Immobilization of Water-Soluble HRP within Poly-N

Article pubs.acs.org/Langmuir

Immobilization of Water-Soluble HRP within Poly‑N‑isopropylacrylamide Microgel Particles for Use in Organic Media Kornelia Gawlitza,† Radostina Georgieva,§,∥ Neslihan Tavraz,‡ Janos Keller,⊥ and Regine von Klitzing*,† †

Stranski-Laboratory for Physical and Theoretical Chemistry and ‡Institute of Chemistry, Technische Universität Berlin, 10623 Berlin, Germany § Institute of Transfusion Medicine, Center for Tumor Medicine, CharitéUniversitätsmedizin Berlin, 10117 Berlin, Germany ∥ Medical Faculty, Department of Medical Physics, Biophysics and Radiology, Trakia University Stara Zagora, 6000 Stara Zagora, Bulgaria ⊥ Department of Interfaces, Max Planck Institute of Colloids and Interfaces, 14476 Potsdam, Germany S Supporting Information *

ABSTRACT: In the present work, the immobilization of enzymes within poly-Nisopropylacrylamide (p-NIPAM) microgels using the method of solvent exchange is applied to the enzyme horseradish peroxidase (HRP). When the solvent is changed from water to isopropanol, HRP is embedded within the polymer structure. After the determination of the immobilized amount of enzyme, an enhanced specific activity of the biocatalyst in isopropanol can be observed. Karl Fischer titration is used to determine the amount of water within the microgel particles before and after solvent exchange, leading to the conclusion that an “aqueous cage” remains within the polymer structure. This represents the driving force for the immobilization due to the high affinity of HRP for water. Beside, confocal laser scanning microscopy (CLSM) images show that HRP is located within the microgel network after immobilization. This gives the best conditions for HRP to be protected against chemical and mechanical stress. We were able to transfer a water-soluble enzyme to an organic phase by reaching a high catalytic activity. Hence, the method of solvent exchange displays a general method for immobilizing enzymes within p-NIPAM microgels for use in organic solvents. With this strategy, enzymes that are not soluble in organic solvents such as HRP can be used in such polar organic solvents.

1. INTRODUCTION Enzymes are highly catalytically active and responsible for the production of all organic molecules necessary in life. Typically, they react under mild conditions (e.g., ambient temperature and pressure). Because of this fact, enzymes showing a high specificity are attractive for use in synthetic chemistry.1 For this application, it is necessary for the enzymes to be stable at different pH values and in different solvents. Substrates as well as the products of catalytic reactions are often soluble in organic solvents. Hence, the use of organic solvents can lead to a higher yield of the product. Because of the fact that organic solvents can alter the native structure of enzymes and lead to a decrease in activity, many methods have been developed to overcome this problem. One method is the immobilization of enzymes within an inorganic or organic support. As a great advantage, the low contact with the carrier leads to residual mobility and flexibility of the enzyme after immobilization.2 In the literature, enzymes have been immobilized into silica,3 reverse micelles,4 and polysaccharides.5 Additionally, microgels made of poly-N-isopropylacrylamide (p-NIPAM) have been also studied for use as enzyme supports.6,7 These waterswellable microgel systems are of high research interest because of their reversible response to external stimuli such as temperature,8−11 pH,12−14 and ionic strength.15,16 p-NIPAM © 2013 American Chemical Society

microgels undergo a volume phase transition (VPT) at around 32 °C that is correlated to a decrease in size with increasing temperature. This behavior makes these polymer particles useful for a wide field of applications (e.g., biosensors,17 enzyme supports,18,19 and drug delivery20,21). The first investigations of the adsorption of proteins at pNIPAM microgels were done in the early 1990s by Kawaguchi et al.22,23 In another study, polymer particles with a core of polystyrene and a shell of p-NIPAM were used to immobilize β-D-glucosidase.24 In other studies, lysozyme and trypsin were immobilized within p-NIPAM microgel particles with a diameter of between 50 and 80 μm where the location of the enzymes within the polymer structure was detectable by confocal laser scanning microscopy (CLSM).25−27 Although enhanced activity is reached, the described studies investigated the activity exclusively in water. Another study presents the immobilization of lipase within p-NIPAM macrogels, leading to enhanced activity in organic solvents.28 To benefit from the low polydispersity and the high surface-to-volume ratio, it is more efficient to use smaller p-NIPAM particles. In previous studies, Received: September 17, 2013 Revised: December 7, 2013 Published: December 9, 2013 16002

dx.doi.org/10.1021/la403598s | Langmuir 2013, 29, 16002−16009

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for 15 min at 9000g. The residues were redispersed in isopropanol and washed three times with centrifugation and redispersion. The redispersion after centrifugation takes around 1 h. To determine the immobilized amount, one part of the sample was redispersed in buffer while the other part was redispersed in isopropanol again for investigations of the catalytic activity. To determine the location of HRP within the polymer particles after immobilization, the enzyme was labeled with FITC according to the literature33 and freeze-dried afterward. A solution of 2 mg of FITCHRP and 10 mg of p-NIPAM in 2 mL of buffer (0.1 M potassium phosphate buffer, pH 7) was stirred overnight and centrifuged for 10 min at 9000g. One part of the sample was redispersed in buffer while the other part was redispersed in isopropanol. 2.3. Characterization Methods. 2.3.1. Light Scattering. Dynamic light scattering (DLS) was used to determine the size of the microgel particles. Therefore, an ALV goniometer setup with a Nd/YAG laser as the light source (λ = 532 nm) was used to record the correlation functions at a constant scattering angle of 60°. The correlation functions were generated using an ALV-5000/E multiple-τ digital correlator and subsequently analyzed by an inverse Laplace transformation (CONTIN34). The molecular weight of the polymer particles was determined by static light scattering (SLS) using an ALV/CGS-3 compact goniometer system equipped with an ALV/LSE-5004 correlator. Scattering angles from 17 to 37° with 2° steps in between were used, and the concentration of the polymer particles was varied from 1 × 10−6 to 9 × 10−6 g/g. The measurements were made at 25 °C using a Huber compatible control thermostat. A He−Ne laser (λ = 634 nm) was used as the light source. 2.3.2. Confocal Laser Scanning Microscopy (CLSM). To get information on the location of HRP after immobilization within the microgel particles, CLSM was used. Roughly, 20 μL of the prepared samples were placed on a coverslip and investigated by applying an Axiovert 200 M inverted microscope with a 100× oil-immersion objective (numerical aperture 1.3) and a Zeiss LSM 510 Meta confocal scanning unit (Zeiss MicroImaging GmbH, Jena, Germany). To record the fluorescence, the 488 nm line of the argon laser for excitation and a 505 nm long-pass emission filter were used. Z stacks were performed with an upward step of 50 nm starting at the surface of the coverslips. Different Z stacks of the samples were analyzed using the LSM 510 software and displayed as an overlay of transmission and fluorescence channels in orthogonal section views. 2.3.3. Circular Dichroism Spectroscopy (CD). CD spectroscopy was used for investigations of the peptide secondary and tertiary structure in solution. CD spectra were recorded on a Jasco J-715 (Japan) spectrometer in a wavelength range from 190 to 475 nm with 0.5 nm step resolution using quartz cuvettes with an optical path length of 0.1 cm. For measurements in the far-UV range (190 to 270 nm), the HRP concentration was kept at 0.3 mg/mL while the enzyme concentration was 10 mg/mL for measurements in the near-UV−vis range (325 to 475 nm). For HRP in isopropanol, 1:1 mixtures of buffer and isopropanol were used as solvents. All measurements were performed at room temperature. Data processing was carried out using the J-700 software package. After subtracting the blank spectra from the sample spectra, the CD signal was transformed to the mean residue molar ellipticity.35 2.3.4. Determination of the Amount of Water in Microgel Particles. Karl Fischer titration was used to determine the water content in p-NIPAM microgel particles before and after solvent exchange. In the case of freeze-dried polymers, a solution of 20 mg of microgels per mL methanol was prepared under ambient conditions and titrated with a mixture of sulfur dioxide, imidazole, and iodine (Hydranal). The end point of the titration was determined with a platinum electrode. The amount of titrant was used to calculate the amount of water in the sample. The water content in pure methanol was also determined to receive the exact value of the water content for the freeze-dried microgels. The determination of the residual water content after solvent exchange was done by dissolving 20 mg of microgel in 1 mL of buffer (0.1 M potassium phosphate buffer, pH 7) followed by two

the enzyme lipase B from Candida antarctica (CalB) was immobilized within p-NIPAM microgel particles by exchanging water for an organic solvent.29 The immobilization led to enhanced activity, and the location of CalB was determined to be within the polymer structure. Additionally, it was shown that an increase in the cross-linker density leads to a biocatalyst with temperature-controllable activity.7,30 In this article, the method of solvent exchange is applied to horseradish peroxidase (HRP). This enzyme is not soluble in organic solvents, but it can use a wide range of organic substrates (e.g., phenols). It catalyzes the synthesis of a variety of organic compounds without using water as a coreactant.31 The challenge is to bring the hydrophilic enzyme and hydrophobic substrate into contact. The novelty of the present study is the transfer of an enzyme that is usually not soluble in organic solvents in an organic phase without limiting the catalytic activity. Therefore, HRP was immobilized within pNIPAM microgel particles with a cross-linker content of 0.25 mol % by changing the solvent from water to isopropanol. Enhanced activity of the immobilized HRP compared to that of nonimmobilized HRP was determined by UV−vis spectroscopy. Besides, CLSM was used to determine the location of HRP within the polymer network after immobilization. As the driving force for immobilization, a residual amount of water was detected within p-NIPAM microgels after solvent exchange using Karl Fischer titration.

2. EXPERIMENTAL SECTION 2.1. Materials. N-Isopropylacrylamide (97%) (NIPAM), horseradish peroxidase (HRP), bovine serum albumin standard (BSA, 2 mg/mL), Bradford reagent, and pyrogallol (≥99%) were purchased from Sigma-Aldrich. Fluorescein-5-isothiocyanat (FITC) and glycerine (ACS, Reag. Ph Eur) were from Merck. Hydrogen peroxide (H2O2, 30% in water) and isopropanol (≥99.5%) were purchased from ChemSolution. N,N′-Methylenebis(acrylamide) (MBA) (≥99.5%), potassium peroxodisulfate (KPS) (≥99%), and HYDRANALComposite 5 were obtained from Fluka. Mercaptoethanol (98%) was from PlusOne, and ammonium persulfate (APS, ≥98%) was from Affymetrix/USB. Tris(hydroxymethyl)aminomethane (TRIS, ≥99.9%), sodium dodecyl sulfate (SDS, ≥99%), glycine (>99%), acrylamide mixture (30% in water), and N,N,N′,N′-tetramethylethylenediamine (TEMED, 99%) were purchased from Roth. NIPAM was purified by recrystallization in n-hexane. Other chemicals were used as received. Water was taken from a three-stage Millipore Milli-Q Plus 185 purification system. 2.2. Preparation Techniques. 2.2.1. Synthesis of p-NIPAM Microgel Particles. p-NIPAM microgel particles with a cross-linker content of 0.25 mol % were synthesized by surfactant-free emulsion polymerization. Because of the possibility of making larger particles visible by CLSM, a temperature ramp according to Meng et al.32 was used. Briefly, 1.8 g of the NIPAM monomer (0.015 mol) and 8 mg of the MBA cross-linker (5 × 10−5 mol) were dissolved in 125 mL of water. After degassing the solution for 1 h at 45 °C, a solution of 1 mL KPS (0.08 M) was added to the mixture under continuous stirring. A temperature ramp of 1 °C per 2 min was applied until the final temperature of 65 °C was reached. The polymerization was completed by stirring overnight at this temperature under an N2 atmosphere. The received microgel particles were purified by filtering over glass wool, dialysis for 2 weeks, and finally freeze-drying at −85 °C under 1 × 10−3 bar for 48 h. After the dried microgels were dissolved in methanol, the residual amount of water was determined by Karl Fischer titration. 2.2.2. Immobilization of HRP within p-NIPAM Microspheres. For the immobilization process, 5 mg of p-NIPAM particles with a crosslinker content of 0.25 mol % and 0.3 mg of HRP were mixed with 1.5 mL of buffer (0.1 M potassium phosphate buffer, pH 7) at room temperature, leading to an initial concentration of 0.06 mg of HRP per mg of p-NIPAM. The solution was stirred overnight and centrifuged 16003


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used cuvette. Therefore, one unit is defined as the formation of 1 μmol of product per min per mL. For calculation, the extinction coefficient of purpurogallin in isopropanol was determined to be 1.976 mL μmol−1 cm−1.

centrifugation steps (10 min at 9000g) and redispersion in 1 mL of isopropanol. The received solutions as well as pure isopropanol were titrated, and the residual amount of water was calculated. All measurements were made in triplicate. 2.3.5. Determination of the Purity of Enzymes by SDS-Page. To determine the purity of the commercially available enzymes, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-Page) was done. First, the separation and stacking gels were prepared by polymerization. The composition of these gels is shown in Table 1.

3. RESULTS Monodisperse p-NIPAM microgel particles with a cross-linker content of 0.25 mol % were obtained by surfactant-free emulsion polymerization according to a previous study.7 The temperature ramp was used to synthesize microgel particles with a diameter of around 1 μm in order to have the possibility to make them visible under a CLSM. In previous work, the swelling curves in water and isopropanol studied by DLS are shown.7 It was observed that the hydrodynamic diameter (Dh) in water is 1.8 μm at 25 °C and decreases to 0.2 μm by increasing the temperature to 40 °C. The use of isopropanol as a solvent results in a decrease in Dh to 1.2 μm at 25 °C, which corresponds to a collapse of 0.6 μm in diameter compared to water. The decrease in size by using isopropanol indicates that water is a better solvent for the microgel particles than isopropanol. The volume phase transition temperature at which the microgel particles collapse is similar in both solvents. The results are summarized in Table 2.

Table 1. Composition of Stacking and Running Gels for 12% SDS Gelsa components

5% stacking gel (mL)

12% running gel (mL)

H2O 30% acrylamide mixture 1.5 M TRIS (pH 8.8) 1.5 M TRIS (pH 6.8) 10% SDS 10% APS TEMED

9.9 1.7

6.9 12.0 7.5

1.25 0.1 0.1 0.01

0.3 0.3 0.012

a

The acrylamide mixture contains acrylamid and bisacrylamide (37.5:1).

HRP was dissolved in buffer (0.1 M potassium phosphate buffer, pH 7) with a concentration of 0.5 mg/mL. The samples were mixed with the Laemmli buffer36 (pH 6,8, 126 mM TRIS-HCl, 20% glycerine, 4% SDS, 0.02% bromophenol blue, 2.5% mercaptoethanol) in a 1:1 ratio and denaturated at a temperature of 95 °C for 5 min. Each sample (10 μL) was deposited on top of the stacking gel. As the molecular mass standard, 10 μL of the PageRuler prestained protein ladder (10 to 170 kDa, Fermentas) was also separated by the gel. After surrounding the gel with a running buffer (3.03 g of TRIS, 14.41 g of glycine, 10 mL of a 10% SDS-solution), 50 V was applied to the system until the samples reached the separation gel, followed by applying a voltage of 100 V for 1 h. After separation, the gel was stained with Coomassie dye (PageBlue protein staining solution, Thermoscientific) on one hand and with silver nitrate on the other hand. After the sample was washed with water, the molecular weight of the enzymes was determined. 2.3.6. Determination of the Immobilized Amount of HRP. The amount of HRP that is immobilized within the p-NIPAM microgel particles was achieved via determination of the total protein content in aqueous solution using the Bradford reagent according to the manufacturer’s instructions.7 UV−vis spectra were measured with the PerkinElmer Lambda 25 UV−vis spectrometer. 2.3.7. Determination of the Catalytic Activity. The catalytic activity of HRP was determined using the oxidation of pyrogallol to purpurogallin in the presence of hydrogen peroxide. The formation of the product can be determined via UV−vis spectroscopy at a wavelength of 420 nm. The immobilized sample (0.4 mL) redispersed in 1.5 mL of isopropanol was mixed with 0.4 mL of a pyrogallol solution in isopropanol (1 mg/mL) and 0.132 mL of hydrogen peroxide (48 μL of H2O2 (35%) in 20 mL of water). For the determination of the activity of nonimmobilized enzyme, 1 mL of a solution of HRP in isopropanol (0.44 mg/mL) was mixed with 0.1 mL of the pyrogallol and hydrogen peroxide solution, respectively. As reference, the same compositions of the reagents were used unless the samples were replaced by isopropanol. Afterward, the solution was measured via UV−vis spectroscopy for 1 min at 420 nm. Because of the linear behavior of the increase in absorption with time, the volume activity can be calculated using eq 1. UV =

ΔEVtotal VSϵd

Table 2. Dh and VPTT of p-NIPAM Microgel Particles with an MBA Content of 0.25 mol % in Water and Isopropanol7 solvent

Dh at 25 °C (nm)

Dh at 40 °C (nm)

VPTT (°C)

water isopropanol

1762 ± 46 1160 ± 62

210 ± 4 70 ± 2

28 27

The molecular weight (Mw) of the synthesized p-NIPAM microgel particles was determined by SLS measurements. A residual water content of around 12 wt % under ambient conditions was determined by Karl Fischer titration and considered for sample preparation. A refractive index increment of dn/dc = 0.167 cm3 g−1 was used for calculation.37 The received Zimm plots lead to a Mw of 3.0 × 1010 g mol−1.7 To determine the immobilized amount of HRP, it is necessary to investigate the purity of the used enzyme. Therefore, SDS-Page was performed using a polyacrylamide gel (12% MBA) with Coomassie dye and silver staining for analysis. Figure 1 shows the received images. The investigations were done for HRP as received with an expected Mw of 44 000 Da. The chromatograms prove that the used peroxidase has a high purity. The investigation results in an intensive band at around 40 kDa after staining with Coomassie dye as well as with silver nitrate. The fact that the determined Mw is in good agreement with the expected value proves the high purity of this enzyme. After characterization of the p-NIPAM microgel particles and HRP, the enzyme was embedded within the polymer network. Therefore, HRP is mixed with polymer particles in a buffer solution. To reach the immobilization, solvent exchange with isopropanol was carried out. A sketch of the immobilization is shown in Figure 2. For characterization of the immobilized system, it is necessary to determine the immobilized amount of HRP. Briefly, the immobilized sample was centrifuged and redispersed in buffer. Assuming that no enzyme was removed by centrifugation, an embedded amount of 12 ± 2.6 μg of HRP per mg of p-NIPAM microgel particles was obtained by

(1)

where UV is the volume activity, ΔE is the change in absorption per minute, Vtotal is the total volume, VS is the volume of the sample, ϵ is the extinction coefficient of purpurogallin, and d is the thickness of the 16004


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mixed with p-NIPAM microgel particles, the solution was centrifuged and redispersed in either buffer or isopropanol. The size of the p-NIPAM microgel particles allows the application of CLSM as an adequate method to investigate if the enzyme molecules are situated within the polymer network or only on the surface. Figure 3 shows the images made by CLSM for the immobilized system in (a) buffer and (b) isopropanol.

Figure 1. Results from SDS-Page after staining with (a) Coomassie dye and (b) silver nitrate for HRP as received and the used protein standard (M).

Figure 3. CLSM images of the residue of p-NIPAM after incubation with HRP redispersed in (a) buffer and (b) isopropanol in fluorescence mode, transmission mode, and as a superimposed image of both.

Images a1 and b1 display the fluorescence of the sample, images a2 and b2 display the transmission of the sample, and images a3 and b3 reflect an overlay of the first two images to show whether the fluorescence signal matches the position of the microgel particles. The upper series shows the images for the sample redispersed in buffer. Here, the fluorescence signal is distributed over the whole scanning area except for some spherically shaped areas (white circles). These areas fit to the position of the polymer particles, indicating that HRP is not situated within the polymer network. The lower series of Figure 3 shows images obtained by measuring the sample after redispersion in isopropanol. Obviously, spherically shaped fluorescence signals are monitored that are exactly at the position of the p-NIPAM microgel particles (white circles). To confirm the assumption made by the fluorescence images, intensity profiles of the shown images were made. To avoid high-frequency noise, a Fourier transform bandpass filter was used. The profiles are shown in the Supporting Information (Figure S1). To receive more detailed information on the distribution of the enzyme within the p-NIPAM microgel particles, the z-stack option of the CLSM was used. Figure 4a displays an orthogonal section view of the measured sample, and Figure 4b shows a schematic explanation of the received image. The immobilized sample in isopropanol was scanned in 32 different x−y planes with a distance of 50 nm in the z direction between them. The image in the center framed by the blue box shows one of the measured x−y planes. The y−z plane of the cut through the sample along the red vertical line in the central x−y image is represented by the righthand red framed box. The upper green box frames the x−z plane of a cut through the sample along the green horizontal line in the central x−y image. To complete the orthogonal section view, the blue lines in the x−z and y−z images display the z position of the x−y plane shown in the center. In all three planes, the fluorescence signal reflects the spherical shape of the particles. This observation can be supported by fluorescence intensity profiles of the x−y images at three different z positions close to the bottom (z = 4), in the middle (z = 16), and at the end (z = 28) of one

Figure 2. Schematic process of the immobilization of HRP within pNIPAM microgel particles at 25 C.

investigating the sample with a Bradford assay. The precise procedure and the used calibration curves are shown in our previous study.7 Using molecular weights of 3.0 × 1010 g mol−1 for p-NIPAM and 4.4 × 104 g mol−1 for HRP, it was determined that ∼8100 enzyme molecules are embedded within 1 microgel particle. For a better understanding of the immobilization process, it is important to determine the water content within the microgel particles after the exchange of water for isopropanol. Therefore, Karl Fischer titration was performed. Because of the fact that p-NIPAM is stored under ambient conditions, there is also water left in the dried polymer particles. To exclude this amount from the determined residual water content after solvent exchange, the same experiments were done for “dry” microgel particles. Table 3 summarizes the determined water Table 3. Water Contents of p-NIPAM after Freeze Drying (Ambient Conditions) and after Solvent Exchange (se) and Amount of Water per p-NIPAM Microgel Particle MBA (mol %)

cH2O (wt %) (dry)

cH2O (wt %) (after se)

mH2O (g per p-NIPAM)

0.25

12.0 ± 0.9

46.6 ± 2.4

2.32 × 10−14

contents of p-NIPAM particles in the dried state (ambient conditions) and after solvent exchange. There is a huge difference in the water content before and after solvent exchange. This indicates that water remains within the polymer network after the exchange of water for isopropanol. To get information on the location of the enzyme molecules within the polymer matrix, HRP was labeled with FITC prior to the immobilization procedure. After the labeled enzyme was 16005


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The far-UV CD spectra give information about the secondary structure of the enzyme. Figure 5a shows two typical bands at 223 and 207 nm for HRP dissolved in buffer solution. When the same enzyme concentration is used in an isopropanol/ buffer (1:1) solution, the band at 209 nm disappears while the band at 223 nm decreases and is shifted to 229 nm. This strongly indicates a change in the secondary structure of HRP in the presence of isopropanol. The near-UV−vis CD spectra show the Soret region and give information about the tertiary structure. Figure 5b shows small changes in the CD signal at a wavelength of 403 nm by adding isopropanol. To get more information about the Soret region, additional UV−vis absorption measurements were made (Figure S3 in the Supporting Information). It is shown that there is a strong change in the absorption maximum at a wavelength of 403 nm. This indicates loosening of the tertiary structure and a change in the microenvironment of the heme in HRP.38

Figure 4. Orthogonal section view of one x−y plane of p-NIPAM particles with immobilized HRP after redispersion in (a) isopropanol and (b) schematic explanation of the blue, red, and green boxes.

particle. The profiles are presented in the Supporting Information (Figure S2). Concerning the application to catalysis, reaching a high enzyme activity is required after the immobilization process. The catalytic activity of HRP was determined using the oxidation of pyrogallol to purpurogallin in the presence of hydrogen peroxide, isopropanol, and HRP. Purpurogallin can be detected via UV−vis spectroscopy at a wavelength of 420 nm. The volume activity (UV) can be calculated from eq 1. To compare the activity between the immobilized and the nonimmobilized enzyme, the activity measurements were performed for both samples. The results are listed in Table 4. Against expectations, also for nonimmobilized HRP in isopropanol, a small amount of product was formed. Although HRP is not soluble in isopropanol, a small amount of water (as a solvent for H2O2) is added to the activity reaction. Hence, some of the enzymes can be dissolved in this aqueous phase during the activity reaction. For a direct comparison of both systems, the specific activity (Usp) has to be determined. In the case of immobilized HRP, the amount of HRP within the microgel particles for the activity measurements is known, and Usp can be calculated. For nonimmobilized HRP, Usp was first calculated with respect to the initial amount of HRP. Because it is not possible to determine the amount of HRP dissolved in the aqueous phase, this value is only a speculation. To get an idea of the enhancement of the activity by immobilization, the specific activity was also calculated by assuming that the same amount of enzyme was present in the immobilized and nonimmobilized systems (Usp(a)). All of the described results are summarized in Table 4. It is shown that an immobilization of HRP within p-NIPAM microgel particles leads to an enhancement in catalytic activity in isopropanol. The use of isopropanol can lead to possible changes in the secondary and tertiary structure of HRP, resulting in aggregation or deactivation. Therefore, we recorded CD spectra of HRP in buffer and isopropanol/buffer (1:1) in the far-UV (λ = 190 to 270 nm) and near-UV−vis (λ = 325 to 475 nm) ranges (Figure 5).

4. DISCUSSION In our previous studies, it has been shown that solvent exchange is an adequate method of immobilizing lipase B from Candida antarctica (CalB) within p-NIPAM microgel particles, resulting in enhanced activity in organic solvents.7,30 It is known that CalB is soluble in a number of organic solvents. It is usually located at the interface between water and the organic phase where it shows catalytic activity. Hence, this enzyme is a suitable model enzyme for making a proof of principle. For industrial applications, there is strong interest in investigating if the method can be transferred to other enzymes. In the present study, the HRP enzyme was used, which is usually insoluble in organic solvents and not active in aqueous environments. Because HRP is responsible for the oxidation of inorganic and organic compounds, it is important to create a method to obtain active HRP in organic solvents. In this study, HRP was successfully immobilized within pNIPAM microgel particles using the method of solvent exchange that is supported by an immobilized number of ∼8100 enzyme molecules per microgel particle. In comparison to the immobilized amount of CalB, the loading efficiency for HRP is much higher (Table 5). The dimensions of peroxidase are supposed to be 6.2 nm × 4.3 nm × 1.2 nm39 and thus slightly smaller than for CalB (3 nm × 4 nm × 5 nm40). Simultaneously, the exchange of water for isopropanol leads to a decrease in size (Figure 3), which keeps the enzyme inside the p-NIPAM particles. One reason for the higher immobilized amount of HRP could be an easier diffusion within the microgel structure for smaller molecules. However, it cannot explain such a considerable difference. To understand the behavior of proteins in different environments and at interfaces, several factors have to be taken into account. First, one always has to consider their molecular structure. Enzymes CalB and HRP are designed to perform their functions in aqueous environment. As shown in Table 3, there is a huge amount of water left in the polymer

Table 4. Volume Activity (UV), Specific Activity (Usp), and Specific Activity with Respect to the Amount of HRP in the Immobilized System (Usp(a)) of Nonimmobilized HRP and HRP Immobilized within p-NIPAM Microgel Particles activity

nonimmobilized

immobilized

UV (μmol min−1 mL−1) Usp (μmol min−1 μg−1) Usp(a) (μmol min−1 μg−1)

2.03 × 10−3 ± 3 × 10−4 5.55 × 10−6 ± 1 × 10−6 6.15 × 10−5 ± 1 × 10−5

2.72 × 10−1 ± 1 × 10−1 1.60 × 10−2 ± 9 × 10−3 1.60 × 10−2 ± 9 × 10−3

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Figure 5. CD spectra of HRP in buffer (solid line) and in isopropanol/buffer 1:1 (dashed line) measured in the (a) far-UV (190 to 270 nm) and (b) near-UV−vis (325 to 475 nm) ranges.

Table 5. Immobilized Amounts of CalB7 and HRP within pNIPAM Particles after Immobilization by Solvent Exchange enzyme

menzyme (μg per mg p-NIPAM)

Nenzyme per p-NIPAM particle

CalB HRP

6 ± 1 × 10−2 12 ± 2.6

5.4 × 103 ± 1 × 101 8.1 × 103 ± 2 × 103

some probability that in our case HRP has a positive charge at neutral pH and is attracted by the slightly negative p-NIPAM particles. For use as biocatalyst and to get information on whether HRP is protected by the surrounding polymer matrix, it is necessary to determine the position of the enzyme molecules within the polymer structure. Figure 3a and the intensity profile (Figure S1a) indicate that no enzyme could be detected within the microgel particles in buffer solution. This can be explained by the hydrophobic segments of the polymer particles and the low affinity of HRP for such structures when an aqueous solution is the surrounding medium. Figure 3b and the corresponding intensity profile (Figure S1b) show that the obtained spherically shaped fluorescence signals fit with the position of the p-NIPAM microgel particles after changing the solvent to isopropanol. This indicates that the labeled enzyme is immobilized either within the polymer network or adsorbed at the surface of the microgel particles. An orthogonal section view is obtained when using the z-stack option of the CLSM (Figure 4a). It shows spherically shaped fluorescence signals in all three planes. To confirm this result, intensity profiles of the x−y measurements at three different z positions are shown (Figure S2). The high fluorescence intensity assigned to the cut through the center of the particle (z = 16) indicates a high enzyme concentration especially within the center of the microgel particles. Because the adsorption of HRP at the surface of the polymer particles would result in a fluorescent capsule, the orthogonal section view and the intensity profiles prove the immobilization of HRP within the polymer network. Hence, the protection of HRP from environmental influences is given by the surrounding polymer matrix. Furthermore, the catalytic activity of the immobilized HRP is one of the most important characteristics. Table 4 shows that the activity of HRP in isopropanol is enhanced by the immobilization within p-NIPAM microgel particles. This can be explained by the fact that HRP is usually highly active in aqueous solution. The remaining aqueous cage leads to a similar environment and a high specific activity. In contrast, CD and UV−vis spectroscopy showed that mixing HRP with isopropanol leads to a change in the secondary and tertiary structure of the enzyme. Hence, it can be assumed that HRP is unfolded by the use of isopropanol and the active center is no longer accessible to the substrate. These results explain the loss in activity for the nonimmobilized HRP. According to the fact that nonimmobilized HRP is nearly insoluble and less active in

network after exchanging the solvent with isopropanol, representing a kind of “aqueous cage” as schematically drawn in Figure 6. The aqueous cage can be assumed to be the main

Figure 6. Schematic drawing of the immobilization by solvent exchange and the remaining aqueous cage within p-NIPAM microgel particles.

driving force for embedding both enzymes within the polymer network. However, metalloenzyme HRP41 is a glycoprotein with large carbohydrate regions (roughly 18% by mass),42−46 which are strongly hydrophilic. HRP also contains a smaller number of hydrophobic amino acids, which additionally contributes to the strong hydrophilic nature of HRP. In contrast, CalB contains a larger number of hydrophobic amino acids. It has been shown by molecular simulations that the surface of CalB is more than 50% hydrophobic and that the flexibility of the enzyme molecule is decreased in organic solvents because of restricted water exchange on the surface.47,48 Electrostatic attraction may also play a role in the higher efficiency of HRP immobilization in the p-NIPAM particles in comparison to CalB. CalB has a very well defined isoelectric point (pI) at 6.0.40 Hence, CalB is slightly negatively charged in water and will rather be repelled by the also slightly negatively charged p-NIPAM particles. HRP is a group of isoenzymes classified into three major groups based on their respective isoelectric points. The most abundant isoenzyme at more than 50% is HRP-C, which belongs to the so-called neutral isoenzymes. The pI values for HRP-C that can be found in the literature vary between 6.5 and 10.49,50 Therefore, there is 16007


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(4) Chen, H.; Liu, L.-H.; Wang, L.-S.; Ching, C.-B.; Yu, H.-W.; Yang, Y.-Y. Thermally Responsive Reversed Micelles for Immobilization of Enzymes. Adv. Funct. Mater. 2008, 18, 95−102. (5) Grunwald, P.; Hansen, K.; Gunßer, W. The Determination of Effective Diffusion Coefficients in a Polysaccharide Matrix Used for the Immobilization of Biocatalysts. Solid State Ionics 1997, 101, 863− 867. (6) Shiroya, T.; Tamura, N.; Yasui, M.; Fujimoto, K.; Kawaguchi, H. Enzyme Immobilization on Thermosensitive Hydrogel Microspheres. Colloids Surf., B 1995, 4, 267−274. (7) Gawlitza, K.; Wu, C.; Georgieva, R.; Wang, D.; AnsorgeSchumacher, M. B.; von Klitzing, R. Immobilization of Lipase B within Micronsized Poly-N-Isopropylacrylamide Hydrogel Particles by Solvent Exchange. Phys. Chem. Chem. Phys. 2012, 14, 9594−9600. (8) Senff, H.; Richtering, W. Temperature Sensitive Microgel Suspensions: Colloidal Phase Behavior and Rheology of Soft Spheres. J. Chem. Phys. 1999, 111, 1705−1711. (9) Kratz, K.; Hellweg, T.; Eimer, W. Structural Changes in PNIPAM Microgel Particles as Seen by SANS, DLS, and EM Techniques. Polymer 2001, 42, 6631−6639. (10) Berndt, I.; Richtering, W. Doubly Temperature Sensitive CoreShell Microgels. Macromolecules 2003, 36, 8780−8785. (11) Stieger, M.; Pedersen, J. S.; Lindner, P.; Richtering, W. Are Thermoresponsive Microgels Model Systems for Concentrated Colloidal Suspensions? A Rheology and Small-Angle Neutron Scattering Study. Langmuir 2004, 20, 7283−7292. (12) Fernández-Nieves, A.; Fernández-Barbero, A.; Vincent, B.; de las Nieves, F. Charge Controlled Swelling of Microgel Particles. Macromolecules 2000, 33, 2114−2118. (13) Kratz, K.; Hellweg, T.; Eimer, W. Influence of Charge Density on the Swelling of Colloidal Poly(N-isopropylacrylamide-co-acrylicacid) Microgels. Colloids Surf., A 2000, 170, 137−149. (14) Hoare, T.; Pelton, R. Highly pH and Temperature Responsive Microgels Functionalized with Vinylacetic Acid. Macromolecules 2004, 37, 2544−2550. (15) Shibayama, M.; Ikkai, F.; Inamoto, S.; Nomura, S.; Han, C. C. pH and Salt Concentration Dependence of the Microstructure of Poly(N-isopropylacrylamide-co-acrylic acid) gels. J. Chem. Phys. 1996, 105, 4358−4366. (16) Karg, M.; Pastoriza-Santos, I.; Rodriguez-González, B.; von Klitzing, R.; Wellert, S.; Hellweg, T. Temperature, pH, and Ionic Strength Induced Changes of the Swelling Behavior of PNIPAMPoly(allylacetic acid) Copolymer Microgels. Langmuir 2008, 24, 6300−6306. (17) Rubio Retama, J.; Sánchez-Paniagua López, M.; Hervás Péreza, J.; Frutos Cabanillas, G.; López-Cabarcos, E.; López-Ruiz, B. Biosensors Based on Acrylic Microgels A Comparative Study of Immobilized Glucose Oxidase and Tyrosinase. Biosens. Bioelectron. 2005, 20, 2268−2375. (18) Park, T. G.; Hoffman, A. S. Immobilization and Characterization of P-Galactosidase in Thermally Reversible Hydrogel Beads. J. Biomed. Mater. Res. 1990, 24, 21−38. (19) Nayak, S.; Lyon, L. A. Weiche Nanotechnologie mit weichen Nanopartikeln. Angew. Chem. 2005, 117, 7862−7886. (20) Hsiuea, G.-H.; Hsub, S.-H.; Yang, C.-C.; Leed, S.-H.; Yang, I.-K. Preparation of Controlled Release Ophthalmic Drops, For Glaucoma Therapy Using Thermosensitive Poly-N-isopropylacrylamide. Biomaterials 2002, 23, 457−462. (21) Eichenbaum, G. M.; Kiser, P. F.; Dobrynin, A. V.; Simon, S. A.; Needham, D. Investigation of the Swelling Response and Loading of Ionic Microgels with Drugs and Proteins: The Dependence on CrossLink Density. Macromolecules 1999, 32, 4867−4878. (22) Kawaguchi, H.; Fujimoto, K.; Mizuhara, Y. Hydrogel Microspheres III. Temperature-Dependent Adsorption of Proteins on PolyN-isopropylacrylamide Hydrogel Microspheres. Colloid Polym. Sci. 1992, 270, 53−57. (23) Fujimoto, K.; Mizuhara, Y.; Tamura, N.; Kawaguchi, H. Interactions between Thermosensitive Hydrogel Microspheres and Proteins. J. Intell. Mater. Syst. Struct. 1993, 4, 184−189.

isopropanol, the embedding within p-NIPAM microgel particles is a promising method and gives a number of advantages. The enzyme can be used in polar organic solvents (e.g., for chemical synthesis) and is protected from the organic solvent and other environmental influences.

5. CONCLUSIONS In the present study, enzyme HRP was successfully immobilized within p-NIPAM microgel particles using a solvent exchange from water to isopropanol. By CLSM, it has been demonstrated that a large quantity of HRP was embedded in the polymer network and not only adsorbed on the surface. This leads to a protected biocatalyst that shows an enhanced activity in isopropanol compared to the nonimmobilized enzyme. It has been proven that the method of solvent exchange can be transferred to other enzymes, which makes it a promising method for creating biocatalysts. Additionally, even enzymes that are usually active only in water (e.g. HRP) can be immobilized within the polymer particles by this method, resulting in highly active catalysts. Hence, as an important result of high impact these immobilized enzymes can be used in chemical synthesis even if polar organic solvents are present. No chemical adjustment of the polymer matrix is needed for the immobilization, which is another advantage of the solvent exchange.

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ASSOCIATED CONTENT

* Supporting Information S

Intensity profiles for the localization of immobilized HRP and UV−vis absorption spectra of HRP in buffer and in a buffer/ isopropanol mixture. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail:[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Changzhu Wu, Marion B. AnsorgeSchumacher, and Helmuth Möhwald for helpful discussions and collaboration regarding the solvent exchange. We thank Thomas Friedrich for the possibility of using SDS-Page in his laboratory. We acknowledge Gerald Brezesinski for the opportunity to use the CD spectrometer at the Max Planck Institute of Colloids and Interfaces. This work was supported by the DFG via the Cluster of Excellence “Unifying Concepts in Catalysis”.

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REFERENCES

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Immobilization of Water-Soluble HRP within Poly-N

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