An economical, simple and high yield procedure for the immobilization/ stabilization of peroxidases from turnip roots

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1 Journal of Scientific & Industrial Research Vol. 63, June 2004, pp An economical, simple and high yield procedure for the immobilization/ stabilization of peroxidases from turnip roots Syed Musthapa M 2, Suhail Akhtar 1, Amjad Ali Khan 1 and Qayyum Husain 1 * 1 Department of Biochemistry, Faculty of Life Science, Aligarh Muslim University, Aligarh Received: 16 October 2003; accepted: 10 March 2004 Ammonium sulphate fractionated peroxidases from turnip roots (Brassica rapa) were entrapped in calcium alginate gels for high yield immobilization of enzymes. Enzymes were cross-linked with 0.5 per cent glutaraldehyde to increase the molecular dimension of the enzyme; this resulted in the loss of 8 per cent of enzyme activity. Alginate entrapped soluble and cross-linked peroxidases exhibited high stability against temperature, ph, urea and storage than the soluble preparation and were markedly more stable as compared to directly entrapped soluble enzyme preparation. Entrapped cross-linked peroxidase preparation showed retention of very high enzyme activity after six repeated uses. Alginate entrapped soluble peroxidase preparation rapidly lost its activity after each use. The results suggested that such preparations have great potential in the construction of bioreactors to be used for the removal of aromatic compounds from polluted wastewaters/industrial effluents. Keywords: Cross-linking, Entrapment, Glutaraldehyde, Immobilization, Peroxidase, Stabilization, Turnip roots IPC Code: Int. Cl. 7 : C 12 N 11/16 Introduction Peroxidases ( ) are ubiquitous proteins having applications in wide range of fields such as medicine, chemical synthesis, and in the analysis of food, chemical, clinical and environmental samples 1,2. More recently, peroxidases have been employed for several novel applications such as detoxification and removal of various organic pollutants like phenols, aromatic amines, and dyes from polluted wastewater 3-6. They have also been used as catalyst in phenolic resin synthesis, fuel and chemical production from wood pulp, production of dimeric alkaloids, bio-bleaching processes, and oxidation/biotransformation of organic compounds 7-9. This great diversity of applications is due to wide substrate specificity of peroxidase catalysis 10. Soluble enzymes have certain inherent limitations and cannot be employed at the large-scale while immobilized enzymes, on the other hand, offer several advantages. However the high cost and low yield of immobilized enzyme preparation are two important limitations in their applications 11,12. Among the techniques used for immobilization, entrapment in natural biopolymers is favored due to various reasons, e.g., non-toxicity of the matrix, variation in the bead size of the gel, and high yield of immobilization 13,14. Horseradish roots represent the traditional source for the commercial production of peroxidases but they are not available in northern India. Turnip can be considered as an alternative source of peroxidases due to its easier availability. Several earlier reports indicate that the biochemical and kinetic properties of turnip peroxidases are quite comparable to the commercially available horseradish peroxidases 15,16. Here for the first time an effort has been made to immobilize turnip peroxidases in calcium alginate beads. The purpose of this study was to reduce the cost of commercially available enzyme for its immobilization and utilization on large-scale. Alginate entrapped peroxidase preparations exhibited very high yield of immobilization and stability against various forms of denaturants and storage. This preparation showed significantly high reusability after six repeated uses. Materials and Methods

2 MUSTHAPA et al.: ECONOMICAL PROCEDURE FOR IMMOBILIZATION OF PEROXIDASES FROM TURNIP ROOTS 541 Materials Sodium alginate and glutaraldehyde used were the products of Koch-Light, England. Bovine serum albumin was obtained from Sigma Chemical Co. USA. o dianisidine HCl was purchased from Centre for Biochemicals, CSIR, India. All other chemicals and reagents used were of analytical grade. Turnip roots were purchased from the local market. Methods Purification of Peroxidases from Turnip Roots Unless otherwise stated, all purification procedures were carried out at 4-7 C. Peroxidases was extracted by homogenization of 100 per cent (w/v) of turnip roots with distilled water. The homogenate was filtered and then centrifuged at 6000 g for 15 min at 4 C. The filtrate was precipitated by solid ammonium sulphate to 75 per cent saturation. After keeping for overnight, the precipitate was collected by centrifugation on Beckman C-21 cooling centrifuge at g for 15 min at 4 C and redissolved in a minimal volume of 0.05 M sodium acetate buffer, ph 5.6 and dialyzed against 0.02 M sodium acetate buffer for 24 h. The precipitate was removed by centrifugation at 11,000 g for 15 min at 4 C. The clear filtrate thus obtained was stored and used as soluble enzyme 16. Cross-linking of Turnip Peroxidases by Using Glutaraldehyde Prior to entrapment in calcium alginate beads, peroxidases were treated with 0.5 per cent (v/v) glutaraldehyde at 4 C for 2 h with constant shaking. Cross-linking was performed in the presence of o-dianisidine HCl, substrate. After the completion of cross-linking the enzyme was washed thrice with acetate buffer and further incubated with 0.1 per cent (v/v) ethanolamine to neutralize the remaining aldehydic groups 17. Entrapment of Soluble and Cross-linked Peroxidases in Calcium Alginate Gel The soluble and cross-linked peroxidases were mixed independently with 5.0 per cent aqueous sodium alginate solution. The resulting mixture was slowly extruded as droplets through a 5.0 ml syringe with attached needle No. 20 into 0.2 M calcium chloride solution. The gel formation was instantaneous and solution was gently stirred for 2 h 18. Beads were then washed and kept in acetate buffer, ph 5.6 at 4 C until used. Effect of Temperature on the Activity of Soluble and Alginate Entrapped Turnip Peroxidases Appropriate amount of soluble and alginate entrapped peroxidases were incubated at 60 C in 0.05 M sodium acetate buffer, ph 5.6. Aliquots of each preparation were removed at different time intervals and activity was measured. The enzyme activity of soluble and alginate entrapped peroxidases was measured at different temperatures under other standard assay conditions. The activity obtained at 40 C was taken as 100 for the calculation of per cent activity. Effect of ph on the Activity of Soluble and Immobilized Turnip Peroxidases The activity of appropriate amount of soluble and immobilized peroxidases was measured in buffers of different ph values. The molarity of each buffer was 0.05 M. The activity was optimum at ph 5.0 for all the three preparations and it was taken for the calculation of per cent activity. Effect of Urea on the Activity of Soluble and Immobilized Turnip Peroxidases Soluble and immobilized peroxidase preparations were incubated in 4.0 M urea dissolved in 0.05 M sodium acetate buffer, ph 5.6. Aliquots were removed at different time intervals and activity was determined. Storage Stability of Soluble and Immobilized Turnip Peroxidases Soluble and entrapped peroxidase preparations were stored at 37 C for 30 d. An appropriate amount of each preparation was taken out in triplicates on alternate days and activity was determined.

3 542 J SCI IND RES VOL 63 JUNE 2004 Reusability of Calcium Alginate Beads Containing Entrapped Soluble and Cross-linked Turnip Peroxidases The reusability of calcium alginate beads containing soluble and cross-linked peroxidases was assayed on alternate days. After each use the beads were resuspended in 0.05 M sodium acetate buffer, ph 5.6. Measurement of Peroxidase Activity Peroxidase activity was determined from a change in the optical density (A 460 nm ) at 37 C by measuring the initial rate of oxidation of o-dianisidine HCl by hydrogen peroxide using the two substrates in saturating concentration. The immobilized preparations were continuously agitated for the entire duration of assay. The assay was highly reproducible with immobilized preparations. The significant absorption of the colour complexes formed on the gel matrix could be observed 19. One unit of peroxidase activity was defined as the amount of enzyme protein that catalyses the oxidation of 1 µmol of o-dianisidine HCl/min at 37 C. Other Assay The protein concentration was determined by the method of Lowry et al. 20. Bovine serum albumin was used as standard. Results and Discussion Recently, immobilized enzymes have got lot of attention for their use as biocatalysts in the removal/biotransformation of toxic aromatic organic compounds present in wastewater 3,4. The use of soluble enzyme is not practical due to huge amount of enzyme required and extreme denaturing conditions encountered during the process. There are different methods, which can be used for immobilization of enzymes. The immobilized enzymes must be obtained by a cost- effective and technologically convenient method. Peroxidases have been found to be much more applicable in the detoxification of industrial effluents containing phenols, aromatic amines, and dyes 4-6. Calcium alginate mediated entrapment has attracted much attention in the detoxification of Figure 1 Thermal denaturation of soluble and alginate entrapped turnip peroxidase

4 MUSTHAPA et al.: ECONOMICAL PROCEDURE FOR IMMOBILIZATION OF PEROXIDASES FROM TURNIP ROOTS 543 phenolic compounds present in the industrial effluents 21. Partial purification of peroxidases from turnip roots was done by the method of Hamed et al. 6. The crude peroxidase preparation was obtained from 5 kg of turnip roots and the initial specific activity was 17.3 units/mg of protein. The ammonium sulphate precipitation followed by dialysis increased the specific activity 2.5-fold over crude enzyme and this preparation was used for entrapment in calcium alginate beads. Immobilization by means of entrapment is a very rapid and simple technique. It has been earlier described that soluble enzyme could be leached out of beads on long standing or use 21,22. In order to overcome the leaching effect of enzymes out of porous gel beads, peroxidases were crosslinked with glutaraldehyde and subsequently entrapped into alginate gel. Cross-linked peroxidase preparation further resulted in loss of 8 per cent of its original activity. Some previous studies have reported that the crosslinking of enzymes provides higher mechanical and operational stability to the enzymes 17,23. Native peroxidase is quite stable to heat inactivation. Immobilization, however, gives additional stability against heat inactivation. As shown in Figure 1, soluble, alginate entrapped soluble and cross-linked enzyme preparations were incubated at 60 C for various times. Incubation of soluble enzyme at this temperature resulted in the loss of nearly 92 per cent activity in 90 min whereas the alginate-entrapped soluble enzyme retained 26 per cent of the initial activity. Moreover the alginate entrapped cross-linked peroxidase preparation exhibited markedly very high stabilization against thermal denaturation and it retained more than 80 per cent of the initial activity. It indicates that such preparations can be used at high temperatures for the treatment of polluted waters. Improvement in thermal stability of alginate entrapped cross-linked preparation may come from multipoint attachment of peroxidases with glutaraldehyde. Earlier findings described that the cross-linking of enzymes with bi-functional or multi-functional agents enhanced its thermal stability. This enhancement in thermal stability is due to formation of several linkages between enzyme and support 18,23. The changes in the activity of soluble and immobilized peroxidases were also compared after incubation for 1 h at various temperatures. Resistance of enzyme to the high temperatures was greatly increased by immobiliza- Figure 2 Temperature activity profile of soluble and immobilized peroxidases

5 544 J SCI IND RES VOL 63 JUNE 2004 Figure 3 ph activity profile of soluble and immobilized peroxidases tion (Figure 2). A large difference in activity of soluble enzyme was observed when the enzyme was preincubated at various temperatures. Soluble enzyme was almost inactivated between C, whereas alginate entrapped enzyme retained about 45 per cent of its original activity at 70 C. Moreover, alginate entrapped cross-linked enzyme preparation was remarkably more stable and retained nearly 70 per cent activity at 70 C. However, temperature optima of the immobilized preparations remained unaltered but cross-linked preparation exhibited higher stability at high temperatures. It was achieved due to strong bond formation between the enzyme molecules, thus it increased the rigidity to the native enzyme structure 10,24,25. Alginate entrapped crosslinked peroxidases retained their structures and remarkably high activity at elevated temperatures. Therefore, such enzyme preparation could be highly useful at relatively high temperatures, which is an important factor for industrial applications. Effect of ph on the activity of soluble and immobilized enzyme is shown in Figure 3. The ph activity profile of the immobilized enzyme has the same ph optima as the soluble peroxidase, while the entrapped cross-linked enzyme was more resistant to ph changes. This phenomenon was not related to the different stabilities at both extremes of the ph values used in the entrapped preparations as shown in Figure 3 neither enzyme preparations showed any inactivation after 1 h at 40 C incubation in the buffer of ph 3.0 or ph But soluble enzyme lost 81 per cent of its original activity at ph 8.0. This predicts that entrapment of enzymes in gel beads provides microenvironment to the enzyme, which may play an important role on the state of protonation of the protein molecules 26,27. Cross-linking further confers additional resistance to the enzyme against extreme conditions of ph 17,24. Urea at a concentration of 4.0 M is a strong denaturant of protein and this irreversibly denatures the soluble enzyme. Cross-linked enzyme entrapped preparation was found to be more superior in stability as compared to the other preparations as shown in Figure 4. Soluble enzyme lost 81 per cent of its initial activity after 1 h incubation in 4.0 M urea while alginate entrapped enzyme retained 46 per cent of the initial activity. It is therefore obvious that the enzymes in beads were completely accessible to urea and it has been known that it can significantly affect the ionic interaction of uncross-linked entrapped enzyme 28. Although the action mechanism of urea on the protein structures has not yet been completely understood, several earlier studies have proposed that pro-

6 MUSTHAPA et al.: ECONOMICAL PROCEDURE FOR IMMOBILIZATION OF PEROXIDASES FROM TURNIP ROOTS 545 tein is unfolded by the direct interaction of urea molecule with a peptide backbone via hydrogen bonding/hydrophobic interaction, which contributes to the maintenance of protein conformation 29. However the treatment of several enzymes with cross-linking reagents has been shown to result in an enhancement of their resistance to denaturation 24. These observations clearly indicate that cross-linking of enzyme protects it from urea-induced denaturation. Soluble and alginate entrapped peroxidase preparations were stored at 4 C and activity was checked on alternate days. As shown in Figure 5, soluble enzyme lost nearly 85 per cent activity in 30 d while entrapped crosslinked enzyme preparation retained over 92 per cent activity under similar experimental conditions. However, entrapped soluble enzyme preparation was rapidly losing its activity due to leaching of the enzyme out of the gel beads 13. Reusability studies of the alginate entrapped enzyme preparation further confirmed the leaching of soluble enzyme out of the gel beads. As shown in Figure 6, alginate entrapped cross-linked enzyme preparation was still active after six repeated uses whereas alginate entrapped soluble enzyme drastically lost its activity after six repeated uses under similar experi- Figure 5 Storage stability of soluble and immobilized turnip peroxidases mental conditions. These effects may be due to higher porosity of the alginate gel resulting in loss of enzyme molecule from the gel 13,22.

7 546 J SCI IND RES VOL 63 JUNE 2004 Figure 4 The effect of urea on the activity of soluble and immobilized peroxidases Davis and Burns 21 have already shown that polyphenoloxidases entrapped in calcium alginate lost their activity due to leaching of the enzymes out of gel beads. We have earlier reported that the leaching effect of the enzymes out of gels can be overcome by entrapping the concanavalin A-glycoenzyme complexes into alginate beads. A column containing alginate-entrapped concanavalin A-invertase complex Figure 6 Reusability of calcium alginate beads containing entrapped soluble and cross-linked turnip peroxidases was successfully operated for the continuous hydrolysis of sucrose for over 4 months. It suggested that the enzymes with high molecular mass could stay for longer period inside the polymeric matrix 18. However glutaraldehyde mediated cross-linking of turnip peroxidases increases the molecular dimension of the enzyme and thus prevents its leaching from the alginate beads those have limited pore size 13,22. This observation is important in biocatalysts, where its reusability determines its economical value. From these results, it is evident that the alginate entrapped cross-linked peroxidase was found

8 MUSTHAPA et al.: ECONOMICAL PROCEDURE FOR IMMOBILIZATION OF PEROXIDASES FROM TURNIP ROOTS 547 to be much more resistant to denaturation, induced by several chemical and physical agents, compared to the preparation containing entrapped uncross-linked peroxidase. Conclusion Several methods have been described for the immobilization of peroxidases but very few can control the amount of immobilized preparation and provide good stability to the enzymes. The present investigation aims to work out an inexpensive, simple and fast procedure for enzyme immobilization, which turns out to be of extreme interest for the use of peroxidases for the removal of phenols and other aromatic compounds from the polluted wastewater. Turnip roots peroxidases were partially purified by using ammonium sulphate fractionation and were easily immobilized in calcium alginate gel directly and after cross-linking with glutaraldehyde. The stability against denaturing agents and heat inactivation of immobilized enzyme are important factors while selecting an appropriate enzymatic system for applications. This study indicates that alginate entrapped crosslinked enzyme preparation has significantly higher stability against physical and chemical denaturants. Such immobilized enzyme preparations may be used to develop bioreactors for the removal of phenolic and other aromatic pollutants present in wastewater, which are derived from agricultural and industrial activities. Acknowledgements Authors are thankful to Council of Scientific and Industrial Research, New Delhi, India for providing financial assistance. References 1 Laborzewsky J & Ginalska, G, Industrial use of soluble or immobilized plant peroxidases, Plant Perox Newslett, 6 (1995) Macek T, Dobransky R, Pespisilova R, Vanek T & Kralova B, Peroxidases from in vitro cultures of different plant species, In Plant peroxidases biochemistry and physiology edited by K G Welinder, S K Rasmussen, C Penel and H Greppin (University of Copenhagen and University of Geneva) 1993, Husain Q & Jan U, Detoxification of phenols and aromatic amines from polluted wastewater by using phenol oxidases, J Sci Ind Res, 59 (1999) Duran N & Esposito E, Potential applications of oxidative enzymes and phenoloxidase-like compounds in wastewater and soil treatment: A review, Appl Catal B: Environ, 28 (2000) Shaffiqu T S, Roy J J, Nair R A & Abraham T E, Degradation of textile dyes mediated by plant peroxidases, Appl Biochem Biotechnol, (2002) Bhunia A, Durani S & Wangikar P P, Horseradish peroxidase catalyzed degradation of industrially important dyes, Biotechnol Bioeng, 72 (2001) Dordick J S, Marletta M A & Klibanov A M, polymerization of phenols catalyzed by peroxidase in non aqueous media, Biotechnol Bioeng, 30 (1987) Ryu K, McEldon J M, Pokora A R, Cyrus W & Dordick J S, Numerical and Monte-Carlo simulation of phenolic polymerization catalyzed by peroxidase, Biotechnol Bioeng, 42 (1993) Xu Y-P, Huang G-L & Yu Y-T, Kinetics of phenolic polymerization catalyzed by peroxidase in organic media, Biotechnol Bioeng, 47 (1995) McEldon J P & Dordick J S, Unusual thermo-stability of soybean peroxidase, Biotechnol Prog, 12 (1996) Tischer W & Kasche V, Immobilized enzymes: crystals or carriers, TIBTECH, 17 (1999) Gupta M N & Mattiasson B, Unique applications of immobilized proteins in Bioanalytical systems, Meth Biochem Anal, 36 (1992) Kierstan M & Bucke C, The immobilization of microbial cells, sub-cellular organelles, and enzymes in calcium alginate gels, Biotechnol Bioeng, 67 (2000) Smidsrφd O & Gudmund S B, Alginate as immobilization matrix for cells, TIBTECH, 8 (1990) Krell H W, Peroxidase: an important enzyme for diagnostic kit. In Biochemical, molecular and physiological aspects of plants peroxidases, edited by J Laborzeswki, H Greppin, C Penel and Th Gaspar (University of Geneva) 1991, pp Hamed R R, Maharem T M, Abdul Fatah M M & Ataya F S, Purification of peroxidase isoenzymes from turnip roots, Phytochemistry, 48 (1998) Husain Q & Saleemuddin M, Immobilization of glycoenzymes using crude concanavalin A and glutaraldehyde, Enzyme Microb Technol, 8 (1986)

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