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Immobilization of polyphenol oxidase in conducting graft copolymers and determination of phenolic amount in red wines with enzyme electrodes
Enzyme and Microbial Technology 39 (2006) 945–948
Immobilization of polyphenol oxidase in conducting graft copolymers and determination of phenolic amount in red wines with enzyme electrodes Huseyin Bekir Yildiz a , Levent Toppare a,∗ , Yesim Hepuzer Gursel b , Yusuf Yagci b a
Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey b Department of Chemistry, Istanbul Technical University, 34469 Istanbul, Turkey
Received 10 December 2005; received in revised form 19 January 2006; accepted 26 January 2006
Abstract Polyphenol oxidase was immobilized in several conducting copolymer matrices. Three different types of poly(methyl methacrylate-co-methyl thienyl methacrylate) matrices were used to obtain copolymers. Immobilization of enzyme was carried out by the entrapment of the enzyme in conducting copolymer matrices during electrochemical polymerization of pyrrole through thiophene moieties of the polymers. Maximum reaction rates, Michaelis–Menten constants, optimum temperature and pH values and operational stabilities of the enzyme electrodes were investigated. Total amount of phenolic compounds in some Turkish red wines was analyzed using these electrodes. © 2006 Elsevier Inc. All rights reserved. Keywords: Polyphenol oxidase; Immobilization; Electrochemistry; Polyphenols; Red wines
1. Introduction Polyphenol oxidase (Tyrosinase, E.C. 1.14.18.1) is a bifunctional enzyme, which catalyzes ortho hydroxylation of monophenols (cresolase activity) and oxidation of catechols to the corresponding ortho-quinones (catecholase activity). oQuinones follow some other enzymatic and nonenzymatic reactions, which result in formation of biopolymers like melanin. This macromolecule, the most famous product of tyrosinase, is the natural pigment of mammalian hair, eye and skin. Undesirable browning of fruits and vegetables during postharvest-handling has also been ascribed to polyphenol oxidase [1]. Wines, particularly red wines, contain numerous biologically active compounds. The most important of which are polyphenols. Nutritional importance of polyphenols is attributed to their antioxidant power. In particular, flavonoids and related phenolics which are naturally found in red wines gained increasing interest [2]. Red wines, have been reported to be more protective and they play a possible role in reducing thrombotic and anthrogenic processes. Polyphenols also contribute substantially to the
∗
Corresponding author. Tel.: +90 312 210 32 51; fax: +90 312 210 12 80. E-mail address: [email protected] (L. Toppare).
0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.01.026
quality of wines and affect their color, flavor, stability and aging behavior [3]. Polymerization based on electrochemical oxidation of a given monomer, in the presence of enzyme, is one of the alternative methods of enzyme immobilization. This results in the formation of a conducting polymer layer containing entrapped enzyme molecules. The electropolymerization is often done in aqueous solutions of pH close to neutral values to immobilize the enzyme without loss of activity [4]. Advantages of immobilization of enzymes in a conducting polymer by electropolymerization are easy one-step procedure, accurate control of the polymer thickness via the electrical charge passed during the film formation process, localization of electrochemical reaction exclusively on the electrode surface allowing precise modification of microelectrodes and surfaces of complex geometry, and the possibility to build up multilayer structures [5–7]. In this study, the immobilization of polyphenol oxidase was performed via entrapment within three types of polypyrrole/poly(methyl methacrylate-co-methyl thienyl methacrylate) (PMMA-co-PMTM) matrices (Scheme 1). These random copolymers having different compositions are coded as MT1, MT2 and MT3 (Table 1). The copolymers were synthesized and characterized previously [4,8]. Optimum conditions for immobilized polyphenol oxidase, such as pH, temperature and kinetic
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H.B. Yildiz et al. / Enzyme and Microbial Technology 39 (2006) 945–948 platinum foils (1 cm2 ) and the reference electrode was Ag/Ag+ (0.01 M). Immobilization was carried out at a constant potential of +1.0 V for 20 min in the presence of 0.2 mg/mL PPO at room temperature [9]. In order to rule out the possibility of enzyme adsorption on copolymer electrodes, blank experiments with no applied potential were carried out. Enzyme electrodes were kept at 4 ◦ C in citrate buffer solution when not in use.
2.3. Determination of PPO activity The activities of free and immobilized PPO were determined by using Besthorn Hydrazone method [10]. A calibration curve of absorbance versus quinone amount was prepared which defines the amount of quinone produced when catechol reacts with iodine. For the activity determination of immobilized PPO, different concentrations of catechol were prepared (3.0 mL) and put in water bath at 25 ◦ C. One milliliter of 3-methyl-2-benzothiozolinone hydrazone (MBTH) interacts with quinones produced by the enzyme to yield red products instead of brown color pigments in the absence of the color reagent [11]. Enzyme electrode was immersed in the solution and shaken for 5 min. One milliliter sulfuric acid and 1 mL acetone were added for a total volume of 6 mL. After mixing, absorbances were measured at 495 nm [9]. To determine the amount of phenolics found in red wines, the above procedure was used by using red wines as the substrate solution instead of catechol.
2.4. Determination of optimum temperature and pH Scheme 1. Synthesis of conducting copolymer of PMMA-co-PMTM/PPy [4]. Table 1 Number average molecular weight and composition of the random copolymers Code
Mn
MT1 MT2 MT3
1.1 × 105 1.1 × 105 1.2 × 105
Copolymer composition (mol%) PMTM
PMMA
17 25 45
83 75 55
parameters (Km and Vmax ) were investigated. The operational stability studies of these enzyme electrodes were done. Total amount of phenolic compounds in red wines was studied by using these electrodes. 2. Experimental
The reaction temperature was changed between 10 and 80 ◦ C while catechol concentration was kept at 10Km for every case at pH 6.5. For pH optimization, pH of the reaction was altered between pH 2 and 11 while catechol solution was kept at 10Km and 25 ◦ C. The activities were determined as previously described.
2.5. Protein determination Protein determination measurements were performed by Bradford’s method [12]. Bradford’s reagent was prepared by mixing 25 mL phosphoric acid, 12.5 mL ethanol and 25 mg Coomassie Brilliant Blue (G-dye). The mixture was diluted to 50 mL with distilled water. During measurements, a solution of Bradford’s reagent was prepared by mixing one volume of stock solution with four volumes of distilled water. For the preparation of protein calibration curve, bovine serum albumin (BSA) was used. Different concentrations of BSA were prepared as 1 mL. Two milliliter of diluted Bradford’s reagent were added to different concentrations of BSA solutions. The absorbance of these solutions was measured at 595 nm. Since the protein entrapped in enzyme electrode could not be measured, we measured the protein amount in the electrolysis solution before and after the electrolysis. The difference gives amount of protein entrapped in the enzyme electrode during the electrolysis.
2.1. Materials 2.6. Operational stability Polyphenol oxidase (Tyrosinase) [E.C. 1.14.18.1] (50,000 units) and sodium dodecyl sulfate were purchased from Sigma (Taufkirchen, Germany). Pyrrole was obtained from Aldrich (Taufkirchen, Germany). Pyrrole was distilled before use. 3-Methyl-2-benzothiozolinone hydrazone (MBTH), acetone and sulfuric acid were also obtained from Sigma. For the preparation of citrate buffer, citric acid and sodium hydroxide were used as received. Catechol was purchased from Sigma. All catechol solutions were prepared in citrate buffer. Potentioscan Wenking POS-73 potentiostat and Shimadzu UV-160 model spectrophotometer were used.
2.2. Immobilization of polyphenol oxidase in PPy/MT1, PPy/MT2, PPy/MT3 and PPy matrices Immobilization process was achieved by electropolymerization of pyrrole on bare or any one of MT1, MT2 and MT3 coated platinum electrodes. The electrolysis solution consisted of 1.2 mg/mL supporting electrolyte (SDS), 0.01 M pyrrole in 10 mL citrate buffer (pH 6.5). Working and counter electrodes were
Operational stability experiments were performed at 25 ◦ C and pH 6.5 by testing the electrode for 40 repetitive operations without washing the electrodes in between the runs.
3. Results and discussion 3.1. Protein determination for enzyme electrodes Free enzyme was found to have an activity of 11.2 mol/ (min mg protein). Results of protein determination experiment show that PPy/PPO electrode entrapped the highest amount of protein (7.4 × 10−3 mg). The other enzyme electrodes MT1/PPy/PPO, MT2/PPy/PPO and MT3/PPy/PPO entrapped 6.8 × 10−3 , 5.3 × 10−3 and 3.4 × 10−3 mg of protein, respectively. It seems that when the polymers contain higher amounts
H.B. Yildiz et al. / Enzyme and Microbial Technology 39 (2006) 945–948
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Table 2 Kinetic parameters for free and immobilized polyphenol oxidase
Free PPO PPy/PPO MT1/PPy/PPO MT2/PPy/PPO MT3/PPy/PPO
Km (mM)
Vmax
4.00 62.0 74.0 88.0 100.0
11.2 mol/(min mg protein) 0.40 mol/(min cm2 ) 0.31 mol/(min cm2 ) 0.22 mol/(min cm2 ) 0.14 mol/(min cm2 )
of thiophene moieties, the polypyrrole chains attached become shorter yielding lower amounts of immobilized enzyme, since pure polypyrrole entraps the highest amount of enzyme. 3.2. Kinetic studies of immobilized PPO Vmax and Michaelis–Menten constants (Km ) for enzyme electrodes were found from Lineweaver–Burk plot. Kinetic constants for immobilized polyphenol oxidase in three matrices are given in Table 2. Both Km and Vmax values of three enzyme electrodes were inversely proportional to thienyl amount in MT1, MT2 and MT3 matrices. When the thiophene moiety is high, the reaction between enzyme and substrate becomes difficult due to the morphology of copolymer matrices [13]. Therefore, Km value of MT3/PPy/PPO enzyme electrode is the highest one among the three enzyme electrodes. Km is a parameter that is inversely proportional to the affinity of enzyme to substrate. Decrease in the rate of reaction was expected because of immobilization. The Vmax values proved this expectation. When the amount of immobilized protein decreases, the Vmax values also decrease. PPy/PPO, MT1/PPy/PPO, MT2/PPy/PPO and MT3/PPy/PPO yielded reaction rates as 0.40, 0.31, 0.22 and 0.14 mol/(min cm2 ), respectively, hence immobilization of enzyme affects the rate of product formation. 3.3. Effect of temperature The effect of temperature between 10 and 80 ◦ C on the relative enzyme activity was investigated and illustrated in Fig. 1. Both free and immobilized PPO in the PPy/PPO electrode showed a maximum activity at about 40 ◦ C. However, maximum enzyme activities for MT1/PPy and MT2/PPy matrices were at
Fig. 2. Effect of pH on polyphenol oxidase activity immobilized in MT1 (); MT2 (); MT3 ().
60 ◦ C and maximum enzyme activity for MT3/PPy matrice was 70 ◦ C. After these temperatures up to 80 ◦ C, they lost 10% of their relative enzyme activity. These matrices showed high stability against temperature. 3.4. Effect of pH The maximum activity was obtained at pH 5 for the free enzyme. The maximum activity was observed at pH 8 for PPy and MT1/PPy, pH 9 for MT2/PPy and pH 10 for MT3/PPy matrices. They are illustrated in Fig. 2. This might be explained by partitioning of protons [14]. Negatively charged groups of matrice will tend to concentrate protons and these causes lowering the pH around the enzyme. Therefore, the pH around the enzyme will be lower than that of the bulk from which the measurement of pH is carried out. Since MT3 is the highest thiophene moiety containing polymer, we expect this polymer is doped heavily with the supporting electrolyte anion compared to MT2 and MT1. MT1/PPy, MT2/PPy and MT3/PPy matrices lost only 25, 15 and 10% of their activity respectively up to pH 11. Moreover, these matrices are stable on the pH range between pH 7 and 10. Hence, these matrices can be used reliably at high pH values for enzyme reactions. 3.5. Operational stability Operational stability is an important consideration for an immobilized enzyme. It was tried to estimate the stability of electrodes in terms of repetitive uses. In 40 successive measurements, the response of the electrodes did not change significantly. The slight increase in the response of enzyme electrodes are related to the swelling of the polymer structure and reorganization of the enzyme molecules in this matrice [15] (Fig. 3). 3.6. Determination of total phenolic amount in red wines
Fig. 1. Effect of incubation temperature on polyphenol oxidase activity in MT1 (); MT2 (); MT3 ().
Turkish red wines, Brand K and Brand D, were used in the analysis of phenolic amount in wines. Total phenolic amount in Turkish wines are reported as 2000–3000 mg/L [16,17]. Polyphenol oxidase enzyme reacts with −OH groups for phenolic compounds. The total −OH groups were obtained via activity determination of enzyme in red wine.
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Table 3 Total phenolics in two different red wines, determined by the three enzyme electrodes
Brand K Brand D a
Free PPO
PPy/PPO
MT1/PPy/PPO
MT2/PPy/PPO
MT3/PPy/PPO
0.004 M −OH, 220 mg/La 0.005 M −OH, 270 mg/La
0.072 M −OH, 4000 mg/La 0.04 M −OH, 2200 mg/La
0.059 M −OH, 3122 mg/La 0.029 M −OH, 1350 mg/La
0.065 M −OH, 3447 mg/La 0.031 M −OH, 1647 mg/La
0.004 M −OH, 3620 mg/La 0.005 M −OH, 1890 mg/La
Total phenolics are expressed as Gallic acid equivalents [18] with 1.5% error limits.
Fig. 3. Operational stability of MT1/PPy (); MT2/PPy (); MT3/PPy ().
Results of phenolic determination by using free PPO enzyme give unrealistic values when compared to three enzyme electrodes. As known from literature, benzoates act as inhibitors for free PPO. Thus, PPO is inhibited by benzoates found naturally in wines, before it completes enzymatic reactions. It was seen in previous studies that PPO was protected by the electrodes and PPO was not affected by the inhibitors found in wines [7,11,12,18]. In these studies the range of phenolic amount was reported as 3000–4000 mg/L for Brand K and 1500–2000 mg/L for Brand D. As seen in Table 3, the values for three matrices are in good agreement with both literature and previous studies. 4. Conclusion This study shows that polyphenol oxidase can be successfully immobilized in MT1/PPy, MT2/PPy and MT3/PPy matrices. As regards to temperature, pH and operational stability these enzyme electrodes yield very good results. It was also shown that, immobilization of polyphenol oxidase enzyme in conducting polymer electrodes can be achieved as an alternative method for the determination of amount of phenolics in red wines. Acknowledgements This was partially supported by DPT-2005K120580 and TUBA grants. Authors greatfully thank Dr. Senem Kiralp for valuable discussions. References [1] Gheibi N, Saboury AA, Haghbeen K, Moosavi-Movahedi AA. Activity and structural changes of mushroom tyrosinase induced by n-alkyl sulfates. Colloids Surf B: Biointerfaces 2005;45:104–7.
[2] Campanella L, Bonanni A, Finotti E, Tomasetti M. Biosensors for determination of total and natural antioxidant capacitiy of red and white wines: comparison with other spectrophotometric and flourimetric methods. Biosens Bioelectron 2004;19:641–51. [3] Luximon-Ramma A, Bahorun T, Crozier A, Zbarsky V, Datla KP, Dexter DT, et al. Characterization of the antioxidant functions of flavonoids and proanthocyanidins in Mauritian black teas. Food Res Int 2005;38:357–67. [4] Yildiz HB, Kiralp S, Toppare L, Yagci Y. Immobilization of invertase in conducting polypyrrole/pmma-co pmtm graft copolymers. J Appl Polym Sci 2005;96:502–7. [5] Cirpan A, Alkan S, Toppare L, Hepuzer Y, Yagci Y. Immobilization of invertase in conducting copolymers of 3-methylthienyl methacrylate. Bioelectrochemistry 2003;59:29–33. [6] Gursel A, Alkan S, Toppare L, Yagci Y. Immobilization of invertase and glucose oxidase in conducting H-type polysiloxane/polypyrrole block copolymers. React Funct Polym 2003;57:57–65. [7] Kiralp S, Cirpan A, Toppare L. Enzyme electrodes for determination of total phenolic capacity of red wines. J Appl Polym Sci 2005;98:521–4. [8] Guner Y, Toppare L, Hepuzer Y, Yagci Y. Conducting graft copolymers of pyrrole and thiophene with random copolymers of methyl methacrylate and 3-methylthienyl methacrylate. Eur Polym J 2004;40:1799– 806. [9] Kiralp S, Toppare L, Yagci Y. Determination of phenolic compounds in wines with enzyme electrodes fabricated by immobilization of polyphenol oxidase in conducting copolymers. Des Monomers Polym 2004;7:3–7. [10] Mazzocco F, Pifferi PG. An improvement of the spectrophotometric method for the determination of tyrosinase catecholase activity by Besthorn’s hydrazone. Anal Biochem 1996;72:643–7. [11] Ortega F, Dominguez E, Jonsson-Pettersson G, Gorton L. Amperometric biosensor for the determination of phenolic compounds using a tyrosinase graphite electrode in a flow injection system. J Biotechnol 1993;31:289–300. [12] Kiralp S, Toppare L, Yagci Y. Immobilization of polyphenol oxidase in conducting copolymers and determination of phenolic compounds in wines with enzyme electrodes. J Int Biol Macromol 2003;33:37–41. [13] Yildiz HB, Kiralp S, Toppare L, Yagci Y. Immobilization of glucose oxidase in conducting graft copolymers and determination of glucose amount in orange juices with enzyme electrodes. J Int Biol Macromol 2005;37:174–8. [14] Yildiz HB, Toppare L. Biosensing approach for alcohol determination using immobilized alcohol oxidase. Biosens Bioelectron, in press. [15] Cirpan A, Alkan S, Toppare L, Cianga I, Yagci Y. Immobilization of cholesterol oxidase in a conducting copolymer of thiophene-3yl acetic acid cholesteryl ester with pyrrole. Des Monomers Polym 2003;6:237–43. [16] Karakaya S, El SN, Tas AA. Antioxidant activity of some foods containing phenolic compounds. Int J Food Sci Nutr 2001;52:501. [17] Sakkiadi AV, Stavrakakis MN, Haroutounian SA. Direct HPLC assay of five biologically interesting phenolic antioxidants in varietal Greek red wines. Lebensm Wiss Technol 2001;34:410–3. [18] Lopez M, Martinez F, Del Valle C, Orte C, Miro M. Analysis of phenolic constituents of biological interest in red wines by high-performance liquid chromatography. J Chromatogr A 2001;922:359–63.
Immobilization of polyphenol oxidase in conducting graft copolymers and determination of phenolic amount in red wines with enzyme electrodes Huseyin Bekir Yildiz a , Levent Toppare a,∗ , Yesim Hepuzer Gursel b , Yusuf Yagci b a
Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey b Department of Chemistry, Istanbul Technical University, 34469 Istanbul, Turkey
Received 10 December 2005; received in revised form 19 January 2006; accepted 26 January 2006
Abstract Polyphenol oxidase was immobilized in several conducting copolymer matrices. Three different types of poly(methyl methacrylate-co-methyl thienyl methacrylate) matrices were used to obtain copolymers. Immobilization of enzyme was carried out by the entrapment of the enzyme in conducting copolymer matrices during electrochemical polymerization of pyrrole through thiophene moieties of the polymers. Maximum reaction rates, Michaelis–Menten constants, optimum temperature and pH values and operational stabilities of the enzyme electrodes were investigated. Total amount of phenolic compounds in some Turkish red wines was analyzed using these electrodes. © 2006 Elsevier Inc. All rights reserved. Keywords: Polyphenol oxidase; Immobilization; Electrochemistry; Polyphenols; Red wines
1. Introduction Polyphenol oxidase (Tyrosinase, E.C. 1.14.18.1) is a bifunctional enzyme, which catalyzes ortho hydroxylation of monophenols (cresolase activity) and oxidation of catechols to the corresponding ortho-quinones (catecholase activity). oQuinones follow some other enzymatic and nonenzymatic reactions, which result in formation of biopolymers like melanin. This macromolecule, the most famous product of tyrosinase, is the natural pigment of mammalian hair, eye and skin. Undesirable browning of fruits and vegetables during postharvest-handling has also been ascribed to polyphenol oxidase [1]. Wines, particularly red wines, contain numerous biologically active compounds. The most important of which are polyphenols. Nutritional importance of polyphenols is attributed to their antioxidant power. In particular, flavonoids and related phenolics which are naturally found in red wines gained increasing interest [2]. Red wines, have been reported to be more protective and they play a possible role in reducing thrombotic and anthrogenic processes. Polyphenols also contribute substantially to the
∗
Corresponding author. Tel.: +90 312 210 32 51; fax: +90 312 210 12 80. E-mail address: [email protected] (L. Toppare).
0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.01.026
quality of wines and affect their color, flavor, stability and aging behavior [3]. Polymerization based on electrochemical oxidation of a given monomer, in the presence of enzyme, is one of the alternative methods of enzyme immobilization. This results in the formation of a conducting polymer layer containing entrapped enzyme molecules. The electropolymerization is often done in aqueous solutions of pH close to neutral values to immobilize the enzyme without loss of activity [4]. Advantages of immobilization of enzymes in a conducting polymer by electropolymerization are easy one-step procedure, accurate control of the polymer thickness via the electrical charge passed during the film formation process, localization of electrochemical reaction exclusively on the electrode surface allowing precise modification of microelectrodes and surfaces of complex geometry, and the possibility to build up multilayer structures [5–7]. In this study, the immobilization of polyphenol oxidase was performed via entrapment within three types of polypyrrole/poly(methyl methacrylate-co-methyl thienyl methacrylate) (PMMA-co-PMTM) matrices (Scheme 1). These random copolymers having different compositions are coded as MT1, MT2 and MT3 (Table 1). The copolymers were synthesized and characterized previously [4,8]. Optimum conditions for immobilized polyphenol oxidase, such as pH, temperature and kinetic
946
H.B. Yildiz et al. / Enzyme and Microbial Technology 39 (2006) 945–948 platinum foils (1 cm2 ) and the reference electrode was Ag/Ag+ (0.01 M). Immobilization was carried out at a constant potential of +1.0 V for 20 min in the presence of 0.2 mg/mL PPO at room temperature [9]. In order to rule out the possibility of enzyme adsorption on copolymer electrodes, blank experiments with no applied potential were carried out. Enzyme electrodes were kept at 4 ◦ C in citrate buffer solution when not in use.
2.3. Determination of PPO activity The activities of free and immobilized PPO were determined by using Besthorn Hydrazone method [10]. A calibration curve of absorbance versus quinone amount was prepared which defines the amount of quinone produced when catechol reacts with iodine. For the activity determination of immobilized PPO, different concentrations of catechol were prepared (3.0 mL) and put in water bath at 25 ◦ C. One milliliter of 3-methyl-2-benzothiozolinone hydrazone (MBTH) interacts with quinones produced by the enzyme to yield red products instead of brown color pigments in the absence of the color reagent [11]. Enzyme electrode was immersed in the solution and shaken for 5 min. One milliliter sulfuric acid and 1 mL acetone were added for a total volume of 6 mL. After mixing, absorbances were measured at 495 nm [9]. To determine the amount of phenolics found in red wines, the above procedure was used by using red wines as the substrate solution instead of catechol.
2.4. Determination of optimum temperature and pH Scheme 1. Synthesis of conducting copolymer of PMMA-co-PMTM/PPy [4]. Table 1 Number average molecular weight and composition of the random copolymers Code
Mn
MT1 MT2 MT3
1.1 × 105 1.1 × 105 1.2 × 105
Copolymer composition (mol%) PMTM
PMMA
17 25 45
83 75 55
parameters (Km and Vmax ) were investigated. The operational stability studies of these enzyme electrodes were done. Total amount of phenolic compounds in red wines was studied by using these electrodes. 2. Experimental
The reaction temperature was changed between 10 and 80 ◦ C while catechol concentration was kept at 10Km for every case at pH 6.5. For pH optimization, pH of the reaction was altered between pH 2 and 11 while catechol solution was kept at 10Km and 25 ◦ C. The activities were determined as previously described.
2.5. Protein determination Protein determination measurements were performed by Bradford’s method [12]. Bradford’s reagent was prepared by mixing 25 mL phosphoric acid, 12.5 mL ethanol and 25 mg Coomassie Brilliant Blue (G-dye). The mixture was diluted to 50 mL with distilled water. During measurements, a solution of Bradford’s reagent was prepared by mixing one volume of stock solution with four volumes of distilled water. For the preparation of protein calibration curve, bovine serum albumin (BSA) was used. Different concentrations of BSA were prepared as 1 mL. Two milliliter of diluted Bradford’s reagent were added to different concentrations of BSA solutions. The absorbance of these solutions was measured at 595 nm. Since the protein entrapped in enzyme electrode could not be measured, we measured the protein amount in the electrolysis solution before and after the electrolysis. The difference gives amount of protein entrapped in the enzyme electrode during the electrolysis.
2.1. Materials 2.6. Operational stability Polyphenol oxidase (Tyrosinase) [E.C. 1.14.18.1] (50,000 units) and sodium dodecyl sulfate were purchased from Sigma (Taufkirchen, Germany). Pyrrole was obtained from Aldrich (Taufkirchen, Germany). Pyrrole was distilled before use. 3-Methyl-2-benzothiozolinone hydrazone (MBTH), acetone and sulfuric acid were also obtained from Sigma. For the preparation of citrate buffer, citric acid and sodium hydroxide were used as received. Catechol was purchased from Sigma. All catechol solutions were prepared in citrate buffer. Potentioscan Wenking POS-73 potentiostat and Shimadzu UV-160 model spectrophotometer were used.
2.2. Immobilization of polyphenol oxidase in PPy/MT1, PPy/MT2, PPy/MT3 and PPy matrices Immobilization process was achieved by electropolymerization of pyrrole on bare or any one of MT1, MT2 and MT3 coated platinum electrodes. The electrolysis solution consisted of 1.2 mg/mL supporting electrolyte (SDS), 0.01 M pyrrole in 10 mL citrate buffer (pH 6.5). Working and counter electrodes were
Operational stability experiments were performed at 25 ◦ C and pH 6.5 by testing the electrode for 40 repetitive operations without washing the electrodes in between the runs.
3. Results and discussion 3.1. Protein determination for enzyme electrodes Free enzyme was found to have an activity of 11.2 mol/ (min mg protein). Results of protein determination experiment show that PPy/PPO electrode entrapped the highest amount of protein (7.4 × 10−3 mg). The other enzyme electrodes MT1/PPy/PPO, MT2/PPy/PPO and MT3/PPy/PPO entrapped 6.8 × 10−3 , 5.3 × 10−3 and 3.4 × 10−3 mg of protein, respectively. It seems that when the polymers contain higher amounts
H.B. Yildiz et al. / Enzyme and Microbial Technology 39 (2006) 945–948
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Table 2 Kinetic parameters for free and immobilized polyphenol oxidase
Free PPO PPy/PPO MT1/PPy/PPO MT2/PPy/PPO MT3/PPy/PPO
Km (mM)
Vmax
4.00 62.0 74.0 88.0 100.0
11.2 mol/(min mg protein) 0.40 mol/(min cm2 ) 0.31 mol/(min cm2 ) 0.22 mol/(min cm2 ) 0.14 mol/(min cm2 )
of thiophene moieties, the polypyrrole chains attached become shorter yielding lower amounts of immobilized enzyme, since pure polypyrrole entraps the highest amount of enzyme. 3.2. Kinetic studies of immobilized PPO Vmax and Michaelis–Menten constants (Km ) for enzyme electrodes were found from Lineweaver–Burk plot. Kinetic constants for immobilized polyphenol oxidase in three matrices are given in Table 2. Both Km and Vmax values of three enzyme electrodes were inversely proportional to thienyl amount in MT1, MT2 and MT3 matrices. When the thiophene moiety is high, the reaction between enzyme and substrate becomes difficult due to the morphology of copolymer matrices [13]. Therefore, Km value of MT3/PPy/PPO enzyme electrode is the highest one among the three enzyme electrodes. Km is a parameter that is inversely proportional to the affinity of enzyme to substrate. Decrease in the rate of reaction was expected because of immobilization. The Vmax values proved this expectation. When the amount of immobilized protein decreases, the Vmax values also decrease. PPy/PPO, MT1/PPy/PPO, MT2/PPy/PPO and MT3/PPy/PPO yielded reaction rates as 0.40, 0.31, 0.22 and 0.14 mol/(min cm2 ), respectively, hence immobilization of enzyme affects the rate of product formation. 3.3. Effect of temperature The effect of temperature between 10 and 80 ◦ C on the relative enzyme activity was investigated and illustrated in Fig. 1. Both free and immobilized PPO in the PPy/PPO electrode showed a maximum activity at about 40 ◦ C. However, maximum enzyme activities for MT1/PPy and MT2/PPy matrices were at
Fig. 2. Effect of pH on polyphenol oxidase activity immobilized in MT1 (); MT2 (); MT3 ().
60 ◦ C and maximum enzyme activity for MT3/PPy matrice was 70 ◦ C. After these temperatures up to 80 ◦ C, they lost 10% of their relative enzyme activity. These matrices showed high stability against temperature. 3.4. Effect of pH The maximum activity was obtained at pH 5 for the free enzyme. The maximum activity was observed at pH 8 for PPy and MT1/PPy, pH 9 for MT2/PPy and pH 10 for MT3/PPy matrices. They are illustrated in Fig. 2. This might be explained by partitioning of protons [14]. Negatively charged groups of matrice will tend to concentrate protons and these causes lowering the pH around the enzyme. Therefore, the pH around the enzyme will be lower than that of the bulk from which the measurement of pH is carried out. Since MT3 is the highest thiophene moiety containing polymer, we expect this polymer is doped heavily with the supporting electrolyte anion compared to MT2 and MT1. MT1/PPy, MT2/PPy and MT3/PPy matrices lost only 25, 15 and 10% of their activity respectively up to pH 11. Moreover, these matrices are stable on the pH range between pH 7 and 10. Hence, these matrices can be used reliably at high pH values for enzyme reactions. 3.5. Operational stability Operational stability is an important consideration for an immobilized enzyme. It was tried to estimate the stability of electrodes in terms of repetitive uses. In 40 successive measurements, the response of the electrodes did not change significantly. The slight increase in the response of enzyme electrodes are related to the swelling of the polymer structure and reorganization of the enzyme molecules in this matrice [15] (Fig. 3). 3.6. Determination of total phenolic amount in red wines
Fig. 1. Effect of incubation temperature on polyphenol oxidase activity in MT1 (); MT2 (); MT3 ().
Turkish red wines, Brand K and Brand D, were used in the analysis of phenolic amount in wines. Total phenolic amount in Turkish wines are reported as 2000–3000 mg/L [16,17]. Polyphenol oxidase enzyme reacts with −OH groups for phenolic compounds. The total −OH groups were obtained via activity determination of enzyme in red wine.
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Table 3 Total phenolics in two different red wines, determined by the three enzyme electrodes
Brand K Brand D a
Free PPO
PPy/PPO
MT1/PPy/PPO
MT2/PPy/PPO
MT3/PPy/PPO
0.004 M −OH, 220 mg/La 0.005 M −OH, 270 mg/La
0.072 M −OH, 4000 mg/La 0.04 M −OH, 2200 mg/La
0.059 M −OH, 3122 mg/La 0.029 M −OH, 1350 mg/La
0.065 M −OH, 3447 mg/La 0.031 M −OH, 1647 mg/La
0.004 M −OH, 3620 mg/La 0.005 M −OH, 1890 mg/La
Total phenolics are expressed as Gallic acid equivalents [18] with 1.5% error limits.
Fig. 3. Operational stability of MT1/PPy (); MT2/PPy (); MT3/PPy ().
Results of phenolic determination by using free PPO enzyme give unrealistic values when compared to three enzyme electrodes. As known from literature, benzoates act as inhibitors for free PPO. Thus, PPO is inhibited by benzoates found naturally in wines, before it completes enzymatic reactions. It was seen in previous studies that PPO was protected by the electrodes and PPO was not affected by the inhibitors found in wines [7,11,12,18]. In these studies the range of phenolic amount was reported as 3000–4000 mg/L for Brand K and 1500–2000 mg/L for Brand D. As seen in Table 3, the values for three matrices are in good agreement with both literature and previous studies. 4. Conclusion This study shows that polyphenol oxidase can be successfully immobilized in MT1/PPy, MT2/PPy and MT3/PPy matrices. As regards to temperature, pH and operational stability these enzyme electrodes yield very good results. It was also shown that, immobilization of polyphenol oxidase enzyme in conducting polymer electrodes can be achieved as an alternative method for the determination of amount of phenolics in red wines. Acknowledgements This was partially supported by DPT-2005K120580 and TUBA grants. Authors greatfully thank Dr. Senem Kiralp for valuable discussions. References [1] Gheibi N, Saboury AA, Haghbeen K, Moosavi-Movahedi AA. Activity and structural changes of mushroom tyrosinase induced by n-alkyl sulfates. Colloids Surf B: Biointerfaces 2005;45:104–7.
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