Enzyme and Microbial Technology 32 (2003) 889894

Immobilization of catalase into chemically crosslinked chitosan beads

Senay Akku s etinus , H. Nursevin ztop
Department of Chemistry, Cumhuriyet University, Sivas 58140, Turkey Received 23 May 2002; received in revised form 13 March 2003; accepted 14 March 2003

Abstract Bovine liver catalase was immobilized into chitosan beads prepared in crosslinking solution. Various characteristics of immobilized catalase such as the pHactivity curve, the temperatureactivity curve, thermal stability, operational stability, and storage stability were evaluated. Among them the pH optimum and temperature optimum of free and immobilized catalase were found to be pH 7.0 and 35 C. The Km value of immobilized catalase (77.5 mM) was higher than that of free enzyme (35 mM). Immobilization decreased in Vmax value from 32,000 to 122 mol (min mg protein)1 . It was observed that operational, thermal and storage stabilities of the enzyme were increased with immobilization. 2003 Elsevier Science Inc. All rights reserved.
Keywords: Catalase; Chitosan beads; Immobilization

1. Introduction The application of enzymes in their native form biochemical, biomedical, biotechnological, and food industrial elds is not always suitable and optimal. Binding of enzymes on a solid support is an advantageous modication of their application. Enzymes are immobilized onto or into a solid matrix to increase their thermostability, operational stability, and recovery. Other benets obtained as well, include better operational control, exibility of reactor design, and ease of product recovery without catalyst contamination. Chitosan could serve as carriers for enzymes and whole cells. Chitosan [poly-(1 4)-2-amino-2-deoxy-d-glucose] is the product of deacetylation of chitin, derived from the exoskeleton of crustacean sand shows enhanced solubility in dilute acids as compared with the parent chitin. Recently, chitosan has attracted great attention, which has been reported to be a promising polymer in medical and biomedical areas. Chitosan is an inexpensive, inert, hydrophilic, biocompatible support, and is thus attractive for enzyme immobilization [1,2]. The presence of amino groups facilitates covalent binding of enzymes. Immobilization can be carried out either by entrapment into chitosan beads or by covalent binding to transparent chitosan lms or by usCorresponding author. E-mail addresses: scetinus@cumhuriyet.edu.tr (S. Akku s etinus), oztop@cumhuriyet.edu.tr (H. Nursevin ztop).

ing glutaraldehyde by formation of the Schiffs base [35]. Immobilized catalase has useful application in the food industry in the removal of hydrogen peroxide from food products after cold pasteurization and in the analytical elds as a component of hydrogen peroxide and glucose biosensor systems [6]. The rst aim of this study was to prepare catalase immobilized chitosan beads and second, to investigate the kinetic parameters, operational, thermal and storage stability of immobilized catalase in batch systems.

2. Materials and methods Chitosan, catalase (hydrogen peroxide oxidoreductase; EC.1.11.1.6) from bovine liver, glutaraldehyde, glyoxal (trimer:dihydrate) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Hydrogen peroxide, tetrasodium pyrophosphate and all other chemicals were obtained from Merck AG (Darmstadt, Germany). 2.1. Preparation of chitosan solution Three grams of chitosan akes were added into 99 ml of distilled water and suspended by magnetic stirring for 10 min. One milliliter of glacial acetic acid was then added and mixing continued for 3 h at room temperature and ltered through a gauze pad. The solution thus obtained was

0141-0229/03/$ see front matter 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0141-0229(03)00065-6

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stored at room temperature and was used within 1 week of preparation. 2.2. Preparation of crosslinking solution To prepare the solution of crosslinking, glyoxal hydrate (10 g) was added to 250 ml of distilled water, heated at 80 C for 10 min, and cooled to room temperature. The solution was mixed with an equal volume of 3% (w/v) tetrasodium pyrophosphate solution (pH 8.0). The solution was used on the same day of preparation. 2.3. Immobilization of catalase into chitosan beads Catalase solution (2 mg ml1 ) was prepared in 0.05 M phosphate buffer (pH 7.0). This freshly prepared catalase solution (0.2 ml) was suspended in 10 ml chitosan solution. This suspension was injected into the crosslinker solution (volume ratio 1:5). The beads were cured for 30 min over ice and washed three times with 0.05 M cold phosphate buffer (pH 7.0). 2.4. Reinforcement of chitosan beads by glutaraldehyde treatment Freshly prepared beads containing catalase were incubated in cold 0.05% (w/v) glutaraldehyde solution in 0.05 M cold phosphate buffer (pH 7.0) for 1 h. The brownish reinforced beads were washed several times by 0.05 M cold phosphate buffer (pH 7.0). 2.5. Activity assays of catalase The activity of catalase was determined spectrophotometrically by the direct measurement of the decrease in the absorbance of hydrogen peroxide at 240 nm due to its decomposition by the enzyme [7]. Activities were carried out at optimum conditions. Approximately 100 mg of catalase immobilized chitosan beads were mixed 10 ml of 10 mM H2 O2 solution in 0.05 M phosphate buffer (pH 7.0) 35 C. After 5 min, the reaction was terminated by removal of the chitosan beads from the reaction mixture. The absorbance of the reaction mixture was determined and the immobilized catalase activity was calculated. The protein content of catalase solution was determined by the method of Bradford [8]. The effect of substrate concentration on the activity was tested by using increasing concentrations of H2 O2 and Vmax and Km values of immobilized and free catalase were determined. The activity assays were carried out over the pH range 312 and temperature range 1565 C to determine the pH and temperature proles of free and immobilized enzyme. Activity of pH proles was determined at various pH in 10 mM H2 O2 solution at 35 C. Activity of temperature proles was determined at indicated temperatures in 10 mM H2 O2 solution (pH 7). The results of pH, temperature of the

medium are presented in a normalized form, with the highest value of each set being assigned the value of 100% activity. 2.6. The thermal stability of free and immobilized catalase The thermal stability of free and immobilized catalase was ascertained by measuring the residual activity of enzyme exposed to various temperatures (2080 C) in 0.05 M phosphate buffer for 2 and 5 h. Activity of samples were performed at optimum conditions. 2.7. The operational stability of free and immobilized catalase The retention of the immobilized enzyme activity was tested as described in activity assays of catalase. After each reaction run, the enzymechitosan beads were removed and washed with 0.05 M phosphate buffer (pH 7.0) to remove any residual substrate within the chitosan beads. They were then reintroduced into fresh reaction medium and enzyme activities were detected at optimum conditions. 2.8. The storage stability of free and immobilized catalase The activity of free and immobilized catalase after storage in 0.05 M phosphate buffer (pH 7.0) at 5 and 25 C was measured in a batch operation mode with the experimental conditions given above.

3. Results and discussion 3.1. The preparation of catalase immobilized chitosan beads and assays of activity The potential use of enzyme and cell immobilized chitosan beads carried out in acidic medium raises a general problem. As chitosan is soluble in acidic solutions, the continuous prolonged exposure of chitosan beads, made via counter-ion precipitation, may result in gel softening and bead disintegration. We used a procedure for the preparation of chemically crosslinked chitosan beads [3]. This procedure is based on the addition of the acidic chitosan solution into a mixture of di-ion (diphosphate) and nontoxic dialdehyde (glyoxal) resulting in chemically crosslinked chitosan beads. Glyoxal crosslinked chitosan beads were exposed to glutaraldehyde solution as a complementary stability curing treatment. As Schiffs base formed between chitosan and glyoxal is essentially reversible, continuous prolonged operation under acidic conditions may result in gradual leakage of glyoxal. Glutaraldehyde irreversible crosslinking via Schiffs base may lead to chitosan beads exhibiting high operational stability. The catalase immobilized chitosan beads

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Fig. 1. The photograph of the catalase immobilized chitosan beads.

obtained under these conditions are regular, transparent and rigid (Fig. 1). To reinforcement, immobilized catalase in chitosan beads were treated glutaraldehyde but it was found that catalase activity was decreased after this operation because crosslinks in chitosan beads may prohibit substrate diffusion to enzyme (Fig. 2). The loss of the activity of catalase immobilized chitosan beads with the solution of 0.05% (w/v) glutaraldehyde less than the other immobilized chitosan beads with various concentration of glutaraldehyde. Thus glutaraldehyde concentration was chosen to be 0.05% (w/v). Initial rates of H2 O2 decomposition were obtained for various levels of H2 O2 solutions for free and immobilized catalase. These data were plotted according to the method of LineweaverBurk (Fig. 3), and kinetic parameters, Km and Vmax were determined. The value of Km was found to be 35 mM whereas the Vmax was calculated as 32,000 mol (min mg protein)1 for free catalase. The Km value was found to be 77.5 mM and the Vmax value was found to be 122 mol (min mg protein)1 for immobilized catalase in chitosan beads. As expected, the Km and Vmax values were signicantly affected after immobilization into chitosan beads. The

Fig. 3. LineweaverBurk plots for: (a) free catalase and (b) immobilized catalase.

change in the afnity 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. So that the Vmax value of immobilized catalase very lower than that of free catalase. The Km value of immobilized catalase bigger than that of free catalase. Comparison of the Km for a given enzyme in both the immobilized and free states provides information about the interaction between the enzyme and its support. An increase in Km once an enzyme has been immobilized, indicates that the immobilized enzyme has an apparent lower afnity for its substrate than that of the free enzyme does, which may be caused by the steric hindrance of the active site by the support, the loss of enzyme exibility necessary for substrate binding, or diffusional resistance to solute transport near the particles of the support. Changes in crosslinking affect bead porosity and therefore diffusion of substrate and products to and from the droplet. Increasing glutaraldehyde concentration may also cause some enzyme denaturation. It may provoke the kinetics of the reaction. 3.2. Effect of temperature on catalytic activity

Fig. 2. The effect of glutaraldehyde concentration on the activity of immobilized catalase.

The temperature dependence of the activities of the free and immobilized catalase were studied in 0.05 M phosphate

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pH of 7.0, but the immobilized enzyme has a broader pH range of high activity. The stability of immobilized catalase at low pH could be attributed to the amino/imine groups, because they can bind to proton in the matrix, resulting in a decrease of local concentration of proton near the enzyme molecules [11]. 3.4. Thermal stability of catalase It is often observed that immobilized enzyme has a higher thermal stability than the corresponding free enzyme because of the reduction of conformational exibility in the immobilized enzyme. The effect of temperature on the stability of free and immobilized catalase is shown in Fig. 6. The activities of free and immobilized enzymes decreased with increase in temperature for both 2 and 5 h preincubation time. At 50 C, free catalase lost about 50% of its activity whereas the immobilized catalase lost about 40% of its activity after 2 h preincubation time. At 45 C, free catalase lost about 50% of its activity whereas the immobilized catalase lost about 30% of its activity after 5 h preincubation time. These results suggested that the thermostability of immobilized catalase becomes signicantly higher than that of free catalase at higher temperature. Immobilization of catalase into chitosan beads is supposed to

Fig. 4. Temperature proles of catalase (, immobilized; , free).

buffer (pH 7.0) in the temperature range 1565 C and temperature proles of free and immobilized catalase are showed in Fig. 4. Optimum temperature was found at about 35 C for free and immobilized catalase. Fig. 4 shows that the loss of the activity of immobilized catalase was lower than that of the free catalase for high temperatures. The support has a protecting effect at the high temperatures at which enzyme deactivation takes place. The conformational exibility of the enzyme affected by immobilization. Immobilization of catalase in chitosan beads caused in an increase in the enzyme rigidity which is commonly reected by increase in stability towards denaturation by raising the temperature [9,10]. 3.3. Effect of pH on enzyme activity The effect of pH on the activity of free and immobilized catalase preparations for hydrogen peroxide degradation 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. 5. Both enzymes showed an optimum

Fig. 5. pH proles of catalase (, immobilized; , free).

Fig. 6. The change of thermal stability of catalase with preincubation time: (a) 2 h and (b) 5 h (, immobilized; , free).

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3.6. Storage stability of catalase Free and immobilized catalase were stored in a phosphate buffer (0.05 M, pH 7.0) at 25 and 5 C, and the activity measurements were carried out for a period of 70 days (Fig. 8). At 25 C, free enzyme lost its all activity within 15 days whereas the immobilized enzyme lost about 50% of its activity during the same period and about 70% after 70 days. At 5 C, free enzyme lost about 50% its activity within 15 days and all its activity within 70 days whereas immobilized enzyme lost the same amount of activity within 45 days and it protected about 40% its activity after 70 days. The decrease in activity was explained as a time-dependent natural loss in enzyme activity, and this was prevented to a signicant degree by immobilization.

Fig. 7. Operational stability of immobilized catalase.

preserve the tertiary structure enzyme, and it protected the enzyme from conformational changes causing effects of the environment. 3.5. Operational stability of catalase Operational stability of the immobilized catalase was determined for 13 successive batch reactions at 35 C for 200 min (Fig. 7). It was observed that immobilized catalase retained about 60% of its activity after 13 cycles.

4. Conclusion Catalase has been successfully immobilized into chitosan beads. The chitosan beads prepared with crosslinking solution and then were treated glutaraldehyde for stability in both alkaline and acidic media. Our experiments have shown that the catalase immobilized into chitosan beads exhibits an improved resistance against thermal and pH denaturation. The operational and storage stability of the immobilized catalase presented in this work may indicate applicability of immobilized catalase for continuous degradation of hydrogen peroxide.

Acknowledgments The authors gratefully acknowledge the nancial support provided by the Cumhuriyet University Research Fund through Project F-86.

References
[1] Yazdani-Pedram M, Retuert J, Quijada R. Hydrogels based on modied chitosan, synthesis and swelling behavior of poly(acrylic acid) graphed chitosan. Macromol Chem Phys 2000;201:923 30. [2] Kumar MNVR. A review of chitin and chitosan applications. React Func Polym 2000;46:127. [3] Freeman A, Dror Y. Immobilization of disguised yeast in chemically crosslinked chitosan bead. Biotechnol Bioeng 1994;44:10838. [4] etinus AS, ztop HN. Immobilization of catalase on chitosan lm. Enzyme Microbiol Technol 2000;26:497501. [5] Ravikumar MNV. Chitin and chitosan bers: a review. Bull Mater Sci 1999;22:90515. [6] Arca MY, ktem HA, ktem Z, Tuncel SA. Immobilization of catalase in poly(isopropyl acrylamide-co-hydroxyethylmethacrylate) thermally reversible hydrogels. Polym Int 1999;48:87984. [7] Aebi H. Catalase. In: Bergmayer HU, editor. Methods of enzymatic analysis. Florida: Verlag Chemie international; 1981. p. 673 84.

Fig. 8. The change of storage stability of catalase with temperature: (a) 25 C and (b) 5 C (, immobilized; , free).

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S . Akku s etinus, H. Nursevin ztop / Enzyme and Microbial Technology 32 (2003) 889894 [10] Abdel-Naby MA. Immobilization of Aspergillus niger NRC 107 xylanase and -xylosidase, and properties of the immobilized enzymes. Appl Biochem Biotechnol 1993;38:6981. [11] Gupta KC, Ravikumar MNV. Preparation, characterization and release proles of pH-sensitive chitosan beads. Polym Int 2000;49: 1416.

[8] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976;72:248. [9] Jiang B, Zhang Y. Immobilization of catalase on crosslinked polymeric hydrogels effect of anion on the activity of immobilized. Eur Polym J 1993;29:12514.