMA/MMA)-PhA, and mp(GMA/MMA)-ABAm beads at 25 °C are presented in Table 2. Values of n > 1 for the all the tested ligands indicate positive co-operativity in binding and heterogeneous nature of adsorption. The magnitude of KF for the adsorbents showed easy adsorption of trypsin from the adsorption medium. Effect of Temperature and pH on the Catalytic Activity. The temperature dependence of activities of free and immobilized trypsin on the p-aminobenzoic acid and phenylalanine functionalized magnetic beads were studied in Tris−HCl buffer (50 mM, pH 7.5) in a temperature range between 20 and 60 °C as shown in Figure 7a. The behavior of the enzyme activity with temperature was as expected: increased temperature caused an increased activity, up to optimum reaction temperature and then overlapped with the

Figure 5. Effect of pH on the adsorption efficiency of trypsin on the different ligands functionalized magnetic beads.

between trypsin and the benzoic acid and phenylalanine ligands was the strongest at around pH 7.5, whereas benzamidine gave the highest adsorption at around pH 8.0. At pH 7.5, the ionic interactions between trypsin molecules and p-aminobenzoic acid or L-phenylalanine ligands may result from both the ionization states of functional groups on the ligands (i.e., primary, secondary amino and carboxyl groups and the amino acid side chains of the trypsin molecules. It should be noted that the large enzyme loading on all the tested supports at increasing pH could be attributed to the high positive charge density on the surface of enzyme and mixed interactive groups of the ligands molecules, which depended on the pH of the medium. On the other hand, significant decreases in the adsorption capacities at low pH values could be explained by the electrostatic repulsion effect between the tested supports and positively charged enzyme molecules.15 Effect of Initial Trypsin Concentration on Adsorption Capacities of Supports. The effect of initial trypsin concentration on the adsorption behavior is presented in Figure 6. As seen in this figure, the amount of the trypsin adsorbed on all the tested supports increased steadily with the increasing of initial trypsin concentration in the medium, then reached a plateau value in each of them. As seen in Figure 6, the experimental trypsin adsorption isotherms are very steep at low trypsin concentration and reached a plateau at about a 1.0 mg/ mL initial trypsin concentration. The maximum enzyme loading on the p-aminobenzoic acid, L-phenylalanine, and paminobenzamidine attached magnetic beads reached 99.6, 84.2, and 75.9 mg/g of beads with enzyme activity recovery of 69.4, 73.2, and 22.9%, respectively. The activity recoveries of the trypsin adsorbed on the p-aminobenzoic acid and L-phenylalanine were higher compared to that of the p-amino136

dx.doi.org/10.1021/ie402656p | Ind. Eng. Chem. Res. 2014, 53, 132−140

Industrial & Engineering Chemistry Research

Article

Table 2. Freundlich and Langmuir Isotherm Parameters at 25 °C for Adsorption of Trypsin on the p-Aminobenzoic Acid, pAminobenzamidin, and L-Phenylalanine Ligands Immobilized Magnetic Beads Freundlich isotherm

Langmuir isotherm

ligand

qexp (mg/g)

KF

n

R2

qm (mg/g)

Kd x 106 (M)

R2

ΔG (kJ/mol)

p-aminobenzoic asit p-aminobenzamidin L-phenylalanine

99.6 75.9 84.2

103.9 77. 6 89.95

2.53 1.44 1.87

0.954 0.980 0.925

106.6 81.2 98.1

4.62 19.40 9.77

0.998 0.970 0.991

−30.4 −26.8 −28.7

and by conformational changes of trypsin during the immobilization procedure. Thus, the protein layers might be preventing the diffusion of the substrates or products into the pore space of the beads. Vmax values were found to be 7345, 5115, and 5387 U/mg of enzyme for free and immobilized trypsin on the mp(GMA/MMA)-ABAc and mp(GMA/MMA)PhA magnetic beads, respectively. The efficiency factor (EF) was used for comparison of Vmax values between free and the immobilized enzyme. The EF was calculated by the following equation: Figure 7. (a)Effect of temperature on catalytic activity of the free and immobilized trypsin preparations and (b) effect of pH on catalytic activity of the free and immobilized trypsin preparations.

EF = Vmax ,immobilized enzyme /Vmax ,free enzyme

The efficiency factors for the immobilized trypsin on the mp(GMA/MMA)-PhA and mp(GMA/MMA)-ABAc beads were calculated to be 0.696 and 0.733, respectively. The amount of trypsin adsorbed on the mp(GMA/MMA)-PhA (99.6 mg/g) was about 16% higher compared to mp(GMA/ MMA)-ABAc (84.2 mg/g) beads. Thus, higher amounts of trypsin were immobilized on pores of mp(GMA/MMA)-PhA, and as a consequence, its pores were blocked at higher degrees. This could cause a restriction of the diffusion of the substrate and the products into and out of the pore spaces of beads. Consequently, a lower catalytic efficiency was observed for magnetic beads loaded with higher amount of enzyme (i.e., mp(GMA/MMA)-PhA). Additionally, the effectiveness factor is lower than 1.0 for both adsorbed enzyme systems, which shows that immobilization via adsorption affected the diffusion of substrates and products. Casein Digestion Efficiency with Trypsin Preparations. Trypsin is commonly used in proteomics experiments to digest proteins into peptides. Trypsin is particularly suited for this purpose because it has a very well-defined specificity. Trypsin specifically hydrolyzes peptide bonds at the carboxyl side of lysine and arginine residues. The usability and efficiency of the immobilized trypsin for protein digestion were tested with a model protein (i.e., casein). Figure 8 presents the action

deactivation of the enzyme preparations. Optimum temperature was found at about 40 °C for free trypsin and at 45 °C for the immobilized trypsin preparations. These results show that immobilized trypsin activities were higher than those of the free trypsin at higher temperature. Additionally, the noncovalent multipoint interactions via amino groups of the enzyme might also reduce the conformational flexibility for the binding to its substrate.28−30 Figure 7b shows the effect of the pH on the BApNA hydrolysis catalyzed by the free and immobilized trypsin on the p-aminobenzoic acid and phenylalanine functionalized magnetic beads. Optimal conversion was obtained at pH 7.5 for the free enzyme, and the optimum pH value for the immobilized enzyme shifted to more alkaline region of about pH 8.0. Additionally, the immobilized trypsin preparations showed higher activity in both the acidic and basic pH regions compared to the free trypsin. This phenomenon is frequently associated with proton donor or acceptor groups in the support material. These groups act by adjusting pH in the surrounding environment of the immobilized enzyme. In the present case, carboxyl groups and secondary amine from p-aminobenzoic acid and L-phenylalanine and primary and secondary amino groups from p-aminobenzamidine provide an environment able to simultaneously donate and accept protons from bulk solution. Other researchers have reported similar observations upon immobilization of trypsin and other enzymes.31−33 Kinetic Parameters. For trypsin enzyme in free-solution form or immobilized on the mp(GMA/MMA)-ABAc and mp(GMA/MMA)-PhA beads, the kinetic parameters Km and Vmax were determined from Lineweaver−Burk plots at constant temperature and pH,while varying the substrate concentration. The calculated Km values were 9.3, 16.8, and 21.7 mM for the free and immobilized trypsin on the mp(GMA/MMA)-ABAc or mp(GMA/MMA)-PhA beads, respectively. These values show 1.81-fold and 2.33-fold decreases in the affinity of the enzyme compared to those of the free form after immobilization on the mp(GMA/MMA)-ABAc and mp(GMA/MMA)PhA beads. The reduced affinity values can be explained by possible diffusional limitations imposed on the flow of substrate and product molecules around the pores of the magnetic beads

Figure 8. Hydrolysis rate of casein by free and immobilized trypsin in batch mode. 137

dx.doi.org/10.1021/ie402656p | Ind. Eng. Chem. Res. 2014, 53, 132−140

Industrial & Engineering Chemistry Research

Article

Regeneration of Supports. For reversible immobilization of enzyme, the supports should be easily regenerated from the immobilized enzyme by desorption after inactivation of immobilized enzyme upon use. The recovered supports can be recharged with the fresh enzyme via adsorption. At alkaline pH conditions (pH 7.5), strong adsorption between trypsin and p-aminobenzoic acid and the phenylalanine ligand functionalized magnetic beads was observed. Although the pH value of the adsorption medium was around 2.5, more than 95% of total adsorbed enzyme on the support was eluted in the presence of 0.5 M NaCl. The interactions between the enzyme and the paminobenzoic acid and phenylalanine functionalized beads in the alkaline pH region could be attributed to high density of charged groups on both the enzyme and the p-aminobenzoic acid and phenylalanine ligand. On the other hand, the significant decrease of the binding interactions at low pH could be due to electrostatic enzyme-support repulsion. Tripsin adsorption capacities were not changed significantly during six successive adsorption−desorption cycles of the p-aminobenzoic acid and phenylalanine ligand functionalized magnetic beads (Figure 10). These results showed that the mp(GMA/MMA)-

of free and the immobilized trypsin preparations (i.e., mp(GMA/MMA)-ABAc and mp(GMA/MMA)-PhA) on casein. In this case, the enzymatic reaction rate was determined by the increase in the absorbance at 280 nm. The increase of absorbance is caused by an increase in the content of small polypeptides that are soluble in trichloroacetic acid. For comparison, the hydrolysis of casein was also performed by free trypsin in solution. Casein hydrolysis rate with free trypsin was higher than those of the immobilized trypsin preparations. The full casein hydrolysis occurred around 150 and 330 min with the free and the immobilized trypsin preparations, respectively. The reduced activity of immobilized trypsin toward high molecular weight substrates has been referred by several researchers in the literature.34−36 The result indicated that the trypsin immobilized on the mp(GMA/MMA)-ABAc and mp(GMA/MMA)-PhA) beads could be practically applied in protein digestion and primary structure identification. The immobilized trypsin preparations could be placed in cartridges and used in automated peptide maps analysis in a continuous system. Thermal Stabilities of the Free and Immobilized Trypsin. Effect of temperature on the stabilities of free and immobilized trypsin preparations are shown in Figure 9. The

Figure 10. Adsorption capacities of p-aminobenzoic acid and phenylalanine ligand immobilized magnetic beads to trypsin after each cycle of cleaning.

ABAc and mp(GMA/MMA)-PhA beads can be repeatedly used in enzyme immobilization without detectable losses in their initial adsorption capacities.

■

Figure 9. Thermal stability of the free and immobilized trypsin at 60 and 70 °C.

CONCLUSION It was shown that the pH and the initial concentration of enzyme had important effects on the adsorption equilibrium. The same magnetic support with different functional ligands was suitable for adsorption of trypsin. It should be noted that the retained activity of an immobilized enzyme is the most important property. It is affected by both the amount of enzyme immobilized on the support and the accessibility of active sites of the enzyme to its substrate. From this point of view, p-aminobenzamidine ligand functionalized magnetic beads retained a small activity compared to the p-aminobenzoic acid and L-phenylalanine ligands. Trypsin immobilized on mp(GMA/MMA)-ABAc and mp(GMA-co-MMA)-PhA beads showed significant activities toward casein, and thus, they can be used for peptide production studies and especially for proteomic applications. The regenerated mp(GMA/MMA)ABAc and mp(GMA/MMA)-PhA beads can be reused for the reversible immobilization of the fresh enzyme. Thus, the

pattern of heat stabilities of the immobilized trypsin preparations indicated that smaller rates of thermal inactivation were observed for the immobilized trypsin on the on the mp(GMA/MMA)-ABAc and mp(GMA/MMA)-PhA beads compared to the free enzyme. At 60 °C, the free enzyme retained 38% of its initial activity after 120 min of heat treatment, whereas the immobilized enzyme on the mp(GMA/ MMA)-ABAc and mp(GMA/MMA)-PhA beads showed significant resistance to thermal inactivation (retaining about 72% and 75% of their initial activities after the same period). Furthermore, incubation at 70 °C inactivated the soluble enzyme after 60 min, but the enzyme immobilized on the mp(GMA/MMA)-ABAc and mp(GMA/MMA)-PhA beads retained 40% and 44% of their initial activities. Increased thermal stability of immobilized trypsin preparations has been reported in the literature.37−39 138

dx.doi.org/10.1021/ie402656p | Ind. Eng. Chem. Res. 2014, 53, 132−140

Industrial & Engineering Chemistry Research

Article

(12) Kang, K.; Kan, C.; Yeung, A.; Liu, D. The properties of covalently immobilized trypsin on soap-free P(MMA-EA-AA) latex particles. Macromol. Biosci. 2005, 5, 344−351. (13) Wang, Y.; Guan, Y.; Yang, Y.; Yu, P.; Huang, Y. Enhancing the stability of immobilized catalase on activated carbon with gelatin encapsulation. J. Appl. Polym. Sci. 2013, 130, 1498−1502. (14) Arica, M. Y.; Soydogan, H.; Bayramoglu, G. Reversible immobilization of Candida rugosa lipase on fibrous polymer grafted and sulfonated p(HEMA/EGDMA) beads. Bioprocess. Biosyst. Eng. 2010, 33, 227−236. (15) Bayramoglu, G.; Gursel, I.; Yilmaz, M.; Arica, M. Y. Immobilization of laccase on itaconic acid grafted and Cu(II) ion chelated chitosan membrane for bioremediation of hazardous materials. J. Chem. Technol. Biotechnol. 2012, 87, 530−539. (16) Kuo, C.-H.; Chen, G.-J.; Twu, Y.-K.; Liu, Y.-C.; Shieh, C.-J. Optimum lipase immobilized on diamine-grafted PVDF membrane and its characterization. Ind. Eng. Chem. Res. 2012, 51, 5141−5147. (17) Andre, J.; Borneman, Z.; Wessling, M. Enzymatic conversion in ion-exchange mixed matrix hollow fiber membranes. Ind. Eng. Chem. Res. 2013, 52, 8635−8644. (18) Arica, M. Y.; Bayramoglu, G. Invertase reversibly immobilized ontopolyethylenimine-grafted poly(GMA-MMA) beads for sucrose hydrolysis. J. Mol. Catal. B: Enzym. 2006, 38, 131−138. (19) Bayramoglu, G.; Altintas, B.; Arica, M. Y. Immobilization of glucoamylase onto polyaniline grafted magnetic hydrogel via adsorption and adsorption/cross-linking. Appl. Microbiol. Biotechnol. 2013, 97, 1149−1159. (20) Siodmiak, T.; Ziegler-Borowska, M.; Marszatt, M. P. Lipaseimmobilized magnetic chitosan nanoparticles for kinetic resolution of (R,S)-ibuprofen. J. Mol. Catal. B: Enzym. 2013, 94, 7−14. (21) Bayramoglu, G.; Arica, M. Y. Preparation of comb-type magnetic beads by surface-initiated ATRP: modification with nitrilotriacetate groups for removal of basic dyes. Ind. Eng. Chem. Res. 2012, 51, 10629−10640. (22) Liu, X.; Chen, X.; Li, Y.; Wang, X.; Peng, X.; Zhu, W. Preparation of superparamagnetic Fe3O4@alginate/chitosan nanospheres for Candida rugosa lipase immobilization and utilization of layer-by-layer assembly to enhance the stability of immobilized lipase. ACS Appl. Mater. Interfaces 2012, 4, 5169−5178. (23) Wang, M.-F.; Mu, Y.-Y.; Qi, W.; Su, R. X.; He, Z. M. Intensive protein digestion using cross-linked trypsin aggregates in proteomics analysis. ChemPlusChem 2013, 78, 407−412. (24) Calleri, E.; Temporini, C.; Gasparrini, F.; Simone, P.; Villani, C.; Ciogli, A.; Massolini, G. Immobilized trypsin on epoxy organic monoliths with modulated hydrophilicity: Novel bioreactors useful for protein analysis by liquid chromatography coupled to tandem mass spectrometry. J. Chromatogr. A 2011, 1218, 8937−8945. (25) Bayramoglu, G.; Tunali, Y.; Arica, M. Y. Immobilization of betagalactosidase onto magnetic poly(GMA-MMA) beads for hydrolysis of lactose in bed reactor. Catal. Commun. 2007, 8, 1094−1101. (26) Sidney, S. Quantitative organic analysis, 3rd ed.; Wiley: New York; 1967. (27) Venturini, G.; Menegatti, E.; Ascenzi, P. Competitive inhibition of nitric oxide synthase by p-aminobenzamidine, a serine proteinase inhibitor. Biochem. Biophys. Res. Commun. 1997, 232, 88−90. (28) Chen, G.-J.; Kuo, C.-H.; Chen, C.-I.; Yu, C.-C.; Shieh, C.-J.; Liu, Y.-C. Effect of membranes with various hydrophobic/hydrophilic properties on lipase immobilized activity and stability. J. Biosci. Bioeng. 2012, 113, 166−172. (29) Fernandez-Lafuente, R.; Rodriguez, V.; Mateo, C.; FernandezLorente, G.; Arminsen, P.; Sabuquillo, P.; Guisan, J. M. Stabilization ofenzymes D-amino acid oxidase/against hydrogen peroxide via immobilizationand post-immobilization techniques. J. Mol. Cat. B: Enzym. 1999, 7, 173−179. (30) Fernandez-Lafuente, R. Lipase from Thermomyces lanuginosus: Uses and prospects as an industrial biocatalyst. J. Mol. Catal. B: Enzym. 2010, 62, 197−212. (31) Bayramoglu, G.; Hazer, B.; Altintas, B.; Arica, M. Y. Covalent immobilization of lipase onto amine functionalized polypropylene

presented method can provide economic advantages for largescale biotechnological applications.

■

ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*M. Y. Arica. Phone: +90 312 709 7878. E-mail: g_ [email protected], [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by Scientific and Technological Research Council of Turkey (TUBITAK), grant number 107T799. The authors would like to thanks to H. Soydogan and B. Altıntas for their technical assistance in the preparation of magnetic beads and adsorption and purification studies of trypsin.

■

REFERENCES

(1) Anirudhan, T. S.; Rejeena, S. R. Adsorption and hydrolytic activity of trypsin on a carboxylate-functionalized cation exchanger prepared from nanocellulose. J. Colloid Interface Sci. 2012, 381, 125− 136. (2) Oktem, H. A.; Bayramoglu, G.; Ozalp, V. C.; Arica, M. Y. Single step purification of recombinant Thermus aquaticus DNA-polymerase using DNA-aptamer immobilized novel affinity magnetic beads. Biotechnol. Prog. 2007, 23, 146−154. (3) Ansari, S. A.; Husain, Q. Potential applications of trypsin immobilized on/in nano materials: A review. Biotechnol. Adv. 2012, 30, 512−523. (4) Sun, L.; Liang, H.; Yuan, Q.; Wang, T.; Zhang, H. Study on a carboxyl-activated carrier and its properties for papain immobilization. J. Chem. Technol. Biotechnol. 2012, 87, 1083−1088. (5) Bayramoglu, G.; Karagoz, B.; Altintas, B.; Arica, M. Y.; Bicak, N. Poly(styrene-divinylbenzene) beads surface functionalized with diblock polymer grafting and multi-modal ligand attachment: Performance of reversibly immobilized lipase in ester synthesis. Bioprocess Biosyst. Eng. 2011, 34, 735−746. (6) Bayramoglu, G.; Erdogan, H.; Arica, M. Y. Studies of adsorption of alkaline trypsin by poly(methacrylic acid) brushes on chitosan membranes. J. Appl. Polym. Sci. 2008, 108, 456−465. (7) Pecova, M.; Sebela, M.; Markova, Z.; Polakova, K.; Cuda, J.; Safarova, K.; Zboril, R. Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion. Nanotechnology 2013, 24, 125102. (8) Peng, G.; Zhao, C.; Liu, B.; Ye, F.; Jiang, H. Immobilized trypsin onto chitosan modified monodisperse microspheres: A different way for improving carrier’s surface biocompatibility. Appl. Surf. Sci. 2012, 258, 5543−5552. (9) Sun, J.; Ma, H.; Liu, Y.; Su, Y.; Xia, W.; Yang, Y. Improved preparation of immobilized trpsin on superparamagnetic nanoparticles decorated with metal ions. Colloids Surf., A 2012, 414, 190−197. (10) Arica, M. Y.; Bayramoglu, G. Polyethyleneimine-grafted poly (hydroxyethyl methacrylate-co-glycidyl methacrylate) membranes for reversible glucose oxidase immobilization. Biochem. Eng. J. 2004, 20, 73−77. (11) Liu, Y. Z.; Feng, Z. C.; Shao, Z. Z.; Chen, X. Chitosan-based membrane chromatography for protein adsorption and separation. Mater. Sci. Eng., C 2012, 32, 1669−1673. 139

dx.doi.org/10.1021/ie402656p | Ind. Eng. Chem. Res. 2014, 53, 132−140

Industrial & Engineering Chemistry Research

Article

membrane and its application in green apple flavor (ethyl valerate) synthesis. Process Biochem. 2011, 46, 372−378. (32) Liu, Q.; Hua, Y.; Kong, X.; Zhang, C.; Chen, Y. Covalent immobilization of hydroperoxide lyase on chitosan hybrid hydrogels and production of C6 aldehydes by immobilized enzyme. J. Mol. Catal. B: Enzym. 2013, 95, 89−98. (33) Wu, X. M.; Zhou, C.; Zhu, S. M.; Cen, T.; Jiang, B.; Shen, S. B. Improved method for immobilization of trypsin to enhance catalytic performance of trypsin in confined nanospaces. J. Chem. Eng. Jpn. 2010, 43, 595−602. (34) Monzo, A.; Sperling, E.; Guttman, A. Proteolytic enzymeimmobilization techniques for MS-based protein analysis. TrAC, Trends Anal. Chem. 2009, 28, 854−864. (35) Miao, A. Z.; Dai, Y.; Ji, Y. H.; Jiang, Y. X.; Lu, Y. Liquidchromatographic and mass-spectrometric identification of lens proteins using microwave-assisted digestion with trypsin-immobilized magnetic nanoparticles. Biomed. Biophys. Res. Commun. 2009, 380, 603−608. (36) Sakikawa, T.; Shimazaki, Y. Enzymolysis of high density lipoprotein with a combination of membrane-immobilized esterase and trypsin. J. Pharm. Biomed. Anal. 2012, 71, 179−182. (37) Hong, J.; Gong, P.; Xu, D.; Dong, L.; Yao, S. Stabilization of αchymotrypsin by covalent immobilization on amine-functionalized superparamagnetic nanogel. J. Biotechnol. 2007, 128, 597−605. (38) Netto, C. G. C. M.; Toma, H. E.; Andrade, L. H. Superparamagnetic nanoparticles as versatile carriers and supporting materials for nzymes. J. Mol. Catal. B: Enzym. 2013, 85−86, 71−92. (39) Karagoz, B.; Bayramoglu, G.; Altintas, B.; Bicak, N.; Arica, M. Y. Poly(glycidyl methacrylate)-polystyrene diblocks copolymer grafted nanocomposite microspheres from surface-initiated atom transfer radical polymerization for lipase immobilization: Application in flavor ester synthesis. Ind. Eng. Chem. Res. 2010, 49, 9655−9665.

140

dx.doi.org/10.1021/ie402656p | Ind. Eng. Chem. Res. 2014, 53, 132−140