Immobilization of catalase inpoly(isopropylacrylamide- co-hydroxyethylmethacrylate) thermallyreversible hydrogelsM Yakup Arıca,1* H Avni Oktem,2 Zeki Oktem3 and S Ali Tuncel41Department of Biology, Kırıkkale University, 71450 Kırıkkale, Turkey2Department of Biological Science, Middle East Technical University, 06531 Ankara, Turkey3Department of Chemistry, Kırıkkale University, 71450 Kırıkkale, Turkey4Chemical Engineering Department, Hacettepe University, 06532 Ankara, Turkey

Abstract: Catalase was entrapped in thermally reversible poly(isopropylacrylamide-co-hydroxyethyl-

methacrylate) (pNIPAM/HEMA) copolymer hydrogels. The thermoresponsive hydrogels, in cylind-

rical geometry, were prepared in an aqueous buffer by redox polymerization. It was observed that upon

entrapment, the activity retention of catalase was decreased between 47 and 14%, and that increasing

the catalase loading of hydrogel adversely affected the activity. The kinetic behaviour of the entrapped

enzyme was investigated in a batch reactor. The apparent kinetic constant of the entrapped enzyme

was determined by the application of Michaelis±Menten model and indicated that the overall reaction

rate was controlled by the substrate diffusion rate through the hydrogel matrix. Due to the

thermoresponsive character of the hydrogel matrix, the maximum activity was achieved at 25°C with

the immobilized enzyme. The Km value for immobilized catalase (28.6mM) was higher than that of free

enzyme (16.5mM). Optimum pH was the same for both free and immobilized enzyme. Operational,

thermal and storage stabilities of the enzyme were found to increase with immobilization.

# 1999 Society of Chemical Industry

Keywords: catalase; thermo-responsive gels; hydrogels; entrapment; immobilization

INTRODUCTIONEnzymes are usually immobilized by chemical and

physical means to increase enzyme stability and enable

long term operation.1±3 Suitable matrices include

hydrogels that are highly compatible for immobiliza-

tion of enzymes due to their hydrophilic nature.

Poly(N-isopropylacrylamide) (pNIPAM) is a hydrogel

and exhibits reversible volume change in response to a

change in temperature. The thermal cycling behaviour

of these hydrogels could be used for controlling on and

off in various applications such as immobilized enzyme

reactors, controlled drug delivery systems and separa-

tion processes.4±7 pNIPAM hydrogels are known to

have a negative thermosensitivity at room tempera-

ture; increasing the temperature results in slow

deswelling that becomes rapid as the temperature

approaches the lower critical solution temperature

(LCST, �32°C). When the LSTC is reached the gel

collapses, losing most of its water content. A change in

the LCST of the gel could be achieved by addition of

different comonomers. Various thermoresponsive gels

were produced by copolymerization of NIPAM with

different acrylate based comonomers, and some of

them have been proposed as support materials for the

immobilization of enzymes and cells.6,8±12 Copoly-

merization of NIPAM with HEMA made suitable

materials for immobilization of enzymes because

their LCST values were higher than that of pure

pNIPAM.11,12 In some cases, the biotechnological and

biomedical applications of synthetic polymers may

require functional groups for coupling of enzymes and

other proteins. The derivation of plain pNIPAM

structure is usually dif®cult without losing the thermo-

sensitivity. However, the presence of hydroxyl groups

on the HEMA structure provides functional groups for

derivation of the copolymer.11,13±15 Immobilized

catalase has useful applications in the food industry

in the removal of hydrogen peroxide from food

products after cold pasteurization and in the analytical

®eld as a component of hydrogen peroxide and glucose

biosensor systems.16±19

In the present study, a series of pNIPAM/HEMA

hydrogels in cylindrical form with different NIPAM/

HEMA ratios were prepared by a redox polymeriza-

tion technique. Catalase was immobilized in thermo-

sensitive pNIPAN/HEMA hydrogels, via physical

entrapment. The effects of the NIPAM/HEMA ratio

and enzyme loading on the immobilization ef®ciency

Polymer International Polym Int 48:879±884 (1999)

* Correspondence to: M Yakup Arıca, Kırıkkale University, Department of Biology, Faculty of Science, 71450 Yahsihan-Kırıkkale, Turkey(Received 9 February 1999; accepted 5 May 1999)

# 1999 Society of Chemical Industry. Polym Int 0959±8103/99/$17.50 879

and on the recovered enzyme activity were studied.

The kinetic parameters and operational, thermal and

storage stability of immobilized enzyme were investi-

gated in batch systems.

EXPERIMENTALMaterialsCatalase (CAT) (hydrogen peroxide oxidoreductase;

EC.1.11.1.6) from bovine liver (250000 unitsmgÿ1

solid) was obtained from Sigma Chemical Co (St

Louis, MO, USA) and used as received. Tetramethy-

lenediamine (TEMED) and 2-hydroxyethyl metha-

crylate (HEMA) were obtained from Sigma Chemical

Co. The latter was distilled under reduced pressure in

the presence of hydroquinone and stored at 4°C until

use. N-Isopropylacrylamide (NIPAM) was obtained

from Aldrich Chemical Corporation (USA) and

recrystallized from hexane before use. Polyethylene

glycol (PEG 4000, Mn 4000), N,N-methylenebisacry-

lamide (BisAA) and potassium persulphate (KPS)

were obtained from BDH Chemicals Ltd (UK) and

used as received. All other chemicals were of reagent

grade and were purchased from Merck AG (Darm-

stadt, Germany).

Preparation of enzyme–gel cylindersCatalase immobilized pNIPAM/HEMA hydrogels in

the cylindrical form were prepared by redox polymer-

ization as previously described.11 The polymerization

was carried out in a cylindrical die (internal diameter

3.2mm, length 85mm) at �4°C under nitrogen

atmosphere for 24h. To check the effect of monomer

ratio on the catalase activity, in the initial polymeriza-

tion mixture three different NIPAM/HEMA mole

ratios were used. N-Isopropylacrylamide (115, 100 or

72mg), 2-hydroxyethyl methacrylate (0.02, 0.04 or

0.07ml), N,N-methylenebisacrylamide (3.3mg),

potassium persulphate (5mg) and polyethylene glycol

4000 (100mg) were dissolved in phosphate buffer

(0.85ml, 0.1M, pH 6.8). The resulting mixture was

equilibrated at �4°C for 30min in a thermostatic

water bath. After this period, catalase (2mg) and

tetramethylenediamine solution (0.1ml, 10% v/v)

were added to this polymerization mixture. The

mixture was diluted to 1.5ml with the same buffer

solution and mixed. It was then transferred into three

glass dies and polymerized as described above. The

thermally reversible pNIPAM/HEMA enzyme gel

(diameter 3.2mm, length 60mm) was removed from

the die by applying pressure and cut into three lengths

of 20mm. The hydrogel cylinders were washed several

times with cold phosphate buffer (0.1M, pH 6.8) and

stored at �4°C in the same buffer until use.

To test the effect of loading on the activity of the

enzyme, the above mentioned technique was used

except that the NIPAM/HEMA mole ratio (74/26)

was ®xed and the amount of catalase was varied

between 1.0 and 5.0mg in the polymerization mixture.

Activity assays of free and immobilized catalaseCatalase activity was determined spectrophotometri-

cally, by direct measurement of the decrease in the

absorbance of hydrogen peroxide at 240nm due to its

decomposition by the enzyme. Hydrogen peroxide

solutions (5±30mM) were used to determine the

activity of both the free and the immobilized enzyme.20

A 4ml aliquot of reaction mixture was preincubated

at 25°C, for 10min and the reaction was started by

adding 50ml of catalase solution (100mg solid per ml).

The decrease in absorbance at 240nm was recorded

for 5min. The rate of change in the absorbance (DA240

minÿ1) was calculated from the initial linear portion

with the help of the calibration curve (the absorbance

of hydrogen peroxide solutions of various concentra-

tions (5±30mM) at 240nm).

The same assay medium was used for the determi-

nation of the activity of the immobilized enzyme. The

enzymatic reaction was started by the introduction of

three catalase immobilized cylindrical gels (diameter

3.2mm, length 20mm) into the assay medium and was

carried out at 25°C with shaking in a water bath

equipped with a temperature control system. After

15min, the reaction was terminated by removal of the

gel cylinders from the reaction mixture. The absor-

bance of the reaction mixture was determined and the

immobilized catalase activity was calculated.

One unit of activity is de®ned as the decomposition

of 1mmol hydrogen peroxide per minute at 25°C and

pH 6.8.

These activity assays were carried out over the pH

range 4.0±8.0 and temperature range 20±60°C to

determine the pH and temperature pro®les for the free

and immobilized enzyme.

The effect of substrate concentration was tested in

the 5±30mM H2O2 concentration range. The results

of pH, temperature and substrate concentration of the

medium are presented in a normalized form, with the

highest value of each set being assigned the value of

100% activity.

Determination of immobilization efficiencyThe amount of protein in the crystalline enzyme

preparation and in the wash solution was determined

using Coomassie Brilliant Blue as described by

Bradford.21 A calibration curve constructed with

bovine serum albumin (BSA) solution (0.02±

0.2mgmlÿ1) was used in the calculation of enzyme

concentration.

Operational stability of immobilized catalase in thebatch systemThe retention of the immobilized enzyme activity was

tested as described in `Activity assays of free and

immoblized catalase'. After each reaction run, the

enzyme±hydrogel cylinders were collapsed and washed

with phosphate buffer (0.1M, pH 6.8) at 35°C for

30min to remove any residual substrate within the

hydrogel matrix. They were then reintroduced into

880 Polym Int 48:879±884 (1999)

MY Arõca et al

fresh reaction medium or stored in the same fresh

buffer at 4°C until the next run.

Storage stabilityThe activity of free and entrapped catalase after

storage in phosphate buffer (0.1M, pH 6.8) at 4°Cwas measured in a batch operation mode with the

experimental conditions given above.

Thermal stability of free and immobilized enzymeThe thermal stability of free and immobilized catalase

was ascertained by measuring the residual activity of

the enzyme exposed to two different temperatures (55

and 65°C) in phosphate buffer (0.1M, pH 6.8) for 3h.

Every 15min three gel rods were removed and assayed

for enzymatic activity as described above. The ®rst

order inactivation rate constants, ki were calculated

from the equation

ln A � ln A0 ÿ kit �1�where A0 is the initial activity and A is the activity after

t min.

Characterization of enzyme gel cylindersThe thermoresponsive behaviour of copolymer hydro-

gels prepared with different NIPAM and HEMA ratios

was determined at various temperatures (12±40°C) in

phosphate buffer (0.1M, pH 6.8) using a gravimetric

procedure described elsewhere.22 The equilibrium

swelling ratio was de®ned as the weight ratio of water

contained within the gel to dry polymer. The swelling

ratios of the gel cylinders were calculated by using the

expression

Swelling ratio�%� � f�Ws ÿW0�=W0g � 100 �2�where W0 and Ws are the weights of gel cylinders

before and after swelling, respectively.

RESULTS AND DISCUSSIONProperties of pNIPAM/HEMAIn this study, a copolymer gel of poly(N-isopropyla-

crylamide-co-hydroxyethylmethacrylate) (pNIPAM/

HEMA) was chosen as the support matrix. Suitable

matrices include hydrogels that are highly compatible

for immobilization of enzymes and cells due to their

hydrophilic nature and high water content to provide

the enzymes with a microenvironment similar to that

in vivo.

The copolymerization of NIPAM with HEMA in

the presence of PEG 4000 as a diluent improved the

hydrogel properties such as crack formation, and

disintegration in the hydrogel structure during swel-

ling and deswelling periods was not observed.

The effect of NIPAM/HEMA mole ratio on the

immobilization ef®ciency, recovered activity and

swelling ratio are presented in Table 1. It was observed

that immobilization was very ef®cient and more than

84% of the enzyme that was added in the polymeriza-

tion mixture was retained. Another observation was

that as the ratio of comonomer (HEMA) increased in

the initial polymerization mixture, the recovered

activity of the catalase was adversely affected. Figure

1 shows the swelling pro®le of the three different

hydrogels. Varying the comonomer ratios results in

signi®cantly different swelling pro®les mainly before

the LCST region. It is also observed that the swelling

ratio of the hydrogel decreased as the ratio of

comonomer (HEMA) in the initial polymerization

mixture increased.

Immobilization and retention of activityThe effect of loading on the activity of catalase

entrapped in pNIPAM/HEMA hydrogel cylinders

was determined by varying the enzyme contents within

the hydrogel cylinders. As presented in Fig 2, the

highest retention of enzyme activity (47%) was

Table 1. Effect of hydrogel compositionon swelling ratio, immobilizationefficiency and recovered activity

Hydrogel

composition

Monomer ratio

NIPAM/HEMA

(mol/mol)

Swelling ratio

(at 25°C)

Immobilization

ef®ciency

(%)

Recovered

activity

(%)

PNIPAM/HEMA1 85/15 6.75 84 37

PNIPAM/HEMA2 74/26 5.2 91 28

PNIPAM/HEMA3 53/47 4.5 97 19

Figure 1. Swelling ratio of pNIPAM/HEMA cylinders as a function oftemperature.

Polym Int 48:879±884 (1999) 881

Immobilization of catalase in pNIPAM/HEMA hydrogels

obtained with the lowest enzyme loading (0.33mg) per

ml of gel. As the enzyme content increased (from 0.33

to 3.33mg per ml of gel), retention of activity

decreased, dropping to a minimum of 14%. A high

enzyme load in the support generally leads to a low

retained activity. This is brought about by over-

saturation of the pore space of the matrix with the

enzyme, as a result of which substrate diffusion is

restricted. With an increase in catalase loading in the

hydrogel cylinders (0.33±3.33mg per ml of gel), the

enzyme activity was also increased (33.6�103 to

110.7�103U per ml of pNIPAM/HEMA hydrogel),

but not at the same rate because of the loss in retained

activity with increased load (Fig 2). As presented in

Table 1, pNIPAM/HEMA2 hydrogel composition

with a loading of 1.3mg enzyme per ml of hydrogel

was found to be optimum and it was used in the rest of

the study.

Kinetic parameters, Michaelis constant Km and

Vmax for free and immobilized catalase, were deter-

mined by varying the concentration of hydrogen

peroxide in the reaction medium. The kinetic proper-

ties of free and immobilized enzymes are presented in

Table 2. For free enzyme Km was found to be

16.5mM, whereas the Vmax value was calculated as

236�103U per mg of protein. Kinetic constants of the

immobilized catalase were also determined in the

batch system. The Km value was found to be 28.6mM.

The Vmax value of immobilized enzyme was estimated

from the data as 84�103U per ml of hydrogel. As

expected, the Km and Vmax values were signi®cantly

affected after entrapment within pNIPAM/HEMA2

hydrogel. The change in the af®nity of the enzyme to

its substrate is probably caused by structural changes

in the enzyme introduced by the immobilization

procedure or by lower accessibility of the substrate to

the active site of the immobilized enzyme.

Effect of temperature on catalytic activityThe temperature dependence of the activities of the

soluble and immobilized catalase were studied in

phosphate buffer (0.1M, pH 6.8) in the temperature

range 20±60°C (Fig 3). The optimum temperature for

the immobilized catalase was at 25°C, which was

15°C lower than that of free enzyme. The temperature

pro®le of the immobilized enzyme showed a steep

increase and decrease around optimum activity due to

the thermally reversible behaviour of the carrier

hydrogel. At low temperature, the hydrogel was in

the swollen state and the pores of the hydrogel were

very open. Therefore, the effective diffusion coef®cient

of substrate was higher due to lower internal mass

transfer resistance. The available surface area for the

substrate diffusion was also higher due to the swollen

gel volume. These two factors provided a higher rate of

substrate diffusion through the hydrogel carrier

according to Fick's law, while the immobilized enzyme

activity was controlled kinetically at low temperature.

Figure 2. Effect of enzyme loading on the entrapped enzyme activityretention and enzyme–hydrogel activity.

Table 2. Properties of free andimmobilized catalase

Form of enzyme

Vmax

(U�10ÿ3mgÿ1 enzyme)

Km

(mM)

Recovered

activity

(%)

Activity

(U�10ÿ3ml gel)

Free catalase 236 16.5 100 ±

Immobilized catalase

(PNIPAM/HEMA2)

65 28.6 28 84

Figure 3. Temperature profiles of free and immobilized catalase.

882 Polym Int 48:879±884 (1999)

MY Arõca et al

As presented in Fig 1, increasing the temperature

caused deswelling of the matrix and then reduced the

activity of the enzyme at 25°C. This reduction of

activity as a response to deswelling can be due to

mechanical pressure of the shrunken network on the

enzyme, or to a reduction in diffusion of the substrate

through the deswollen hydrogel, and also to a decrease

in the surface area for substrate diffusion with

shrinking of the hydrogel volume. At 25°C, the

kinetics and above mentioned factors should be

balanced, yielding a maximum enzyme activity for

the enzyme±hydrogel system. Thus, the temperature

dependent activity of immobilized enzyme is opposite

to that of free enzyme which exhibits higher activity at

higher temperature. Similar observations have also

been reported for other thermally reversible hydrogel

carriers.4,11,12

Effect of pH on enzyme activityThe effect of pH on the activity of free and

immobilized catalase for hydrogen peroxide degrada-

tion was studied at various pH values at 35°C. The

reactions were carried out in acetate and phosphate

buffers and the results are presented in Fig 4. The

immobilized enzyme system has the same optimum as

the free enzyme (around pH 7.0). The free enzyme

gave a signi®cantly broader pro®le in the acidic region

(between pH 4.0 and 6.0) than the immobilized

enzyme. The pH pro®le of the immobilized enzyme

was slightly broader in the alkaline region with respect

to the free enzyme. These results are possibly due to

secondary interactions (eg ionic and polar interactions,

hydrogen bonding) between the enzyme and the

hydrogel carrier. Other researchers have reported

Figure 5. Operational stability of immobilized catalase in a batch system.

Figure 4. pH profiles of free and immobilized catalase. Figure 6. Influence of temperature on the stability of free and immobilizedcatalase.

Figure 7. Storage stability of free and immobilized catalase.

Polym Int 48:879±884 (1999) 883

Immobilization of catalase in pNIPAM/HEMA hydrogels

similar observations upon immobilization of catalase

and other enzymes.13,23±25

Operational stability of immobilized catalase in abatch systemOperational stability of the immobilized catalase was

determined for 20 successive batch reactions at 25°Cfor 2h. The initial hydrogen peroxide concentration

was 10mM in phosphate buffer (pH 6.8, 0.1M). The

results presented in Fig 5 shows that the immobilized

enzyme activity remained almost the same as the

original activity after six cycles. After that, a steady

decrease in degradation capability of the immobilized

catalase was observed, and this loss reached 42% after

20 cycles of batch operation. Because catalase is stable

at 25°C, the loss in the immobilized enzyme activity

during batch operation is not thermal deactivation but

could result from poisoning brought about by the

substrate.

Thermal stabilityThe effect of temperature on the stability of free and

immobilized enzyme is shown in Fig 6. At 55°C, the

free enzyme lost all its initial activity after a 240min

incubation period, while the immobilized catalase

showed signi®cant resistance to thermal inactivation

(retaining about 38% of its initial activity after the

same period). At 65°C free and immobilized catalase

lost all of their initial activity after 120min and

210min, respectively. The thermal inactivation rate

constants for free and immobilized preparation at

65°C were calculated as ki(free) 1.73�10ÿ2minÿ1 and

ki(immobilized) 9.84�10ÿ3minÿ1, respectively. These

results suggest that the thermostability of immobilized

catalase becomes signi®cantly higher at higher tem-

perature. If the heat stability of enzymes increased

upon immobilization, the potential application of

these enzymes would be extended. Increased thermal

stability has been reported for a number of immobi-

lized enzymes, and the polymer network is supposed to

preserve the tertiary structure of enzyme. In addition,

it has also been reported that hydrogel carriers such as

Sephadex, Sepharose, poly(2-hydroxyethyl methacry-

late) and poly(acrylamide) protect enzymes from

thermal inactivation and yield higher thermal stabi-

lities.20,26 On the basis of these observations,

pNIPAM/HEMA2 is a hydrophilic network which

induces higher thermal stability compared to that of its

free counterpart.

Storage stabilityFree and immobilized catalase preparations were

stored in phosphate buffer (0.1M, pH 6.8) at 4°Cand the activity of the enzymes was determined

weekly. The activity measurements were carried out

for a period of 70 days (Fig 7). The free enzyme lost all

its activity within 20 days whereas the immobilized

enzyme lost about 17% of its activity during the same

period, and about 78% after 70 days. The decrease in

activity was explained as a time-dependent natural loss

in enzyme activity, and this was prevented to a

signi®cant degree by immobilization.

CONCLUSIONSCatalase immobilized thermally reversible pNIPAM/

HEMA hydrogels have been prepared by redox

polymerization. The swelling capacity of the hydrogels

can be adjusted by changing the NIPAM/HEMA mole

ratio in the initial polymerization mixture. As pre-

viously mentioned, controlling and changing the

immobilized enzyme activity could be achieved by

regulating the temperature because the hydrogel

response to temperature is reversible. The enzyme±

hydrogels could be used many times without any

deformation during swelling and deswelling cycles.

Immobilization of guest biomolecules in thermally

reversible hydrogel networks provides the design for a

system that produces the required response at a given

temperature.

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