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

†Stranski-Laboratory for Physical and Theoretical Chemistry and ‡Institute of Chemistry, Technische Universitat Berlin, 10623 Berlin,Germany§Institute of Transfusion Medicine, Center for Tumor Medicine, ChariteUniversitatsmedizin 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-N-isopropylacrylamide (p-NIPAM) microgels using the method of solvent exchange isapplied to the enzyme horseradish peroxidase (HRP). When the solvent is changedfrom water to isopropanol, HRP is embedded within the polymer structure. After thedetermination of the immobilized amount of enzyme, an enhanced specific activity ofthe biocatalyst in isopropanol can be observed. Karl Fischer titration is used todetermine 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 highaffinity of HRP for water. Beside, confocal laser scanning microscopy (CLSM) images show that HRP is located within themicrogel network after immobilization. This gives the best conditions for HRP to be protected against chemical and mechanicalstress. We were able to transfer a water-soluble enzyme to an organic phase by reaching a high catalytic activity. Hence, themethod of solvent exchange displays a general method for immobilizing enzymes within p-NIPAM microgels for use in organicsolvents. With this strategy, enzymes that are not soluble in organic solvents such as HRP can be used in such polar organicsolvents.

1. INTRODUCTION

Enzymes are highly catalytically active and responsible for theproduction of all organic molecules necessary in life. Typically,they react under mild conditions (e.g., ambient temperatureand pressure). Because of this fact, enzymes showing a highspecificity are attractive for use in synthetic chemistry.1 For thisapplication, it is necessary for the enzymes to be stable atdifferent pH values and in different solvents. Substrates as wellas the products of catalytic reactions are often soluble inorganic solvents. Hence, the use of organic solvents can lead toa higher yield of the product. Because of the fact that organicsolvents can alter the native structure of enzymes and lead to adecrease in activity, many methods have been developed toovercome this problem. One method is the immobilization ofenzymes within an inorganic or organic support. As a greatadvantage, the low contact with the carrier leads to residualmobility 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, microgelsmade of poly-N-isopropylacrylamide (p-NIPAM) have beenalso studied for use as enzyme supports.6,7 These water-swellable microgel systems are of high research interest becauseof their reversible response to external stimuli such astemperature,8−11 pH,12−14 and ionic strength.15,16 p-NIPAM

microgels undergo a volume phase transition (VPT) at around32 °C that is correlated to a decrease in size with increasingtemperature. This behavior makes these polymer particlesuseful for a wide field of applications (e.g., biosensors,17 enzymesupports,18,19 and drug delivery20,21).The first investigations of the adsorption of proteins at p-

NIPAM microgels were done in the early 1990s by Kawaguchiet al.22,23 In another study, polymer particles with a core ofpolystyrene and a shell of p-NIPAM were used to immobilizeβ-D-glucosidase.24 In other studies, lysozyme and trypsin wereimmobilized within p-NIPAM microgel particles with adiameter of between 50 and 80 μm where the location of theenzymes within the polymer structure was detectable byconfocal laser scanning microscopy (CLSM).25−27 Althoughenhanced activity is reached, the described studies investigatedthe activity exclusively in water. Another study presents theimmobilization of lipase within p-NIPAM macrogels, leading toenhanced activity in organic solvents.28 To benefit from the lowpolydispersity and the high surface-to-volume ratio, it is moreefficient to use smaller p-NIPAM particles. In previous studies,

Received: September 17, 2013Revised: December 7, 2013Published: December 9, 2013

Article

pubs.acs.org/Langmuir

© 2013 American Chemical Society 16002 dx.doi.org/10.1021/la403598s | Langmuir 2013, 29, 16002−16009

the enzyme lipase B from Candida antarctica (CalB) wasimmobilized within p-NIPAM microgel particles by exchangingwater for an organic solvent.29 The immobilization led toenhanced activity, and the location of CalB was determined tobe within the polymer structure. Additionally, it was shown thatan increase in the cross-linker density leads to a biocatalyst withtemperature-controllable activity.7,30

In this article, the method of solvent exchange is applied tohorseradish peroxidase (HRP). This enzyme is not soluble inorganic solvents, but it can use a wide range of organicsubstrates (e.g., phenols). It catalyzes the synthesis of a varietyof organic compounds without using water as a coreactant.31

The challenge is to bring the hydrophilic enzyme andhydrophobic substrate into contact. The novelty of the presentstudy is the transfer of an enzyme that is usually not soluble inorganic solvents in an organic phase without limiting thecatalytic activity. Therefore, HRP was immobilized within p-NIPAM microgel particles with a cross-linker content of 0.25mol % by changing the solvent from water to isopropanol.Enhanced activity of the immobilized HRP compared to that ofnonimmobilized HRP was determined by UV−vis spectrosco-py. Besides, CLSM was used to determine the location of HRPwithin the polymer network after immobilization. As thedriving force for immobilization, a residual amount of water wasdetected within p-NIPAM microgels after solvent exchangeusing Karl Fischer titration.

2. EXPERIMENTAL SECTION2.1. Materials. N-Isopropylacrylamide (97%) (NIPAM), horse-

radish peroxidase (HRP), bovine serum albumin standard (BSA, 2mg/mL), Bradford reagent, and pyrogallol (≥99%) were purchasedfrom 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 fromChemSolution. N,N′-Methylenebis(acrylamide) (MBA) (≥99.5%),potassium peroxodisulfate (KPS) (≥99%), and HYDRANAL-Composite 5 were obtained from Fluka. Mercaptoethanol (98%)was from PlusOne, and ammonium persulfate (APS, ≥98%) was fromAffymetrix/USB. Tris(hydroxymethyl)aminomethane (TRIS,≥99.9%), sodium dodecyl sulfate (SDS, ≥99%), glycine (>99%),acrylamide mixture (30% in water), and N,N,N′,N′-tetramethylethy-lenediamine (TEMED, 99%) were purchased from Roth. NIPAM waspurified by recrystallization in n-hexane. Other chemicals were used asreceived. Water was taken from a three-stage Millipore Milli-Q Plus185 purification system.2.2. Preparation Techniques. 2.2.1. Synthesis of p-NIPAM

Microgel Particles. p-NIPAM microgel particles with a cross-linkercontent of 0.25 mol % were synthesized by surfactant-free emulsionpolymerization. Because of the possibility of making larger particlesvisible by CLSM, a temperature ramp according to Meng et al.32 wasused. Briefly, 1.8 g of the NIPAM monomer (0.015 mol) and 8 mg ofthe MBA cross-linker (5 × 10−5 mol) were dissolved in 125 mL ofwater. After degassing the solution for 1 h at 45 °C, a solution of 1 mLKPS (0.08 M) was added to the mixture under continuous stirring. Atemperature ramp of 1 °C per 2 min was applied until the finaltemperature of 65 °C was reached. The polymerization was completedby stirring overnight at this temperature under an N2 atmosphere. Thereceived 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, theresidual 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 cross-linker content of 0.25 mol % and 0.3 mg of HRP were mixed with 1.5mL of buffer (0.1 M potassium phosphate buffer, pH 7) at roomtemperature, leading to an initial concentration of 0.06 mg of HRP permg of p-NIPAM. The solution was stirred overnight and centrifuged

for 15 min at 9000g. The residues were redispersed in isopropanol andwashed three times with centrifugation and redispersion. Theredispersion after centrifugation takes around 1 h. To determine theimmobilized amount, one part of the sample was redispersed in bufferwhile the other part was redispersed in isopropanol again forinvestigations of the catalytic activity.

To determine the location of HRP within the polymer particles afterimmobilization, the enzyme was labeled with FITC according to theliterature33 and freeze-dried afterward. A solution of 2 mg of FITC-HRP and 10 mg of p-NIPAM in 2 mL of buffer (0.1 M potassiumphosphate buffer, pH 7) was stirred overnight and centrifuged for 10min at 9000g. One part of the sample was redispersed in buffer whilethe 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 ofthe microgel particles. Therefore, an ALV goniometer setup with aNd/YAG laser as the light source (λ = 532 nm) was used to record thecorrelation functions at a constant scattering angle of 60°. Thecorrelation functions were generated using an ALV-5000/E multiple-τdigital correlator and subsequently analyzed by an inverse Laplacetransformation (CONTIN34).

The molecular weight of the polymer particles was determined bystatic light scattering (SLS) using an ALV/CGS-3 compact goniometersystem equipped with an ALV/LSE-5004 correlator. Scattering anglesfrom 17 to 37° with 2° steps in between were used, and theconcentration 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 Hubercompatible control thermostat. A He−Ne laser (λ = 634 nm) was usedas the light source.

2.3.2. Confocal Laser Scanning Microscopy (CLSM). To getinformation on the location of HRP after immobilization within themicrogel particles, CLSM was used. Roughly, 20 μL of the preparedsamples were placed on a coverslip and investigated by applying anAxiovert 200 M inverted microscope with a 100× oil-immersionobjective (numerical aperture 1.3) and a Zeiss LSM 510 Meta confocalscanning unit (Zeiss MicroImaging GmbH, Jena, Germany). Torecord the fluorescence, the 488 nm line of the argon laser forexcitation and a 505 nm long-pass emission filter were used. Z stackswere performed with an upward step of 50 nm starting at the surfaceof the coverslips. Different Z stacks of the samples were analyzed usingthe LSM 510 software and displayed as an overlay of transmission andfluorescence channels in orthogonal section views.

2.3.3. Circular Dichroism Spectroscopy (CD). CD spectroscopywas used for investigations of the peptide secondary and tertiarystructure in solution. CD spectra were recorded on a Jasco J-715(Japan) spectrometer in a wavelength range from 190 to 475 nm with0.5 nm step resolution using quartz cuvettes with an optical pathlength of 0.1 cm. For measurements in the far-UV range (190 to 270nm), the HRP concentration was kept at 0.3 mg/mL while the enzymeconcentration was 10 mg/mL for measurements in the near-UV−visrange (325 to 475 nm). For HRP in isopropanol, 1:1 mixtures ofbuffer and isopropanol were used as solvents. All measurements wereperformed at room temperature. Data processing was carried out usingthe J-700 software package. After subtracting the blank spectra fromthe sample spectra, the CD signal was transformed to the mean residuemolar ellipticity.35

2.3.4. Determination of the Amount of Water in MicrogelParticles. Karl Fischer titration was used to determine the watercontent in p-NIPAM microgel particles before and after solventexchange. In the case of freeze-dried polymers, a solution of 20 mg ofmicrogels per mL methanol was prepared under ambient conditionsand titrated with a mixture of sulfur dioxide, imidazole, and iodine(Hydranal). The end point of the titration was determined with aplatinum electrode. The amount of titrant was used to calculate theamount of water in the sample. The water content in pure methanolwas also determined to receive the exact value of the water content forthe freeze-dried microgels.

The determination of the residual water content after solventexchange was done by dissolving 20 mg of microgel in 1 mL of buffer(0.1 M potassium phosphate buffer, pH 7) followed by two

Langmuir Article

dx.doi.org/10.1021/la403598s | Langmuir 2013, 29, 16002−1600916003

centrifugation steps (10 min at 9000g) and redispersion in 1 mL ofisopropanol. The received solutions as well as pure isopropanol weretitrated, and the residual amount of water was calculated. Allmeasurements 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, sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-Page) wasdone. First, the separation and stacking gels were prepared bypolymerization. The composition of these gels is shown in Table 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 mixedwith 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 themolecular mass standard, 10 μL of the PageRuler prestained proteinladder (10 to 170 kDa, Fermentas) was also separated by the gel. Aftersurrounding the gel with a running buffer (3.03 g of TRIS, 14.41 g ofglycine, 10 mL of a 10% SDS-solution), 50 V was applied to thesystem until the samples reached the separation gel, followed byapplying a voltage of 100 V for 1 h. After separation, the gel wasstained with Coomassie dye (PageBlue protein staining solution,Thermoscientific) on one hand and with silver nitrate on the otherhand. After the sample was washed with water, the molecular weight ofthe enzymes was determined.2.3.6. Determination of the Immobilized Amount of HRP. The

amount of HRP that is immobilized within the p-NIPAM microgelparticles was achieved via determination of the total protein content inaqueous solution using the Bradford reagent according to themanufacturer’s instructions.7 UV−vis spectra were measured withthe 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 topurpurogallin in the presence of hydrogen peroxide. The formation ofthe product can be determined via UV−vis spectroscopy at awavelength of 420 nm. The immobilized sample (0.4 mL) redispersedin 1.5 mL of isopropanol was mixed with 0.4 mL of a pyrogallolsolution in isopropanol (1 mg/mL) and 0.132 mL of hydrogenperoxide (48 μL of H2O2 (35%) in 20 mL of water). For thedetermination of the activity of nonimmobilized enzyme, 1 mL of asolution of HRP in isopropanol (0.44 mg/mL) was mixed with 0.1 mLof the pyrogallol and hydrogen peroxide solution, respectively. Asreference, the same compositions of the reagents were used unless thesamples were replaced by isopropanol. Afterward, the solution wasmeasured via UV−vis spectroscopy for 1 min at 420 nm. Because ofthe linear behavior of the increase in absorption with time, the volumeactivity can be calculated using eq 1.

=Δ

ϵU

EVV dV

total

S (1)

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

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

3. RESULTSMonodisperse p-NIPAM microgel particles with a cross-linkercontent of 0.25 mol % were obtained by surfactant-freeemulsion polymerization according to a previous study.7 Thetemperature ramp was used to synthesize microgel particleswith a diameter of around 1 μm in order to have the possibilityto make them visible under a CLSM. In previous work, theswelling curves in water and isopropanol studied by DLS areshown.7 It was observed that the hydrodynamic diameter (Dh)in water is 1.8 μm at 25 °C and decreases to 0.2 μm byincreasing the temperature to 40 °C. The use of isopropanol asa solvent results in a decrease in Dh to 1.2 μm at 25 °C, whichcorresponds to a collapse of 0.6 μm in diameter compared towater. The decrease in size by using isopropanol indicates thatwater is a better solvent for the microgel particles thanisopropanol. The volume phase transition temperature at whichthe microgel particles collapse is similar in both solvents. Theresults are summarized in Table 2.

The molecular weight (Mw) of the synthesized p-NIPAMmicrogel particles was determined by SLS measurements. Aresidual water content of around 12 wt % under ambientconditions was determined by Karl Fischer titration andconsidered for sample preparation. A refractive index incrementof dn/dc = 0.167 cm3 g−1 was used for calculation.37 Thereceived Zimm plots lead to a Mw of 3.0 × 1010 g mol−1.7

To determine the immobilized amount of HRP, it isnecessary to investigate the purity of the used enzyme.Therefore, SDS-Page was performed using a polyacrylamidegel (12% MBA) with Coomassie dye and silver staining foranalysis. Figure 1 shows the received images. The investigationswere done for HRP as received with an expected Mw of 44 000Da.The chromatograms prove that the used peroxidase has a

high purity. The investigation results in an intensive band ataround 40 kDa after staining with Coomassie dye as well aswith silver nitrate. The fact that the determined Mw is in goodagreement with the expected value proves the high purity ofthis 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 buffersolution. To reach the immobilization, solvent exchange withisopropanol was carried out. A sketch of the immobilization isshown 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 redis-persed in buffer. Assuming that no enzyme was removed bycentrifugation, an embedded amount of 12 ± 2.6 μg of HRPper mg of p-NIPAM microgel particles was obtained by

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

components 5% stacking gel (mL) 12% running gel (mL)

H2O 9.9 6.930% acrylamide mixture 1.7 12.01.5 M TRIS (pH 8.8) 7.51.5 M TRIS (pH 6.8) 1.2510% SDS 0.1 0.310% APS 0.1 0.3TEMED 0.01 0.012

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

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

solvent Dh at 25 °C (nm) Dh at 40 °C (nm) VPTT (°C)

water 1762 ± 46 210 ± 4 28isopropanol 1160 ± 62 70 ± 2 27

Langmuir Article

dx.doi.org/10.1021/la403598s | Langmuir 2013, 29, 16002−1600916004

investigating the sample with a Bradford assay. The preciseprocedure and the used calibration curves are shown in ourprevious 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 wasdetermined that ∼8100 enzyme molecules are embeddedwithin 1 microgel particle.For a better understanding of the immobilization process, it

is important to determine the water content within themicrogel particles after the exchange of water for isopropanol.Therefore, Karl Fischer titration was performed. Because of thefact that p-NIPAM is stored under ambient conditions, there isalso water left in the dried polymer particles. To exclude thisamount from the determined residual water content aftersolvent exchange, the same experiments were done for “dry”microgel particles. Table 3 summarizes the determined water

contents of p-NIPAM particles in the dried state (ambientconditions) and after solvent exchange. There is a hugedifference in the water content before and after solventexchange. This indicates that water remains within the polymernetwork 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 tothe immobilization procedure. After the labeled enzyme was

mixed with p-NIPAM microgel particles, the solution wascentrifuged and redispersed in either buffer or isopropanol. Thesize of the p-NIPAM microgel particles allows the applicationof CLSM as an adequate method to investigate if the enzymemolecules are situated within the polymer network or only onthe surface. Figure 3 shows the images made by CLSM for theimmobilized system in (a) buffer and (b) isopropanol.

Images a1 and b1 display the fluorescence of the sample,images a2 and b2 display the transmission of the sample, andimages a3 and b3 reflect an overlay of the first two images toshow whether the fluorescence signal matches the position ofthe microgel particles. The upper series shows the images forthe sample redispersed in buffer. Here, the fluorescence signal isdistributed over the whole scanning area except for somespherically shaped areas (white circles). These areas fit to theposition of the polymer particles, indicating that HRP is notsituated within the polymer network. The lower series of Figure3 shows images obtained by measuring the sample afterredispersion in isopropanol. Obviously, spherically shapedfluorescence signals are monitored that are exactly at theposition of the p-NIPAM microgel particles (white circles). Toconfirm the assumption made by the fluorescence images,intensity profiles of the shown images were made. To avoidhigh-frequency noise, a Fourier transform bandpass filter wasused. 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-stackoption of the CLSM was used. Figure 4a displays an orthogonalsection view of the measured sample, and Figure 4b shows aschematic 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 directionbetween them. The image in the center framed by the blue boxshows one of the measured x−y planes. The y−z plane of thecut through the sample along the red vertical line in the centralx−y image is represented by the righthand red framed box. Theupper green box frames the x−z plane of a cut through thesample along the green horizontal line in the central x−y image.To complete the orthogonal section view, the blue lines in thex−z and y−z images display the z position of the x−y planeshown in the center. In all three planes, the fluorescence signalreflects the spherical shape of the particles. This observationcan be supported by fluorescence intensity profiles of the x−yimages 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 1. Results from SDS-Page after staining with (a) Coomassiedye and (b) silver nitrate for HRP as received and the used proteinstandard (M).

Figure 2. Schematic process of the immobilization of HRP within p-NIPAM microgel particles at 25 C.

Table 3. Water Contents of p-NIPAM after Freeze Drying(Ambient Conditions) and after Solvent Exchange (se) andAmount of Water per p-NIPAM Microgel Particle

MBA(mol %)

cH2O (wt %)(dry)

cH2O (wt %)(after se)

mH2O (g perp-NIPAM)

0.25 12.0 ± 0.9 46.6 ± 2.4 2.32 × 10−14

Figure 3. CLSM images of the residue of p-NIPAM after incubationwith HRP redispersed in (a) buffer and (b) isopropanol influorescence mode, transmission mode, and as a superimposedimage of both.

Langmuir Article

dx.doi.org/10.1021/la403598s | Langmuir 2013, 29, 16002−1600916005

particle. The profiles are presented in the SupportingInformation (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 theoxidation of pyrogallol to purpurogallin in the presence ofhydrogen peroxide, isopropanol, and HRP. Purpurogallin canbe detected via UV−vis spectroscopy at a wavelength of 420nm. The volume activity (UV) can be calculated from eq 1. Tocompare the activity between the immobilized and thenonimmobilized enzyme, the activity measurements wereperformed for both samples. The results are listed in Table 4.Against expectations, also for nonimmobilized HRP inisopropanol, a small amount of product was formed. AlthoughHRP is not soluble in isopropanol, a small amount of water (asa solvent for H2O2) is added to the activity reaction. Hence,some of the enzymes can be dissolved in this aqueous phaseduring the activity reaction. For a direct comparison of bothsystems, the specific activity (Usp) has to be determined. In thecase of immobilized HRP, the amount of HRP within themicrogel particles for the activity measurements is known, andUsp can be calculated. For nonimmobilized HRP, Usp was firstcalculated with respect to the initial amount of HRP. Because itis not possible to determine the amount of HRP dissolved inthe aqueous phase, this value is only a speculation. To get anidea of the enhancement of the activity by immobilization, thespecific activity was also calculated by assuming that the sameamount of enzyme was present in the immobilized andnonimmobilized systems (Usp(a)). All of the described resultsare summarized in Table 4. It is shown that an immobilizationof HRP within p-NIPAM microgel particles leads to anenhancement in catalytic activity in isopropanol.The use of isopropanol can lead to possible changes in the

secondary and tertiary structure of HRP, resulting inaggregation or deactivation. Therefore, we recorded CD spectraof 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).

The far-UV CD spectra give information about the secondarystructure of the enzyme. Figure 5a shows two typical bands at223 and 207 nm for HRP dissolved in buffer solution. Whenthe same enzyme concentration is used in an isopropanol/buffer (1:1) solution, the band at 209 nm disappears while theband at 223 nm decreases and is shifted to 229 nm. Thisstrongly indicates a change in the secondary structure of HRPin the presence of isopropanol. The near-UV−vis CD spectrashow the Soret region and give information about the tertiarystructure. Figure 5b shows small changes in the CD signal at awavelength of 403 nm by adding isopropanol. To get moreinformation about the Soret region, additional UV−visabsorption measurements were made (Figure S3 in theSupporting Information). It is shown that there is a strongchange in the absorption maximum at a wavelength of 403 nm.This indicates loosening of the tertiary structure and a changein the microenvironment of the heme in HRP.38

4. DISCUSSION

In our previous studies, it has been shown that solventexchange is an adequate method of immobilizing lipase B fromCandida antarctica (CalB) within p-NIPAM microgel particles,resulting in enhanced activity in organic solvents.7,30 It isknown that CalB is soluble in a number of organic solvents. It isusually located at the interface between water and the organicphase where it shows catalytic activity. Hence, this enzyme is asuitable model enzyme for making a proof of principle. Forindustrial applications, there is strong interest in investigating ifthe method can be transferred to other enzymes. In the presentstudy, the HRP enzyme was used, which is usually insoluble inorganic solvents and not active in aqueous environments.Because HRP is responsible for the oxidation of inorganic andorganic compounds, it is important to create a method toobtain active HRP in organic solvents.In this study, HRP was successfully immobilized within p-

NIPAM microgel particles using the method of solventexchange that is supported by an immobilized number of∼8100 enzyme molecules per microgel particle. In comparisonto the immobilized amount of CalB, the loading efficiency forHRP 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 (3nm × 4 nm × 5 nm40). Simultaneously, the exchange of waterfor isopropanol leads to a decrease in size (Figure 3), whichkeeps the enzyme inside the p-NIPAM particles. One reasonfor the higher immobilized amount of HRP could be an easierdiffusion 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 betaken into account. First, one always has to consider theirmolecular structure. Enzymes CalB and HRP are designed toperform their functions in aqueous environment. As shown inTable 3, there is a huge amount of water left in the polymer

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

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

activity nonimmobilized immobilized

UV (μmol min−1 mL−1) 2.03 × 10−3 ± 3 × 10−4 2.72 × 10−1 ± 1 × 10−1

Usp (μmol min−1 μg−1) 5.55 × 10−6 ± 1 × 10−6 1.60 × 10−2 ± 9 × 10−3

Usp(a) (μmol min−1 μg−1) 6.15 × 10−5 ± 1 × 10−5 1.60 × 10−2 ± 9 × 10−3

Langmuir Article

dx.doi.org/10.1021/la403598s | Langmuir 2013, 29, 16002−1600916006

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

driving force for embedding both enzymes within the polymernetwork. However, metalloenzyme HRP41 is a glycoproteinwith large carbohydrate regions (roughly 18% by mass),42−46

which are strongly hydrophilic. HRP also contains a smallernumber of hydrophobic amino acids, which additionallycontributes to the strong hydrophilic nature of HRP. Incontrast, CalB contains a larger number of hydrophobic aminoacids. It has been shown by molecular simulations that thesurface of CalB is more than 50% hydrophobic and that theflexibility of the enzyme molecule is decreased in organicsolvents because of restricted water exchange on the sur-face.47,48

Electrostatic attraction may also play a role in the higherefficiency of HRP immobilization in the p-NIPAM particles incomparison to CalB. CalB has a very well defined isoelectricpoint (pI) at 6.0.40 Hence, CalB is slightly negatively charged inwater and will rather be repelled by the also slightly negativelycharged p-NIPAM particles. HRP is a group of isoenzymesclassified into three major groups based on their respectiveisoelectric points. The most abundant isoenzyme at more than50% is HRP-C, which belongs to the so-called neutralisoenzymes. The pI values for HRP-C that can be found inthe literature vary between 6.5 and 10.49,50 Therefore, there is

some probability that in our case HRP has a positive charge atneutral pH and is attracted by the slightly negative p-NIPAMparticles.For use as biocatalyst and to get information on whether

HRP is protected by the surrounding polymer matrix, it isnecessary to determine the position of the enzyme moleculeswithin the polymer structure. Figure 3a and the intensity profile(Figure S1a) indicate that no enzyme could be detected withinthe microgel particles in buffer solution. This can be explainedby the hydrophobic segments of the polymer particles and thelow affinity of HRP for such structures when an aqueoussolution is the surrounding medium. Figure 3b and thecorresponding intensity profile (Figure S1b) show that theobtained spherically shaped fluorescence signals fit with theposition of the p-NIPAM microgel particles after changing thesolvent to isopropanol. This indicates that the labeled enzymeis immobilized either within the polymer network or adsorbedat the surface of the microgel particles. An orthogonal sectionview is obtained when using the z-stack option of the CLSM(Figure 4a). It shows spherically shaped fluorescence signals inall three planes. To confirm this result, intensity profiles of thex−y measurements at three different z positions are shown(Figure S2). The high fluorescence intensity assigned to the cutthrough the center of the particle (z = 16) indicates a highenzyme concentration especially within the center of themicrogel particles. Because the adsorption of HRP at thesurface of the polymer particles would result in a fluorescentcapsule, the orthogonal section view and the intensity profilesprove the immobilization of HRP within the polymer network.Hence, the protection of HRP from environmental influences isgiven by the surrounding polymer matrix.Furthermore, the catalytic activity of the immobilized HRP is

one of the most important characteristics. Table 4 shows thatthe activity of HRP in isopropanol is enhanced by theimmobilization within p-NIPAM microgel particles. This canbe explained by the fact that HRP is usually highly active inaqueous solution. The remaining aqueous cage leads to asimilar environment and a high specific activity. In contrast, CDand UV−vis spectroscopy showed that mixing HRP withisopropanol leads to a change in the secondary and tertiarystructure of the enzyme. Hence, it can be assumed that HRP isunfolded by the use of isopropanol and the active center is nolonger accessible to the substrate. These results explain the lossin activity for the nonimmobilized HRP. According to the factthat nonimmobilized HRP is nearly insoluble and less active in

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 p-NIPAM Particles after Immobilization by Solvent Exchange

enzyme menzyme (μg per mg p-NIPAM) Nenzyme per p-NIPAM particle

CalB 6 ± 1 × 10−2 5.4 × 103 ± 1 × 101

HRP 12 ± 2.6 8.1 × 103 ± 2 × 103

Figure 6. Schematic drawing of the immobilization by solventexchange and the remaining aqueous cage within p-NIPAM microgelparticles.

Langmuir Article

dx.doi.org/10.1021/la403598s | Langmuir 2013, 29, 16002−1600916007

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

5. CONCLUSIONS

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

■ ASSOCIATED CONTENT

*S Supporting InformationIntensity profiles for the localization of immobilized HRP andUV−vis absorption spectra of HRP in buffer and in a buffer/isopropanol mixture. This material is available free of charge viathe Internet at http://pubs.acs.org/.

■ AUTHOR INFORMATION

Corresponding Author*E-mail:klitzing@chem.tu-berlin.de.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We acknowledge Changzhu Wu, Marion B. Ansorge-Schumacher, and Helmuth Mohwald for helpful discussionsand collaboration regarding the solvent exchange. We thankThomas Friedrich for the possibility of using SDS-Page in hislaboratory. We acknowledge Gerald Brezesinski for theopportunity to use the CD spectrometer at the Max PlanckInstitute of Colloids and Interfaces. This work was supportedby the DFG via the Cluster of Excellence “Unifying Conceptsin Catalysis”.

■ REFERENCES(1) Ogino, H.; Ishikawa, H. Enzymes Which Are Stable in thePresence of Organic Solvents. J. Biosci. Bioeng. 2001, 91, 109−116.(2) Ansorge-Schumacher, M. B. In Handbook of HeterogeneousCatalysis; Ertl, G., Knozinger, H., Schuth, F., Weitkamp, J., Eds.; Wiley-VCH: Weinheim, Germany, 2008; Immobilization of BiologicalCatalysts, p 644.(3) Lei, J.; Fan, J.; Yu, C.; Zhang, L.; Jiang, S.; Tu, B.; Zhao, D.Immobilization of Enzymes in Mesoporous Materials: Controlling theEntrance to Nanospace. Microporous Mesoporous Mater. 2004, 73,121−128.

(4) Chen, H.; Liu, L.-H.; Wang, L.-S.; Ching, C.-B.; Yu, H.-W.; Yang,Y.-Y. Thermally Responsive Reversed Micelles for Immobilization ofEnzymes. Adv. Funct. Mater. 2008, 18, 95−102.(5) Grunwald, P.; Hansen, K.; Gunßer, W. The Determination ofEffective Diffusion Coefficients in a Polysaccharide Matrix Used forthe 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.; Ansorge-Schumacher, M. B.; von Klitzing, R. Immobilization of Lipase B withinMicronsized Poly-N-Isopropylacrylamide Hydrogel Particles bySolvent Exchange. Phys. Chem. Chem. Phys. 2012, 14, 9594−9600.(8) Senff, H.; Richtering, W. Temperature Sensitive MicrogelSuspensions: 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 PNIPAMMicrogel Particles as Seen by SANS, DLS, and EM Techniques.Polymer 2001, 42, 6631−6639.(10) Berndt, I.; Richtering, W. Doubly Temperature Sensitive Core-Shell Microgels. Macromolecules 2003, 36, 8780−8785.(11) Stieger, M.; Pedersen, J. S.; Lindner, P.; Richtering, W. AreThermoresponsive Microgels Model Systems for ConcentratedColloidal Suspensions? A Rheology and Small-Angle NeutronScattering Study. Langmuir 2004, 20, 7283−7292.(12) Fernandez-Nieves, A.; Fernandez-Barbero, A.; Vincent, B.; de lasNieves, F. Charge Controlled Swelling of Microgel Particles.Macromolecules 2000, 33, 2114−2118.(13) Kratz, K.; Hellweg, T.; Eimer, W. Influence of Charge Densityon the Swelling of Colloidal Poly(N-isopropylacrylamide-co-acryl-icacid) Microgels. Colloids Surf., A 2000, 170, 137−149.(14) Hoare, T.; Pelton, R. Highly pH and Temperature ResponsiveMicrogels 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 ofPoly(N-isopropylacrylamide-co-acrylic acid) gels. J. Chem. Phys. 1996,105, 4358−4366.(16) Karg, M.; Pastoriza-Santos, I.; Rodriguez-Gonzalez, B.; vonKlitzing, R.; Wellert, S.; Hellweg, T. Temperature, pH, and IonicStrength Induced Changes of the Swelling Behavior of PNIPAM-Poly(allylacetic acid) Copolymer Microgels. Langmuir 2008, 24,6300−6306.(17) Rubio Retama, J.; Sanchez-Paniagua Lopez, M.; Hervas Pereza,J.; Frutos Cabanillas, G.; Lo pez-Cabarcos, E.; Lo pez-Ruiz, B.Biosensors Based on Acrylic Microgels A Comparative Study ofImmobilized Glucose Oxidase and Tyrosinase. Biosens. Bioelectron.2005, 20, 2268−2375.(18) Park, T. G.; Hoffman, A. S. Immobilization and Characterizationof P-Galactosidase in Thermally Reversible Hydrogel Beads. J. Biomed.Mater. Res. 1990, 24, 21−38.(19) Nayak, S.; Lyon, L. A. Weiche Nanotechnologie mit weichenNanopartikeln. 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 GlaucomaTherapy Using Thermosensitive Poly-N-isopropylacrylamide. Bioma-terials 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 ofIonic Microgels with Drugs and Proteins: The Dependence on Cross-Link Density. Macromolecules 1999, 32, 4867−4878.(22) Kawaguchi, H.; Fujimoto, K.; Mizuhara, Y. Hydrogel Micro-spheres III. Temperature-Dependent Adsorption of Proteins on Poly-N-isopropylacrylamide Hydrogel Microspheres. Colloid Polym. Sci.1992, 270, 53−57.(23) Fujimoto, K.; Mizuhara, Y.; Tamura, N.; Kawaguchi, H.Interactions between Thermosensitive Hydrogel Microspheres andProteins. J. Intell. Mater. Syst. Struct. 1993, 4, 184−189.

Langmuir Article

dx.doi.org/10.1021/la403598s | Langmuir 2013, 29, 16002−1600916008

(24) Welsch, N.; Wittemann, A.; Ballauff, M. Enhanced Activity ofEnzymes Immobilized in Thermoresponsive Core-Shell Microgels. J.Phys. Chem. B 2009, 113, 16039−16045.(25) Johansson, C.; Hansson, P.; Malmsten, M. Mechanism ofLysozyme Uptake in Poly(acrylic acid) Microgels. J. Phys. Chem. B2009, 113, 6183−6193.(26) Johansson, C.; Gernandt, J.; Bradley, M.; Vincent, B.; Hansson,P. Interaction between Lysozyme and Colloidal Poly(NIPAM-co-acrylic acid) Microgels. J. Colloid Interface Sci. 2010, 347, 241−251.(27) Mansson, R.; Frenning, G.; Malmsten, M. Factors AffectingEnzymatic Degradation of Microgel-Bound Peptides. Biomacromole-cules 2013, 14, 2317−2325.(28) Wack, H.; Nellesen, A.; Schwarze-Benning, K.; Deerberg, G.Hydrogel Composites with Temperature Induced Phase Transition forBiocatalysis. J. Chem. Technol. Biotechnol. 2011, 86, 519.(29) Bai, S.; Wu, C.; Gawlitza, K.; von Klitzing, R.; Ansorge-Schumacher, M. B.; Wang, D. Using Hydrogel Microparticles toTransfer Hydrophilic Nanoparticles and Enzymes to Organic Mediavia Stepwise Solvent Exchange. Langmuir 2010, 26, 12980−12987.(30) Gawlitza, K.; Wu, C.; Georgieva, R.; Ansorge-Schumacher, M.B.; von Klitzing, R. Temperature Controlled Activity of Lipase B fromCandida Antarctica after Immobilization within p-NIPAM MicrogelParticles. Z. Phys. Chem. 2012, 226, 749−759.(31) Ryu, K.; Dordick, J. S. How Do Organic Solvents AffectPeroxidase Structure and Function? Biochemistry 1992, 31, 2588−2598.(32) Meng, Z.; Smith, M. H.; Lyon, L. A. Temperature-ProgrammedSynthesis of Micron-Sized Multi-Responsive Microgels. Colloid Polym.Sci. 2009, 287, 277−285.(33) Nargessi, R. D.; Smith, D. S. Fluoremetric Assays for Avidin andBiotin. Methods Enzymol. 1986, 122, 67−72.(34) Provencher, S. W. A Constrained Regularization MethodRegularization Method for Inverting Data Represented by LinearAlgebraic or Integral Equations. Comput. Phys. Commun. 1982, 27,213−227.(35) Fears, K. P. Adsorption-Induced Changes in Enzyme BioactivityCorrelated with Adsorbed Protein Orientation and Conformation;ProQuest: 2009.(36) Laemmli, U. K. Cleavage of Structural Proteins during theAssembly of the Head of Bacteriophage T4. Nature 1970, 227, 680−685.(37) Gao, J.; Hu, Z. Optical Properties of N-IsopropylacrylamideMicrogel Spheres in Water. Langmuir 2002, 18, 1360−1367.(38) Tong, J.; Luxenhofer, R.; Yi, X.; Jordan, R.; Kabanov, A. V.Protein Modification with Amphiphilic Block Copoly(2-oxazoline)s asa New Platform for Enhanced Cellular Delivery. Mol. Pharmaceutics2010, 7, 984−992.(39) Zhang, J.; Chi, Q.; Dong, S.; Wang, E. In Situ ElectrochemicalScanning Tunnelling Microscopy Investigation of Structure forHorseradish Peroxidase and Its Electrocatalytic Property. Bioelec-trochem. Bioenerg. 1996, 39, 267−274.(40) Uppenberg, J.; Hansen, M. T.; Patkar, S.; Jones, T. A. TheSequence, Crystal Structure Determination and Refinement of TwoCrystal Forms of Lipase B from Candida antarctica. Structure 1994, 2,293−308.(41) Veitch, N. C. Horseradish Peroxidase: A Modern View of aClassic Enzyme. Phytochemistry 2004, 65, 249−259.(42) Welinder, K. G. Amino Acid Sequence Studies of HorseradishPeroxidase. Eur. J. Biochem. 1979, 95, 483−502.(43) Welinder, K. G. Covalent Structure of the GlycoproteinHorseradish Peroxidase. FEBS Lett. 1976, 72, 19−23.(44) Clarke, J.; Shannon, L. M. The Isolation and Characterization ofthe Glycopeptides from Horseradish Peroxidase Isoenzymes C.Biochim. Biophys. Acta 1976, 421, 428−442.(45) Yang, B. Y.; Gray, J. S. S.; Montgomery, R. The Glycans ofHorseradish Peroxidase. Carbohydr. Res. 1996, 287, 203−212.(46) Mogharrab, N.; Ghourchian, H.; Amininasab, M. StructuralStabilization and Functional Improvement of Horseradish Peroxidase

upon Modification of Accessible Lysines: Experiments and Simulation.Biophys. J. 2007, 92, 1192−1203.(47) Trodler, P.; Pleiss, J. Modeling Structure and Flexibility ofCandida antarctica Lipase B in Organic Solvents. BMC Struct. Biol.2008, 8, 1−10.(48) Li, C.; Tan, T.; Zhang, H.; Feng, W. Analysis of theConformational Stability and Activity of Candida antarctica Lipase Bin Organic Solvents: Insight from Molecular Dynamics and QuantumMechanics/Simulations. J. Biol. Chem. 2010, 285, 28434−28441.(49) Aibara, S.; Yamashita, H.; Mori, E.; Kato, M.; Morita, Y.Isolation and Characterization of Five Neutral Isoenzymes ofHorseradish Peroxidase. J. Biochem. 1982, 92, 531−539.(50) Lavery, C. B.; Macinnis, M. C.; Macdonald, M. J.; Williams, J.;Spencer, C. A.; Burke, A. A.; Irwin, D. J.; D’Cunha, G. B. Purificationof Peroxidase from Horseradish (Armoracia rusticana) Roots. J. Agric.Food Chem. 2010, 58, 8471−8476.

Langmuir Article

dx.doi.org/10.1021/la403598s | Langmuir 2013, 29, 16002−1600916009