ARTIFICIAL CELLS, BLOOD SUBSTITUTES, AND BIOTECHNOLOGY

Vol. 32, No. 3, pp. 443–452, 2004

Catalase Immobilization in Cellulose Acetate

Beads and Determination of its Hydrogen

Peroxide Decomposition Level by using a

Catalase Biosensor

Hatice Yildiz, Erol Akyilmaz,* and Erhan Dinckaya

Department of Biochemistry, Faculty of Science, Ege University,

Bornova-Izmir, Turkey

ABSTRACT

Catalase enzyme (EC 1.11.1.6) was immobilized by entrapping in

cellulose acetate beads. This organic matrix is highly resistant to

mechanical stability and can be used under various conditions. Initial

studies were conducted to examine the immobilization ability of

catalase on the matrix previously activated with a series of reagent

normally and the best results were obtained with the beads activated

with Ce(SO4)2. In the optimization studies of the immobilized enzyme

optimum pH and temperature were found as pH:7.0 (Tris–HCl,

50mM) and 35�C. In the characterization studies of the immobilized

*Correspondence: Erol Akyilmaz, Department of Biochemistry, Faculty of

Science, Ege University, Bornova-Izmir 35100, Turkey; E-mail: akyilmaz@

sci.ege.edu.tr.

443

DOI: 10.1081/LABB-200027507 1073-1199 (Print); 1532-4184 (Online)

Copyright & 2004 by Marcel Dekker, Inc. www.dekker.com

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enzyme some parameters such as storage and thermal stability

were investigated. Finally, the immobilized enzyme was used for

the decomposition of hydrogen peroxide in milk samples and

also by using a catalase biosensor prepared the decomposition

level of hydrogen peroxide was detected.

Key Words: Immobilization; Catalase; Cellulose acetate;

Biosensor; Hydrogen peroxide.

INTRODUCTION

Using of enzymes in nature form for biochemical, biotechnological,biomedical, and the other areas such as environmental, food industriesis not generally suitable for both economical and the product yield. So,immobilization of enzymes by using some different chemical and physicalmethods and using them in the immobilized form especially in medicine,industry, and biotechnological process is the most widespread way(Cetinus and Oztop, 2003; Mosbach, 1988; Tarhan and Uslan, 1990).Hydrogen peroxide is very important agent and commonly used in manyindustrial and biotechnological process for waste water treatment andsterilization (Tarhan and Akertek, 1995). In the diary industry itis used for cold pasteurization of the milk as an antibacterial agentto prevent the microbial contamination. So, determination of hydrogenperoxide is a very important and necessary for human health. Forthis purpose some methods based on HPLC (Nakoshima et al., 1994),enzymatic-spectrophotometric (Demmano et al., 1996), and also biosen-sors (Akgol and Dinckaya, 1999; Wang et al., 1995; Stein and Hain,1995) were developed. Hydrogen peroxide is substrate of catalase enzymeand it is used in the diary process in order to prevent microbiologicalcontamination. At the end of the process all of the hydrogen peroxideis degraded into O2 and H2O by catalase enzyme. So the immobilizationof catalase is very important for both economical and yielded usage ofthe enzyme.

The aim of the present work was to immobilize the catalase byentrapping in cellulose acetate beads. For the optimization of theimmobilization procedure some parameters such as effects of pH andtemperature were investigated. The immobilized enzyme was used forthe decomposition of hydrogen peroxide in milk samples and also byusing a catalase biosensor prepared according to Akgol and Dinckaya(1999) the decomposition level of hydrogen peroxide was detected.In this method dissolved oxygen concentration which was related to

444 Yildiz, Akyilmaz, and Dinckaya

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hydrogen peroxide decomposition determined by using a YSI 57 Modeloxygenmeter.

MATERIALS AND METHODS

Materials

Catalase [E.C 1.11.1.6] from bovine liver, cellulose acetate, bovineserum albumin (BSA), and glutaraldehyde (25%) were obtained fromSigma (St. Louis, USA). Hydrogen peroxide (30%), Ce(SO4)2, and allother chemicals used in the experiments were purchased from Merck(Darmstadt, Germany).

Apparatus

In the spectrophotometric analyses a Jasco V-500 uv/vis spectro-photometer (Japan) were used. In the biosensor experiments a YSI 57Model oxygenmeter (YSI, Yellow Springs, Ohio) combined with a YSI5739 Model dissolved oxygen (DO) probes based on amperometricconsist of Au (cathode), Ag-AgCl (anode), half saturated KCl (electro-lyte), a teflon membrane which is selective for oxygen, and an ultra-thermostat (Colora, Germany) were also used.

Preparation of Cellulose Acetate Beads

For this purpose 900mg cellulose acetate was dissolved in 5mL ofacetone and then the solution was added drop-wise into hegzane solutionby using a suitable syringe at 25�C.

Immobilization of Catalase in Cellulose

Acetate Beads

Catalase was immobilized in cellulose acetate beads prepared byusing both entrapping and cross-linking processs together. For theimmobilization of catalase 700 piece of beads (approximately 1 g) wereadded to 100mM of Ce(SO4)2 solution prepared in 0.1NHNO3 withstirring for 2 h. At the end of the time, the beads were removed fromthe solution by a decantation process and washed with distilled water.

Catalase Immobilization in Cellulose Acetate Beads 445

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And then the beads were added to 10mL of BSA solution (10mg/mL)prepared in borate buffer (pH:9.0, 0.1M) and stirred again for 30min.40mg of sodium cyanidborhydrid was added to the solution andcontinued to stirring for 2 h. The beads were treated decantation processagain and washed with distilled water. After this step the beads weretreated with 2.5mL of catalase enzyme solution (11200U/mL) and stirredfor 10min. Glutaraldehyde solution (2.5%) was added to the mixtureand continued to stirring for 5min. Finally, the beads were removed fromthe mixture by a decantation process and washed.

Activation of the Cellulose Acetate Beads by Ce(SO4)2

In the activation of the cellulose acetate beads prepared, catalase wasbound to cellulose acetate beads by covalent bonds. In this case, hydroxylgroups of cellulose acetate were oxidized to aldehyde groups by Ce(SO4)2(0.1M prepared in 0.1MHNO3) and activated. After activation step,spacer arms were formed by occurring Schiff bases between aldehydegroups and –NH2 groups of BSA. Catalase and BSA were immobilizedtogether by using a cross-linking agent, glutaraldehyde.

Measurement of Free and Immobilized Catalase Activity

Activity mesurement of free and immobilized catalase was made for10.5mM hydrogenperoxide at 240 nm by using a uv/vis spectropho-tometer according to Aebi (1974). Activity of catalase immobilized wasaccepted as mmol/dk g (immobilized beads). The molar extinctioncoefficient is 40 cm2/mmol at this wavelength.

Preparation of Catalase Biosensor

Catalase (9324.2 IU/mL) and 225 bloom gelatin (33.3mg/mL) weremixed at 38�C in distilled water. Two hundred microliters of the mixedsolution was spread over dissolved oxygen (DO) probe membraneand allow to dry at 4�C for 1 h. Finally, it was immersed in 2.5%glutaraldehyde in 50mM phosphate buffer (pH:7.5) for 3min. Thebiosensor prepared contained 1650.3 IU/cm2 and 5.9mg/cm2 gelatin inthe bioactive layer.

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RESULTS AND DISCUSSION

Optimization Studies

In this section of the study effect of pH; temperature, buffer system,and buffer concentration on the free and immobilized catalyzes wereinvestigated.

Effect of pH

For the determination of the effect of pH on the free and immobi-lized catalase phosphate buffers (50mM) that is in the pH range 6.0–8.0were used. Figure 1 shows the results obtained. According to the figurethe highest activity was obtained in pH:7.0 for free and immobilizedcatalase. Therefore, the pH profile of the immobilized catalase was muchbroader with respect to the free enzyme. By using different buffer systemssuch as Tris–HCl, phosphate, and phosphate/citrate (pH:7.0, 50mM)the most suitable buffer system for the enzyme activity was determined.Results obtained from the experiments showed that phosphate buffersystem gave the most suitable results for both free and the immobilizedcatalase. After detection of the most suitable buffer system phosphatebuffers at varying concentrations (25, 50, 100, and 200mM) wereprepared and tested for the maximum enzyme activity. According to the

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pH

Act

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Figure 1. Effect of pH on free and immobilized catalase, -�-: free catalase,

-�-: immobilized catalase (pH 6.0–8.0: phosphate buffers, 50mM,T 35�C).

Catalase Immobilization in Cellulose Acetate Beads 447

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results, although free enzyme shown the maximum activity at 100mMimmobilized catalase shown the maximum activity when 50mM concen-tration of the buffer used.

Effect of Temperature

Determination of the effect of temperature on free and the immobi-lized catalase activity was investigated in phosphate buffer in a tempera-ture range 10–40�C (Fig. 2).

From the figure it can be said that the optimum temperature for bothfree and immobilized catalase in the working conditions is found as 35�C.This is the optimum temperature of free enzyme given in literatures andenzyme handbooks so immobilization procedure didn’t show an effecton the optimum temperature of the enzyme. There was no importantobservation in activity change of free enzyme by increasing of thetemperature. Therefore, increase in the temperature resulted in an activityincrease for the immobilized enzyme.

Characterization Studies

In order to investigate characterization of the free and immobilizedcatalase enzyme some parameters such as thermal and pH stabilityexperiments were done.

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Figure 2. Effect of temperature on free and immobilized catalase, -�-: free

catalase, -�-: immobilized catalase (pH 7.0: phosphate buffer, 50mM).

448 Yildiz, Akyilmaz, and Dinckaya

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Detection of Thermal Stability of the Free andImmobilized Catalase

In this study the free and immobilized catalase were incubated inphosphate buffer (pH:7.0, 50mM) at different temperature (20–50�C)for 18 h. At the end of the time under the optimum conditions theactivities of the enzymes were assayed. Figure 3 shows the resultsobtained. According to the figure increase temperature from 20 to 40resulted in increase in activity of the immobilized enzymes, so it can besaid that immobilization was gained a thermal stability to the enzymerelated to increase its conformational stability by the immobilization. Onthe other hand, there was a negative effect in the activity of free enzymeby increase in the temperature.

Detection of pH Stability of the Free andImmobilized Catalase

For the determination of pH stability free and immobilized catalasewere incubated in buffers that is in the pH range 4.5–9.0 for 18 h at 20�C.At the end of the time the activity measurements of the enzymes weremade under the optimum assay conditions (Fig. 4). As it is shown fromthe figure pH stability of the both enzymes shown a similarity. Both free

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Figure 3. Determination of thermal stability of free and immobilized catalase,

-�-: free catalase, -�-: immobilized catalase.

Catalase Immobilization in Cellulose Acetate Beads 449

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and the immobilized catalase gave an optimum at pH:7.0, however, theimmobilized catalase gave a much broader pH stability between pH:6.5and pH:7.5 with respect to the free enzyme. The results obtained can bethought as an effect of micro environment of the enzyme and pH on theenzyme stability.

By using Catalase Biosensor Determination of HydrogenPeroxide Decomposition in Milk Samples

In the experiments a YSI Model 57 oxygenmeter and 5739 Modeldissolved oxygen probe covered with a oxygen sensitive teflon membranewere used. Biosensor measurements were done in the steady-statecondition at 35�C. Milk samples contained 0.03 and 0.1% hydrogenperoxide were prepared. Before the treatment of the milk samples by theimmobilized catalase the hydrogen peroxide concentration of the samples(0.03 and 0.1%) were determined by using the catalase biosensorprepared. The milk samples were treated with the immobilized catalase(450 beads obtained 27.39U/50mL) for 70min by stirring. During thistime, in the beginning and per 20min samples treated with immobilizedcatalase were taken from the reaction medium and hydrogen peroxidedecomposition level of the milk samples were determined by using

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Figure 4. Determination of pH stability of free and immobilized catalase, -�-:

free catalase, -�-: immobilized catalase.

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catalase biosensor. Addition of the milk samples contained hydrogen

peroxide in to the reaction cell caused a rapid current increase due

to hydrogen peroxide destruction in the enzyme layer on the biosensor

(Fig. 5). According to the results given in the figure, it was detected

that 78% of hydrogen peroxide of sample 1 (0.03%) and 22% of

hydrogen peroxide of sample 2 (0.1%) decomposed in first 10min.

During from 10 to 70min there was no change in the decomposition

level for sample 2. This results from catalase enzyme denaturation

occurred in high concentration of hydrogen peroxide. On the other

hand, at the end of the 70min all hydrogen peroxide of sample 2 was

decompozed.From all the results obtained from the biosensor it is obvious that

catalase enzyme immobilized in cellulose acetate beads is very useful and

efficient and the immobilized enzyme can be used in all enzymatic,

biochemical and industrial applications needed to catalase.

ACKNOWLEDGMENT

This study was supported by Ege University Research Fund.

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Figure 5. Determination of decomposition level of hydrogen peroxide in milk

samples by using the catalase biosensor, -�-: Sample 1 contained 0.03% H2O2,

-�-: Sample 2 contained 0.1% H2O2.

Catalase Immobilization in Cellulose Acetate Beads 451

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chemically crosslinked chitosan beads. Enzyme and Microbial

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catalases and their application in pasteurization of milk with

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stability of immobilised catalase. Process Biochemistry 25:14–18.Wang, F., Schubert, F., Rinneberg, H. (1995). A fluorometric rote assay

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