Surface-Initiated Ring-Opening Polymerization of Poly(2-methyl-2

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Surface-Initiated Ring-Opening Polymerization of Poly(2-methyl-2oxazoline) from Poly(bromoethyl methacrylate/methyl methacrylate) Microspheres and Modification into PEI: Immobilization of α‑Amylase by Adsorption and Cross-Linking Gulay Bayramoglu,*,†,‡ Bunyamin Karagoz,§ Niyazi Bicak,§ and M. Yakup Arica‡ †

Department of Chemistry, Faculty of Sciences, and ‡Biochemical Processing and Biomaterial Research Laboratory, Gazi University, 06500 Teknikokullar, Ankara, Turkey § Department of Chemistry, Istanbul Technical University, Maslak 34469, Istanbul, Turkey ABSTRACT: Bromide-functionalized microspheres were prepared by the suspension polymerization of bromoethyl methacrylate (BEMA), methyl methacrylate (MMA), and ethylene glycol dimethacrylate (EGDMA). The microspheres were employed as a solid macroinitiator for the ring-opening polymerization of 2-methyl 2-oxazoline. The surface brushes were then converted into poly(ethylenimine) (PEI) by simple hydrolysis with HCl and then NaOH as inferred from chemical analyses and FTIR spectra. The microspheres were used for the immobilization of α-amylase through adsorption and covalent cross-linking. The optimum pH for free enzyme was 6.0 and shifted by about 1.0 unit to the acidic pH range upon immobilization. Compared to free α-amylase, the immobilized enzyme preparations were found to exhibit better tolerance to variations in pH and temperature, as well as improved storage stabilities. The activity retentions of the adsorbed and cross-linked enzyme preparations were found to be 69% and 53%, respectively. Furthermore, the cross-linked enzyme was reused for six consecutive cycles without any activity loss.

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Immobilization can be defined as the fixation of biological materials (e.g., proteins, organelles, or microorganisms) through covalent or noncovalent bonds on solid supports.20−27 The immobilization of enzymes on the surface of support materials might have a higher commercial potential than entrapment or encapsulation. α-Amylase (EC 3.2.1.1; 1,4-α-Dglucan glucanohydrolase), produced from the genus Bacillus, has been extensively studied for industrial applications in the sugar, brewing, alcohol, and detergent industries.24,28−30 αAmylase is used to hydrolyze α-glycoside bonds of polysaccharides, such as starch and glycogen. The hydrolyzed products are widely applied in the food, paper, and textile industries. Amylases have not been entrapped in matrixes as frequently as the other enzymes because they can act on macromolecular substrates such as starch. Entrapment or encapsulation generally reduces the recovered enzyme activity as a result of the restriction of diffusion of the high-molecularweight substrate. On the other hand, immobilization of this enzyme on the surface of supports through adsorption and/or covalent linkage can increase the stability of the enzyme through multipoint interactions and decrease mass-transfer limitations. Additionally, the enzyme-support can be easily separated from the reaction medium by simple filtration.31−33 In the present work, we synthesized bromoethyl-functionalized methacrylate-based cross-linked microspheres by suspension polymerization. The bromoethyl surface groups were

INTRODUCTION

Solid-supported resins have attracted great attention because of their potential applications in many different roles such as catalysts, biomolecules, and reagent carriers.1−3 Expensive biomolecules and catalyst immobilization systems have crucial importance because of the easy purification of the reaction medium, enabling it to be used many times by regeneration. In particular, enzyme immobilization on a solid support increases the stability of the enzyme to external conditions such as pH and temperature.4−6 Many different approaches are used to obtain polymer-based cross-linked microspheres. Generally, dispersion systems such as suspension, emulsion, and precipitation polymerization systems are applied for synthesizing microspheres.7−9 The crucial task is to anchor functional groups on the surface of the solid support for suitable conjugations with either enzymes or biomolecules. In the literature, mainly two different approaches are used for obtaining functional groups on support surfaces. The first of these is postmodification of the support surfaces, but this approach is neither suitable for obtaining dense functionalization nor easy for tethering functional groups on surfaces.10−13 The second is direct polymerization with the desired functional monomer. The advantage of this approach is that the density of the functionality on the surface can be arranged by hand, although some functionality is embedded in the support materials.14 Many different functional monomers, such as glycidyl methacrylate, chloromethylstyrene, and hydroxyethyl methacrylate, have been used in direct polymerization reactions for obtaining functionalized cross-linked microspheres.7,15−19 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 14263

June 17, 2014 August 19, 2014 August 25, 2014 August 25, 2014 dx.doi.org/10.1021/ie502428q | Ind. Eng. Chem. Res. 2014, 53, 14263−14271

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solution (4 mL, 65%) and mixed with AgNO3 solution (4 mL, 1.0 mol L−1). The resulting white AgBr precipitate was filtered, washed with water, and dried at 40 °C under reduced pressure. The obtained dry AgBr weighed 0.068 ± 0.0018 g, indicating 1.45 mmol of bromide per gram of cross-linked polymer. Grafting of 2-Methyl 2-oxazoline onto the Surface of the p(BEMA/MMA) Microspheres through SurfaceInitiated Ring-Opening Polymerization. Surface-initiated ring-opening polymerization (SI-ROP) was carried out as follows: p(BEMA/MMA) microspheres (6 g), dried acetonitrile (30 mL), and freshly distilled 2-methyl 2-oxazoline (15 mL) were mixed in a flame-dried flask (100 mL) under a nitrogen atmosphere. The reaction vessel was sealed, and the reaction was conducted at 110 °C under gentle stirring (400 rpm) for 24 h. At the end of the reaction, the mixture was cooled and poured into distilled water. After filtration, the microspheres were washed with distilled water (3 × 40 mL) and ethanol (30 mL) to remove any residual reactant. Finally, the microspheres were dried at 50 °C under reduced pressure for 24 h. The dry product weighed 10.44 g, implying a minimum grafting yield of 74%. For precise assignment of the grafting yield, the amine content of its hydrolysis product was determined by acid titration (see below). Hydrolysis of Grafted Poly(oxazoline) Brushes. The p(BEMA/MMA) microspheres with grafted poly(oxazoline) brushes [p(BEMA/MMA)-g- p(oxazoline)] (about 10g) were transferred to a flask (100 mL) containing HCl solution (40 mL, 4.0 mol L−1), and the solution was boiled for 48 h under a reflux condenser with stirring at a constant rate. After the completion of the reaction period, the mixture was filtered and washed several times with water to remove impurities. Then, the hydrolyzed solid product was allowed to interact with NaOH solution (25 mL, 4 mol L−1) for 24 h. The resulting product was filtered, washed with distilled water (5 × 100 mL), and dried under reduced pressure at 40 °C for 24 h. The dry product weighed 7.2 g. The resulting microspheres hereafter are denoted as p(BEMA/MMA)-g-PEI microspheres for short. The weight measurements and FTIR spectra showed that approximately all of the oxazoline units on the microspheres were converted into ethylene imine groups. In other words, full hydrolysis was accomplished easily by the present method. The amine content of the hydrolysis product was estimated by acid titration, in which 0.2 g of the dry product was swelled in distilled water (5 mL), and HCl solution (5 mL, 4.0 mol L−1) was added to this mixture. The mixture was stirred for 24 h at room temperature and filtered. The filtrate and washings were combined and made up to 50 mL in a volumetric flask. Ten milliliters of this solution was titrated with NaOH solution (0.1 mol L−1), and the secondary amine content was found to be 6.875 mmol per gram of hydrolyzed polymer. Assuming full hydrolysis, this value implies 0.296 g of graft layer per 0.704 g of core microsphere. Because the bromide groups of the starting polymer were hydrolyzed to OH groups, 1.0 g of the initial polymer with 1.45 mmol of bromide would correspond to 0.908 g. The grafting degree prior to hydrolysis would be 6.875/(0.704/0.908) = 8.86 mmol g−1. Considering the 85 g mol−1 mass of the oxazoline repeating unit, this corresponds to 75.4% grafting. Immobilization Studies. The p(BEMA/MMA)-g-PEI microspheres were used for the immobilization of α-amylase through adsorption and cross-linking. The effect of pH on the adsorption capacity of the p(BEMA/MMA)-g-PEI microspheres for α-amylase was studied at various pH values in

then used as initiation sites for the ring-opening polymerization of 2-methyl 2-oxazoline. Subsequently, oxazoline brushes were hydrolyzed to obtain linear PEI as in our previous study.34 This work thus demonstrates that poly(bromoethyl methacrylate/ methyl methacrylate) [p(BEMA/MMA)] microspheres are suitable for the simple preparation of amine-functionalized surface brushes. The size and structure of the microspheres were characterized using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and analytical methods. The immobilization of α-amylase was realized by two different methods (i.e., adsorption and crosslinking). Subsequently, the thermal, operational, and storage stabilities and kinetic behaviors of both immobilized α-amylase preparations were determined batchwise. In addition, the crosslinked immobilized α-amylase was applied for the hydrolysis of starch in a continuous reactor.

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EXPERIMENTAL SECTION Materials. Bromoethyl methacrylate (BEMA) was synthesized as described in the literature35 and redistilled under a vacuum before use. Methyl methacrylate (MMA, 99%), and ethylene glycol dimethacrylate (EGDMA, 99%) were distilled prior to use. 2,2-Azobis(isobutyronitrile) (AIBN, 98%) was purified by recrystallization from methanol. 2-Methyl 2oxazoline was freshly distilled over CaH2 under reduced pressure just before use. α-Amylase from Bacillus sp. (type IIA, lyophilized powder, ≥1500 units/mg off protein), starch, maltose, 3,5-dinitrosalicylic acid (DNSA), poly(N-vinylpyrrolidone) (MW = 10.000), methanol, and toluene were all obtained from Sigma-Aldrich (St. Louis, MO). Preparation of Cross-Linked p(BEMA/MMA) Microspheres. The p(BEMA/MMA) microspheres were prepared by the suspension polymerization technique as described in the literature.34 For this purpose, BEMA (12.1 g or 62.5 mmol), MMA (16.25 g or 162.5 mmol, as a diluting comonomer), EGDMA (4.95 g or 25 mmol, as the cross-linker), AIBN (0.16 g or 0.975 mmol, as the initiator), and toluene (45 mL, porogen) were added successively to a round-bottom flask (500 mL) equipped with a mechanical stirrer. Meanwhile, an aqueous solution of water (300 mL) containing anhydrous sodium sulfate (5.0 g) and poly(vinylpyrrolidone) (1.0 g) was prepared in a beaker. Then, the mixture was transferred to a dropper and added dropwise to the flask under a nitrogen atmosphere with continuous stirring at 1200 rpm. The nitrogen stream was stopped after the addition process was completed, and the flask was mounted in a thermostatted oil bath and heated to 60 °C for 8 h. The reaction mixture was poured into water (1.0 L), and the product “microspheres” were filtered, washed with water (2 × 300 mL) and methanol (2 × 50 mL), and dried under a vacuum at 60 °C for 16 h. The pearl-like microspheres weighed 31 g. The product was fractionated by sieving and determined to be mostly (85%) in the 125−420-μm size range, and the 125−210-μm size fraction was used in the following reactions. Determination of the Bromide Content of the Microspheres. Into a 100 mL flask equipped with a reflux condenser were added p(BEMA/MMA) microspheres (0.5 g) and NaOH in methanol (2 mol L−1, 25 mL). The mixture was heated to 70 °C for 24 h under continuous stirring. The reaction mixture was cooled, filtered, and washed with water (2 × 10 mL). The filtrate and washings were combined in a 50 mL volumetric flask and diluted to this volume with distilled water. Then, 25 mL of this solution was neutralized with nitric acid 14264

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either acetate (20.0 mL, 50 mmol L−1, pH 4.0−5.5) or phosphate (20.0 mL, 5.0 mmol L−1, pH 6.0−8.0) buffer. In these α-amylase adsorption experiments, the initial α-amylase concentration was 2.0 mg mL−1, and adsorption studies were conducted at 25 °C with microspheres (1.0 g) under continuous stirring for 2.0 h. After this period, the α-amylaseadsorbed microspheres were removed from the enzyme solution by suitable centrifugation and washed with the same fresh buffer solution three times. For immobilization of α-amylase through cross-linking, αamylase-adsorbed p(BEMA/MMA)-g-PEI microspheres were cross-linked with glutaraldehyde (0.5%, v/v) at 22 °C for 6.0 h. Unreacted glutaraldehyde was washed sequentially with acetate buffer (50 mmol L−1, pH 5.5) and then phosphate buffer (50 mmol L−1, pH 6.0). Then, the enzyme-immobilized microspheres were treated with ethylenediamine 0.2% (v/v) at 22 °C for 1.0 h to block free aldehyde groups on the enzymeimmobilized microspheres, and finally, the microspheres were removed from the enzyme solution by centrifugation and washed several times with fresh buffer solution. Protein measurements were performed by Bradford’s method.36 For the calibration curve, different concentrations of α-amylase were used, and the absorbances of these solutions were measured at 595 nm using a UV−vis spectrophotometer (PG Instrument Ltd., model T80+; PRC). Activity Assays of α-Amylase. The activities of both the free and immobilized α-amylase preparations were determined by measuring the amount of reducing ends formed after enzymatic hydrolysis of starch in the medium according to the method described by Robyt and Whelan.37 The assay mixture (100 mL) contained sodium potassium tartrate (25 g), NaOH (1.6 g), and DNSA (1.0 g) in distilled water. The assay mixture (2.5 mL) and enzymatic hydrolyzed starch sample (0.1 mL) were mixed and incubated in boiling water for 10 min. After the mixture had cooled, distilled water (2.4 mL) was added, and the absorbance of the formed red color was measured using a UV/ vis spectrophotometer (PG Instrument Ltd., model T80+) at 595 nm. A standard curve was prepared using maltose solutions (0.1 mL) of different concentrations. Determination of the Optimum Temperature and pH for Enzyme Preparations. The effects of temperature on the activities of the free and immobilized enzyme preparations were studied in the range of 20−80 °C in batch operation mode with a soluble starch concentration of 10.0 g L−1 in phosphate buffer (50 mmol L−1, pH 6.0). The effects of pH on the activities of free and immobilized enzyme preparations were investigated at 60 °C. The concentration of the soluble starch was 10.0 g/L, and it was prepared in acetate buffer (50 mmol L−1) in the pH range of 3.0−5.5 and in phosphate buffer (50 mmol L−1) in the pH range of 6.0−8.0. The kinetic parameters (i.e., Km and Vmax values) of the enzyme preparations were determined by measuring the initial rates of the reaction with starch (5−30 g L−1) in phosphate buffer (50 mmol L−1, pH 6.0) at 60 °C. Thermal and Storage Stability of Free and Immobilized Enzymes. The thermal stabilities of free and immobilized α-amylase preparations were determined by measuring the residual activity of the enzyme exposed to two different temperatures (i.e., 70 and 80 °C) in phosphate buffer (50 mmol L−1, pH 6.0) for 2 h. After every 15-min time interval, a 0.1-g sample of microspheres was removed and assayed for enzymatic activity, as described above. The half-life (t1/2) was calculated according to the equation t1/2 = (ln 2)/ki.

The first-order inactivation rate constant, ki, was calculated from the equation ln A t = ln A 0 − k it

(1)

where A0 is the initial activity and At is the activity after time t (in days). The storage stabilities of free and immobilized α-amylase preparations were determined after storage in phosphate buffer (50 mmol L−1, pH 6.0) at 4 °C for 60 days. Operational Stability of the Immobilized α-Amylase Preparations. To evaluate the operational stability of the immobilized α-amylase preparations, both α-amylase-immobilized preparations were used for the hydrolysis of soluble corn starch in a batch reactor. The same enzyme-immobilized microspheres were washed with phosphate buffer (50 mmol L−1, pH 6.0) after use for 30 min in the hydrolysis reaction. They were then transferred to a fresh reaction medium and used to determine the remaining immobilized enzyme activity. The residual activity was defined as the fraction of total hydrolytic activity recovered after immobilization on the p(BEMA/MMA)-g-PEI microspheres compared with the same quantity of free enzyme. One unit of α-amylase activity is defined as the amount of enzyme that produces reducing ends equal to 1.0 μmol of glucose in 1.0 min at 60 °C and pH 6.0. The results in terms of dependence on pH, temperature, storage, and repeated use are presented in normalized form with the highest value of each set being assigned the value of 100% activity. Characterization of the p(BEMA/MMA) Microspheres. FTIR spectra were recorded on a Perkin-Elmer FT-IR Spectrum One B spectrometer. The surface morphologies of the pristine and grafted microspheres were observed by scanning electron microscopy (SEM). Micrographs of the samples were recorded with a JEOL 1540 instrument at 10 kV after they had been coated with a thin layer of gold. The surface area of the p(BEMA/MMA)-g-PEI microspheres was measured by the BET method using a surface area apparatus.38 The repeatability of each data set was analyzed using a statistical package in Excel for Windows as described previously.13

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RESULTS AND DISCUSSION Properties of the p(BEMA/MMA) Microspheres. p(BEMA/MMA) microspheres were synthesized by suspension polymerization in the presence of EGDMA and AIBN as the cross-linker and radical initiator, respectively (Figure 1A). The bromide content of the microspheres was found to be 1.45

Figure 1. (A) Synthesis of cross-linked p(BEMA/MMA) microspheres. (B) Preparation of surface-grafted poly(N-acetyl ethylenimine) brushes and their hydrolysis. 14265

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mmol/g by a titrimetric method. Surface-tethered poly(Nacetyl ethylenimine) brushes were obtained using bromoethyl groups on the microspheres as initiation sites through SI-ROP in acetonitrile solution. A simple acid hydrolysis of the grafted polymer resulted in the complete conversion of PEI brushes (Figure 1B). This approach furnishes a simple way to obtain secondary-amine-functionalized surface brushes. Figure 2A shows the FTIR spectrum of pristine bromoethylfunctionalized microspheres, in which methacrylate ester

Figure 3. SEM micrographs: (A) p(BEMA/MMA) and (B) p(BEMA/ MMA)-g-PEI microspheres.

Immobilization Protocols with p(BEMA/MMA)-g-PEI Microspheres. Amylases are frequently used in the food industry for the hydrolysis of starch and dextrin substrates to accelerate the conversion of these macromolecular substrates to dextrin and maltose. In this work, α-amylase from Bacillus sp. was immobilized onto the p(BEMA/MMA)-g-PEI microspheres by two different methods (i.e., adsorption and crossliking) to compare the usefulness of the resulting immobilized enzyme preparations. The pH value of the medium affects the surface charge distributions of both α-amylase and the p(BEMA/MMA)-g-PEI microspheres. The effect of pH on the α-amylase adsorption capacity on the p(BEMA/MMA)-gPEI microspheres was investigated at pH values between 4.0 and 8.0. The maximum amount of enzyme was adsorbed at around pH 6.0 and gave the highest adsorption capacity [34.3 mg of enzyme (g of microspheres)−1]. The isoelectric point (pI) of α-amylase is around 5.4. The observed maximum adsorption at pH 6.0 can be explained based on the facts that the surface of the PEI-functionalized microspheres is expected to be positively charged and the enzyme has a negative charge net charge at pH 6.0. The effect of the initial enzyme concentration on the immobilization efficiency of the p(BEMA/MMA)-g-PEI microspheres was studied at pH 6.0 and 25 °C for concentrations of α-amylase in the medium between 0.5 and 3.0 mg mL−1. The effects of the enzyme loading and retention of α-amylase activity are presented in Figure 4A. The maximum adsorption capacity was found to be 35.8 mg g−1, and the amount of enzyme adsorbed on the microspheres reached a plateau value at around 2.0 mg mL−1. As depicted in Figure 4, an increase in the initial enzyme concentration in the medium led to an increase in the amount of immobilized enzyme on the microspheres; on the other hand, the retained enzyme activity decreased with increasing enzyme loading. It should be noted that the highest retention of enzyme activity was obtained with the lowest enzyme loading [9.7 mg (g of microspheres)−1]. As the enzyme loading increased [from 9.7 to 35.8 mg (g of microspheres)−1], the activity retention decreased from 83% to 69%. This could be due to the overloading of enzyme

Figure 2. FTIR spectra: (A) pristine bromoethyl-functionalized microspheres, (B) surface-tethered poly(N-acetyl ethylenimine) brushes, (C) after hydrolysis into poly(ethylene imine).

vibrations were observed at 1730, 1225, and 1150 cm−1. These bands are associated with the stretching vibrations of carbonyl and C−O−C bonds. After surface grafting of the microspheres, a carbonyl stretching vibration band of the Nacetyl group appears at 1625 cm−1 in Figure 2B, indicating the presence of poly(N-acetyl ethylenimine) brushes in the resulting product. This band disappears after the hydrolysis (Figure 2C), implying the complete hydrolysis of the acetamido group. The bands at 3320 and 1550 cm−1 in this figure can be ascribed to the stretching and in-plane bending vibrations, respectively, of the amino group. The overall result implies that those modification steps result in the formation of PEI brushes on the microspheres. The p(BEMA/MMA) microspheres were grafted with 2-methyl-2-oxazoline through SI-ROP. The grafting yield attained was around 75% within 24 h at 110 °C under bulk conditions. The synthesis pathway and chemistry of the p(BEMA/MMA)−polyethylenimine microspheres are depicted in Figure 1. The specific surface areas of the p(BEMA/MMA) and p(BEMA/MMA)-g-PEI microspheres were measured by the BET method and were found to be 6.45 and 4.58 m2/g of microspheres, respectively. The decrease in the specific surface area after the grafting of the beads can be attributed to the decrease in the porosity of the grafted microspheres. Electron microscopy was used to observe the morphological differences between the bare and grafted microspheres. The surface morphologies of the p(BEMA/MMA) and p(BEMA/ MMA)-g-PEI microspheres are presented in panels A and B, respectively, of Figure 3. The SEM micrograph of the PEIfunctionalized microspheres (Figure 3B) indicates a smooth surface, whereas the bare p(BEMA/MMA) microspheres exhibit an irregular porous surface (Figure 3A). In the case of the polymer-grafted microspheres, the porous surface structure disappeared and was filled with the grafted polymer. 14266

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spheres were transferred to phosphate buffer (pH 7.5, 50 mmol L−1) containing glutaraldehyde (0.5%). The cross-linking reaction with glutaraldehyde was carried out at 22 °C for 6.0 h in an orbital shaker at 150 rpm. With this approach, crosslinked α-amylase aggregates were obtained on the p(BEMA/ MMA)-g-PEI microspheres. It should be noted that immobilization of α-amylase on the PEI-functionalized support through this hybrid method can provide a more stable cross-linked enzyme that is also attached at the same time to the microspheres by covalent bonds. The chemistry of immobilization is presented in Figure 5. The bifunctional coupling agent

Figure 4. (A) Effect of initial α-amylase concentration on immobilization efficiency and retained enzyme activity. (B) Langmuir isotherm plots for the adsorption of α-amylase on p(BEMA/MMA)-gPEI microspheres.

molecules around the pore spaces of the microspheres and the restricted diffusion of large substrate “starch” molecules into the pore space of the support for enzymatic reaction. The Langmuir isotherm model is frequently used to describe adsorption data.13 The data for the adsorption of α-amylase at 25 °C were processed in accordance with the linearized form of the Langmuir isotherm equations39,40 Ceq /qeq = Ceq /qm + 1/(K aqm)

Figure 5. Schematic representations of enzyme immobilization methods.

(i.e., glutaraldehyde) provides a linkage between the amino groups of the enzyme and the support. Hence, α-amylase also has several available surface carbonyl groups for immobilization. With this approach, these groups were not used because the enzyme contains aspartic and glutamic acid residues that are essential for catalytic activity. Additionally, the terminal carboxyl residue of α-amylase is located at vicinity of the active site, and use of this residue might prevent the binding of large starch molecules to the active sites.27 In the cross-linking method, the relative activity of α-amylase on the p(BEMA/MMA)-g-PEI microspheres was found to be 677 U (g of microspheres)−1 [enzyme loading of 34.3 mg of enzyme (g of microspheres)−1], and the activity yield of the cross-linking method was around 53%. On the other hand, the retained activity of the adsorbed α-amylase was about 896 U (g of microspheres)−1. A significant drop in retained activity was observed when adsorption was combined with glutaraldehyde cross-linking on the microspheres. A low relative activity shows that most of the protein molecule was attached to the microspheres in an inactive form.15 In contrast, the relative activity of the adsorbed enzyme was about 69%, which was higher than that of the cross-linked amylase (53%). The decrease in the relative activity of the cross-linked α-amylase might be due to a decrease in the flexibility of the enzyme

(2)

where qeq is the equilibrium adsorption amount (mg of enzyme/g of microspheres); Ceq is the equilibrium concentration of enzyme (mg mL−1); and the parameters qm and Ka (= b) represent the maximum adsorption capacity and the adsorption equilibrium constant, respectively. The amount of adsorbed enzyme based on the microsphere weight (qeq) was plotted against the free enzyme concentration (Ceq) in the medium at equilibrium (Figure 4B). The data were fitted to the Langmuir isotherm equation. The parameters qm and Ka were found to be 37.1 mg (g of microspheres)−1 and 9.17 × 105 M−1, respectively. The maximum amount of experimentally adsorbed enzyme was found to be 35.8 mg g−1, which was very close to the calculated Langmuir adsorption capacity (qm) of 37.1 mg g−1 with a high regression coefficient (R2) value of 0.997. The high value of the regression coefficient obtained using the Langmuir isotherm model suggests that the Langmuir isotherm model accurately fits the experimental data. This implies that the adsorption of α-amylase on the p(BEMA/MMA)-g-PEI microspheres follows the Langmuir model, which indicates monolayer-type adsorption.41 After adsorption of α-amylase on the p(BEMA/MMA)-g-PEI microspheres as described above, the enzyme-adsorbed micro14267

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molecules, which prevents them from taking the active conformation for binding the substrate.13,38 Similar results were also reported for several othehr enzymes immobilized through glutaraldehyde coupling.42,43 Effects of pH and Temperature on Free and Immobilized α-Amylase Activities. The activities of free and immobilized α-amylase preparations were measured at different pH values. As shown in Figure 6, free α-amylase

Figure 7. Effects of temperature on the catalytic activities of the free and immobilized α-amylase preparations.

with the free and both immobilized enzyme preparations. The kinetic parameters for the free and immobilized enzyme preparation were calculated from the Lineweaver−Burk equation. For the free amylase, the Km and Vmax values were 6.35 g L−1 and 1327 U (mg of protein)−1, respectively, for corn starch. The Vmax values for adsorbed and cross-linked α-amylase preparations were found to be 916 and 703 U (mg of protein)−1, respectively. The Km values for adsorbed and covalently immobilized α-amylase were found to be 9.23 and 16.54 g L−1, respectively. The change in observed Km value of an enzyme for its substrate can be caused by conformational changes in the enzyme molecules introduced by the immobilization method.17,32 It should be noted that the changes in the kinetic parameters for covalently immobilized α-amylase were more pronounced than those for the adsorbed enzyme. This might be due to aggregate formation on the microspheres after glutaraldehyde cross-linking and could contribute to a decrease in the affinity of the enzyme for the large substrate. Thermal and Storage Stabilities of the α-Amylase Preparations. The thermal stabilities of the free and immobilized α-amylases were determined by measuring the residual activities of the samples incubated for predetermined times in phosphate buffer (50 mmol L−1, pH 6.0) at 70 and 80 °C (Figure 8). At 70 °C, the free α-amylase retained about 33% of its initial activity after a 120-min incubation period. However, the adsorbed and covalently linked enzymes maintained 69% and 83%, respectively, of their initial activities after the same incubation period. At 80 °C, the adsorbed and cross-linked enzymes retained about 42% and 52%, respectively, of their initial activities after a 120-min incubation period, whereas the free enzyme lost all of its initial activity after 75 min. Both immobilized α-amylases were inactivated much more slowly than their free counterpart. The half-life values at 70 °C were determined to be 86, 224, and 447 min for the free, adsorbed, and covalently linked enzymes, respectively; the corresponding values at 80 °C were 11, 83, and 127 min. The thermal inactivation rate constants (ki) were calculated to 8.06 × 10−3, 3.09 × 10−3, and 1.55 × 10−3 at 70 °C and 6.14 × 10−2, 8.28 × 10−3, and 5.44 × 10−3 at 80 °C for free, adsorbed, and cross-linked enzymes, respectively. These results suggest that the thermostability of the immobilized α-amylase preparations increased considerably as a result of immobilization onto the PEI-functionalized microspheres. It should be

Figure 6. Effects of pH on the catalytic activities of the free and immobilized α-amylase preparations.

reached a maximum activity at pH 6.0, whereas the optimal pH for both immobilized α-amylase preparations rose to 5.0. The immobilized α-amylases (i.e., both adsorbed and covalently bonded) on PEI-functionalized microspheres were less sensitive to pH changes in the acidic region compared to the free enzyme. It should be noted that α-amylase immobilized on an ionic polymer bed consisting of fibrous PEI chains should create a solid buffering effect, generating a different pH microenvironment for the immobilized α-amylases than that experienced by the free enzyme in the bulk solution. Additionally, an alkaline microenvironment should form around the generated PEI chains of the microspheres. Therefore, the optimum pH range of the medium can extend widely, and the optimum pH for the immobilized enzymes can also shift to a more acidic range as a result of the presence of a large quantity of amino groups. The observed pH shift was consistent with the related literature.26,31 The effects of temperature on the free and immobilized enzyme activities for corn starch hydrolysis were investigated in the temperature range from 20 to 80 °C. As shown in Figure 7, the optimal reaction temperature of 65 °C for both immobilized α-amylase preparations was 5 °C higher than that for the free enzyme. Whereas the immobilized α-amylase preparations were more stable than those of the free enzyme, in addition, the covalently immobilized enzyme on PEI-functionalized microspheres was more stable than the adsorbed enzyme. The temperature stabilization of α-amylase molecules upon immobilization could be promoted through multipoint interactions of enzyme with the PEI chains. These multipoint interactions between PEI and enzyme resulting from ionic and/ or covalent bounding through glutaraldehyde coupling could limit the deformation of the enzyme when subjected to increasing temperature.15,32 Kinetic Parameters of the Free and Immobilized Enzymes. Enzymatic hydrolysis of starch was carried out 14268

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decrease in activity is explained as a time-dependent natural loss in enzyme activity. Operational Stability of Immobilized α-Amylase. The operational stabilities of adsorbed covalently linked α-amylase were studied in a batch reactor using soluble corn starch as the substrate. The repeated use of the adsorbed and covalently linked enzyme preparations was studied throughout 12 successive runs at 60 °C (data not shown). The performance of the covalently linked enzyme in the reactor can be divided in two phases. In the first six cycles, the catalytic activity of the covalently linked enzyme remained at 100%, whereas in the second phase (from 7 to 12 cycles), the retained activity decreased by about 13%. On the other hand, the retained activity of the adsorbed α-amylase decreased by about 21% after six runs. At the end of 12 run cycles, this value was found to be 54%. The higher stability of the cross-linked α-amylase on PEIgrafted microspheres might be due to multipoint covalent linkages. Therefore, the use of cross-linked α-amylase for largescale applications can provide a stable starch hydrolysis reaction without much loss of enzymatic activity.

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CONCLUSIONS In this work, the surface-initiated ring-opening polymerization of 2-methyl-2-oxazoline on the surface of poly(bromoethyl methacrylate/methyl methacrylate) microspheres was realized. The grafted poly(2-methyl-2-oxazoline) chains were modified to PEI. The polyionic support was used for the immobilization of α-amylase through adsorption and cross-linking. Both presented immobilization methods could be applied in a wider range of temperatures and pH values than the free enzyme. The thermal, storage, and operational stabilities of the immobilized preparations were higher than those of free enzyme, which demonstrates that a stable three-dimensional structure of enzyme was created after immobilization on p(BEMA/MMA)-g-PEI microspheres. The observed stabilities of the immobilized enzyme preparation could be due to the protection of the active three-dimensional structure through ionic and/or multipoint binding to the p(BEMA/MMA)-g-PEI microspheres. The values obtained for the kinetic constants indicate that the covalently linked enzyme changed the affinity of the enzyme toward large starch substrates in a more pronounced manner. Both immobilized enzyme preparations were used in a batch system for the hydrolysis of corn starch, and the retained activities of the adsorbed and cross-linked αamylases were found to be 54% and 13%, respectively, after consecutive runs in a batch system. The high operational stability obtained with the covalently linked enzyme indicates that this preparation could successfully be used in a continuous system for the production of maltose and dextrin from starch. Finally, the p(BEMA/MMA)-g-PEI microspheres have desirable properties and could be used in various biomolecule immobilization processes.

Figure 8. Thermal stabilities of the free and immobilized α-amylase preparations at 70 and 80 °C.

noted that the covalently immobilized enzyme was more resistant than the free and adsorbed α-amylase against heat as a denaturing agent. If the thermal stability of an enzyme were enhanced by immobilization, the potential utilization of such enzymes would be extensive.43−45 The lifespan results (Figure 9) showed that the activities of the adsorbed and covalently linked α-amylases were more

Figure 9. Life spans of free and immobilized α-amylase stored at 4 °C for 60 days.

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AUTHOR INFORMATION

Corresponding Author

stable under the given storage conditions than that of the free enzyme over 60 days of storage. As can be seen from this figure, the free and the immobilized enzymes preserved all of their initial activity during the early storage period, and then their activities gradually decreased over time. The free enzyme lost all of its activity within 40 days. The adsorbed and covalently linked enzymes preserved about 35% and 57%, respectively, of their initial activities during the 60-day storage period. This

*E-mail: [email protected]. Tel.: +90-555-709-7878. Notes

The authors declare no competing financial interest.

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