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Zhengang Zhao, Licai Zhu, Shujuan Yu, Michael Saska
Partial purification and characterization of polyphenol oxidase from sugarcane (Saccharum officinarum L.) Partielle Reinigung und Charakterisierung von Polyphenoloxidase aus Zuckerrohr (Saccharum officinarum L.)
Polyphenol oxidase (PPO) of sugarcane was extracted by using 0.02 mol/L phosphate buffer at pH = 6.8 containing 1.5% polyvinylpolypyrrolidone and 0.5% Triton X-100, and then partially purified by 80% ammonium sulfate fractionation, dialysis, and column chromatography on DEAE-Toyopearl 650M, Sephadex G-100. PPO activity was purified 37.6-fold with a recovery of 18.4%. The PPO showed activity to catechol, chlorogenic acid, 4-methylcatechol, caffeic acid and ferulic acid, but not to l-tyrosine. Optimum conditions (pH value and temperature) for PPO were determined using the five substances. PPO activity is quite thermostable between 20 and 30 °C. After heating for 10 min at 80 °C 90% of the activity is lost. Km and Vmax values of PPO were calculated for each substrate and the best substrate of PPO was chlorogenic acid. PPO was markedly inhibited by metal ions (Cu2+, Al3+, and Mg2+) at 1 and 10 mmol/L, and strongly inhibited by NaHSO3 and ascorbic acid at 1 mmol/L. Key words: polyphenol oxidase, sugarcane, enzyme inhibitors
Zuckerrohr-Polyphenoloxidase (PPO) wurde in 0,02 mol/L Phosphat-Puffer (pH = 6,8) mit 1,5 % Polyvinylpolypyrrolidon und 0,5 % Triton X-100 extrahiert. Die partielle Reinigung erfolgt im ersten Schritt durch 80 % Ammoniumsulfat-Fraktionierung, gefolgt Chromatographie über DEAE-Toyopearl 650M und Sephadex G-100. PPO wurde 37,6-fach angereichert bei einer Ausbeute von 18,4 %. PPO zeigte Aktivität gegenüber Catechin, Chlorogensäure, 4Methylcatechol, Kaffeesäure und Ferulasäure, aber nicht bei l-Tyrosin. pH- und Temperatur-Optima wurden für diese fünf Substrate bestimmt. Die PPO-Aktivität ist thermostabil zwischen 20 und 30 °C. Nach Wärmebehandlung bei 80 °C für 10 min geht 90 % der Aktivität verloren. Km (MichaelisKonstante) und Vmax (maximale Umsatzgeschwindigkeit) der PPO wurden für jedes Substrat ermittelt, Chlorogensäure erwies sich als das beste Substrat. Die Aktivität wurde deutlich gehemmt durch Metallionen (Cu2+, Al3+ und Mg2+) bei Konzentrationen von 1 und 10 mmol/L und stark gehemmt durch NaHSO3 und Ascorbinsäure bei 1 mmol/L. Stichwörter: Polyphenoloxidase, Zuckerrohr, Enzyminhibierung
1
Introduction
Cane sugar color is a complex mix of pigments, some extracted from the sugarcane and some formed during processing. The presence of these colored components impedes crystallization and results in lower sugar yields, poorer sugar quality and high cost of removal. Sugarcane juice tends to change color immediately after extraction. Previous studies have shown that enzymatic browning contributes significantly to color formation in sugarcane juice. Polyphenol oxidase (PPO; EC 1.14.18.1) has been implicated as a leading enzyme that acts upon the phenolic compounds present resulting in enzymatic browning in sugarcane juice [1]. PPO is a widely distributed copper-containing enzyme. It catalyzes the hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to o-quinones. The quinone products of PPO react with a number of functional groups, such as amines, thiols, and phenolics, and form complex brown, red or black pigments [2, 3]. In previous literature, the contribution of enzymatic browning
to color formation in sugarcane juice has been studied [4–8]. As a continuation of enzymatic browning studies in sugarcane juice, the extraction, purification and partial characterization of PPO from sugarcane will be reported, including the optimum pH value and temperature, enzyme kinetics, ion strength effects and inhibition.
2 2.1
Materials and methods Plant material
Samples of sugarcane variety Y 93159 were obtained from a farm in zhangJiang, Guang Dong province (China). The samples were sealed and stored at –20 °C until used in the experiments.
2.2
Enzyme extraction and purification
The crude enzyme extraction method used was based on the same method reported earlier [9], with some modification. Sugar Industry 136 (2011) No. 5 | 296–301
Technology/Technologie A manual stainless steel crusher was used to extract the juice. After extraction, 500 mL cane juice was quickly mixed with 500 mL of 0.02 mol/L phosphate buffer (pH = 6.8) containing 1.5% polyvinylpolypyrrolidone (PVPP) and 0.5% Triton X-100. The suspension was filtered through four layers of cheesecloth and the filtrate centrifuged at 12,000 g for 15 min at 4 °C in a refrigerated centrifuge. The supernatant was used as crude PPO. The crude enzyme was adjusted to 80% saturation with solid ammonium sulfate and then centrifuged at 12,000 g for 30 min at 4 °C. The precipitate was dissolved in a small amount of 0.02 mol/L phosphate buffer (pH = 6.8) and dialyzed at 4 °C in the same buffer for 24 h with four changes of the buffer during dialysis. After dialysis, the extract was loaded onto a column of DEAEToyopearl 650M (1.5 × 10 cm), pre-equilibrated with the dialysis buffer. The column was washed with 2–3× its volumes of the initial buffer and PPO eluted by a linear gradient formed by 0–0.4 mol/L NaCl in 0.02 mol/L sodium-phosphate buffer, pH = 6.8, at a flow rate of 1.0 mL/min. Fractions of 5 mL were collected and assayed for PPO activity. The fractions showing high PPO activity were pooled, lyophilized and redissolved in a small volume of 0.02 mol/L sodium phosphate buffer (pH = 6.8). Then, the concentrate was loaded onto a Sephadex G-100 (2.1 × 30 cm) column, pre-equilibrated with 0.02 mol/L phosphate buffer (pH = 6.8), and eluted with 0.02 mol/L phosphate buffer (pH = 6.8) at a flow rate of 0.2 mL/ min. Fractions of 5 mL were collected in tubes and PPO activity and quantitative protein measurements were carried out.
2.3
Enzyme activity assay
The determination of PPO activity was performed by a UV762 spectrophotometer (Lingguang, Shanghai, China). The reaction was carried out in a 1 cm light path quartz cuvette. PPO activity was assayed at a given wavelength (dependent on the specific quinine product of different substrates) and 25 (±1) °C, as described previously [9], with some modifications. In each measurement, the volume of solution in a quartz cuvette was kept constant at 3 mL. The reaction mixture contained 2.0 mL of 0.2 mol/L phosphate buffer (pH = 6.8), 0.7 mL of 0.05 mol/L substrate and 0.3 mL of enzyme extract. One unit of PPO activity was defined as the amount of enzyme that caused an increase in absorbance of 0.001 under the conditions of the assay.
2.4
echol, caffeic acid, ferulic acid and l-tyrosine as substrates. These compounds were chosen because they are common phenols in plant tissues. Catechol was used at 5 mmol/L; 4methylcatechol, chlorogenic acid, caffeic acid and ferulic acid were used at 2 mmol/L, l-tyrosine was used at 1 mmol/L. Various buffers (0.1 mol/L citrate / 0.2 mol/L phosphate for pH = 3.0–5.5, 0.2 mol/L phosphate for 5.5–7.0, and Tris-HCl for 7.0–10.0) were used for the effect of pH value. Then, the tests were carried out at the optimum pH value activity to determine the optimal temperature. PPO activity was assayed at various reaction temperatures (10–80 °C) using the six different substrates indicated above. The optimum pH values and temperature obtained from this assay were used in subsequent experiments.
2.6
Thermal stability was determined by incubating the partially purified PPO at different temperatures (20–80 °C) for varying periods of time, up to 60 min, in a temperature-controlled water bath; cooling in ice for 3 min and, after cooling, residual activity was measured at the optimum pH value and temperature by using catechol as substrate. The stability of the enzyme was expressed as % residual activity and was calculated by comparison with untreated enzyme.
2.7
Effect of various compounds and ion strength
The effect of various compounds on enzyme activity were studied in the presence (final concentration 1.0 or 10.0 mmol/L) and absence of different ionic compounds (CuSO4, CaCl 2, ZnCl 2, ALCl 3, MnCl 2, MgSO 4, NiSO 4, NaCl, EDTA, K 2SO 4, and BaCl2) or inhibitors (l-cysteine, ascorbic acid, NaHSO 3, b-cyclodextrin, and citric acid) in the assay medium. PPO activity was measured using catechol as substrate, under optimum conditions.
2.8
Enzyme kinetics and substrate specificity
Kinetic parameters (Km and Vmax) were determined using five substrates (catechol, chlorogenic acid, 4-methylcatechol, caffeic acid, and ferulic acid) in varying concentrations and in optimum conditions. Kinetic parameters were determined using the Lineweaver-Burk double reciprocal plot.
Determination of proteins 2.9
The protein content was determined according to Bradford’s dye binding method, using bovine serum albumin (BSA) as a standard [10].
2.5
Heat inactivation of PPO
Effect of pH value and temperature
The pH value and temperature optima of sugarcane PPO were determined using catechol, chlorogenic acid, 4-methylcatNo. 5 (2011) Sugar Industry 136 | 296–301
Statistical analysis
All determinations were conducted at least in triplicate and the results expressed as mean ± standard deviation (S.D.). Analysis of variance (ANOVA) and the Fisher’s least significant difference (LSD) test was performed with the SPSS 15.0 software program. P < 0.05 was selected as the decision for significant differences.
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3 3.1
Results and discussion Extraction and purification of PPO
Sugarcane is rich in phenolic compounds, including phenolic acids, polyphenols, and flavonoids [11–14]. The main problem in the extraction of PPO from sugarcane is the occurrence of these phenolic compounds which react with PPO resulting in its inactivation. In order to improve PPO recovery, PVPP and Triton X-100 were used during extraction. PVPP is a phenoladsorbing agent which is added to the extraction media to bind the phenols and prevent polymerization and reduction of PPO activity [15]. PPO is a membrane-bound protein and can require for solubilization the use of detergent Triton X-100. Therefore, Triton X-100 was also used to improve the activation of latent PPO [16]. Further purification of the PPO was achieved by 80% ammonium sulfate fractionation. This fractionation has been used by some authors to purify PPO from plant tissues [17, 18]. After ammonium sulfate precipitation, the PPO activity level in the dialyzed enzyme extract decreased to 49.9% from 100%.
Table 2: Optimum pH and temperature of sugarcane PPO Substrate Wavelength ConcenOptimum (nm) tration pH value (mmol/L) Catechol 420 5 6.0 Chlorogenic acid 400 2 5.0 4-methylcatechol 420 2 6.5 Caffeic acid 400 2 6.0 Ferulic acid 400 2 6.5 l-tyrosine 420 1 –
Optimum temperature (°C) 40 25 30 30 25 –
Table 1 summarizes the PPO purification showing an overall recovery of 18.4% and a 37.6-fold enrichment. Figure 1 shows the elution profile of proteins and PPO activity from DEAEToyopearl 650M (top) and Sephadex G-100 (down). Activity fractions in 15–22 eluates from DEAE-Toyopearl 650M were pooled, dialyzed and loaded on to Sephadex G-100 column and, fractions 12–18 PPO eluted as a single peak from Sephadex G-100 were pooled. A 37.6-fold purification of PPO with 18.4% yield was achieved, using (NH4)2SO4, DEAE-Toyopearl 650M and Sephadex G-100.
3.2
Effect of pH value and temperature
Optimum pH value and temperature of the PPO are shown in Table 2. Partly purified sugarcane PPO has a pH value optimum of 6.0 with catechol and caffeic acid, 6.5 with 4-methylcatechol and ferulic acid, and 5.0 with chlorogenic acid, respectively. At pH values above 8.0 and below 3.0, PPO activity decreased very rapidly and it was completely inhibited at pH < 3. The rapid inactivation of the enzyme at pH < 3 may be due to denaturing of PPO. These results were similar to the optimum pH value for PPO extracted from shrimp carapace [19]. The pH value is a determining factor in the expression of enzymatic activity, and various plant sources have different pH optima for different substrates [20–22]. The effect of temperature on sugarcane PPO activity was studied from 10 to 80 °C (Table 2). The optimum temperature for PPO is substrate-dependent. The enzyme showed the highest activity at 40 °C with catechol, 30 °C with 4-methylcatechol and caffeic acid, 25 °C with chlorogenic acid and ferulic acid.
3.3
Fig. 1: Elution profile of proteins and PPO activity from DEAE-Toyopearl 650M (top) and Sephadex G-100 (down). Each fraction (5 mL) was monitored at A 280 nm, for protein detection
Table 1: Summary of PPO purification in sugarcane Steps Total volTotal Total proume (mL) activity (U) tein (mg) Crude extract 570 54,456 753 80% (NH4)2SO4 170 27,166 144 DEAE-Toyopearl 32 17,860 14.8 Sephadex G-100 24 10,020 3.7
Specific activity (U/mg) 72 189 1,207 2,708
Effect of thermal stability
Figure 2 shows the thermal stability of partially purified sugarcane PPO at varying temperatures. It was found that the enzyme was stable at 20 °C and 30 °C for 60 min. Treatment for 60 min at 40, 50, 60 and 70 °C reduced PPO activity to: 85 ± 4%, 56 ± 4%, 21 ± 4%, and 4 ± 3%, respectively. The enzyme was unstable at temperatures above 70 °C. IncubaFold Yield (%) tion of partially purified PPO at 80 °C for 10 min caused 90% loss of activity. 1 100 It was completely inactivated after treat2.6 49.9 ment at 80 °C for 20 min. 16.8 32.8 The PPO of table grape [23] and banana 37.6 18.4 [24] showed similar thermal stabilities. Sugar Industry 136 (2011) No. 5 | 296–301
Technology/Technologie effective inhibitor was found to be NaHSO3, followed by ascorbic acid. l-cysteine, b-cyclodextrin and, citric acid showed low inhibition at the low concentrations but were strongly inhibitory at the higher concentrations. The most effective inhibitors were ascorbic acid and sodium metabisulfite for anamur banana [28], tropolone for eggplant [20] and desert truffle [29], and ascorbic acid and l-cysteine for chrysanthemum [30]. Inhibition of PPO activity by citric acid is attributed to be a typical metal ion chelator effect, and it also inhibits enzyme activity by lowering the pH value of the medium. The sulphites (NaHSO3) and amino acids (l-cysteine) inhibit polymerization of quinones by reducing quinones back to phenols or by reacting directly with the enzyme [31]. Ascorbic acid is active at the level of PPO products because it reduces the initial quinone formed by the enzyme back to the phenolic substrates, whereby it gets oxidized [32]. Fig. 2: Thermal stability of sugarcane PPO at varying temperatures with catechol as substrate
3.5 3.4
Effects of ion strength and inhibitors
The effects of various compounds and ion strength on the enzyme activity are shown in Table 3. The enzyme activity is markedly inhibited by metal ions (Cu2+, Al3+, and Mg2+) at 1 and 10 mmol/L, and weakly inhibited by EDTA and metal ions (Na+, Zn2+, Mn2+, Ni2+, K+, and Ba2+) at 1 mmol/L. The PPO of artichoke heads [25] and celery root [26] responded similarly to these compounds. Please note, that the effect of iron salts in relation to polyphenolics and color formation has been recognized since at least 1916 (M.A. Schneller: The Coloring Matter of Cane Juice. Louisiana Bulletin No. 157, Louisiana State University and A&M C o l l e g e , B ato n R o u g e , Au g u s t Table 3: Effect of various compounds and ion 1916). It is unforstrength on sugarcane PPO activity tunate that the Compounds Relative activity (%) effect of iron on 1 mmol/L 10 mmol/L PPO activity was None 100ab ± 0 100 ± 0ab n o t m e a s u re d . CuSO4 71 ± 7c 65 ± 4c Iron reacts with CaCl2 103 ± 5a 97 ± 2ad polyphenols, ZnCl2 99 ± 3ab 89 ± 5e increasing color, AlCl3 81 ± 7d 28 ± 7f but its effect on be e MnCl2 94 ± 3 92 ± 4d PPO activity has MgSO4 93 ± 4be 80 ± 7g not been investiNiSO4 91 ± 5ek 90 ± 5de NaCl 99 ± 2ab 99 ± 1ab gated, as far as the EDTA 96 ± 2ae 81 ± 7g current authors K2SO4 95 ± 5ae 92 ± 4de are aware. BaCl2 94 ± 3be 90 ± 3de Ascorbic acid, lAscorbic acid 19 ± 5f 0 ± 0h cysteine, NaHSO3, l-cysteine 70 ± 8gc 6 ± 3hi citric acid, and NaHSO3 10 ± 4h 0 ± 0h b-cyclodextrin Citric acid 80 ± 8id 30 ± 5f were examined to b-cyclodextrin 75 ± 5cd 9 ± 3i determine their Values represent the mean ±standard error of three replicate samples. a–k Mean values in the potential for inhisame column with the same letter are not signibition of PPO ficantly different (p # 0.05) activity. The most No. 5 (2011) Sugar Industry 136 | 296–301
Substrate specificity
Monophenol and diphenol substrates were tested for substrate specificity of sugarcane PPO. The kinetic parameters for five different substances are summarized in Table 4. Km and Vmax were calculated from the Lineweaver-Burk graphs using these substrates at various concentrations under optimum conditions. Substrate specificities were evaluated by using the Vmax/Km ratio as catalytic efficiency. Vmax/Km indicated that the order of suitability as substrate for sugarcane PPO is chlorogenic acid > 4-methylcatechol > caffeic acid > catechol > ferulic acid. Sugarcane PPO had a great affinity for chlorogenic acid. As seen in the Table 4, PPO showed activity with the diphenols and polyphenolic substrates, whereas the enzyme was unable to oxidize l-tyrosine (monophenolic substrate), suggesting the absence of cresolase activity. Similar results were found for aubergine [33] and latex of Hevea brasiliensis [34].
4
Conclusions
In this paper, maximum PPO activity was obtained by using 0.02 mol/L phosphate buffer at pH = 6.8 containing 1.5% PVPP and 0.5% Triton X-100, and then partly purified by 80% ammonium sulfate fractionation, dialysis, and column chromatography on DEAE-Toyopearl 650M, Sephadex G-100. As for sugarcane juice, the affinity of PPO was highest for chlorogenic acid. The optimum temperature for PPO is substrate-dependent and the enzyme showed the highest activity at 40 °C with catechol, 30 °C with 4-methylcatechol and caffeic acid, 25 °C with chlorogenic acid and ferulic acid. PPO activ-
Table 4: Substrate specificity of sugarcane PPO Substance Km (mmol) Vmax (mol/min) Catechol Chlorogenic acid 4-methylcatechol Caffeic acid Ferulic acid
10.17 ± 0.12 2.59 ± 0.23 3.70 ± 0.15 4.48 ± 0.09 12.73 ± 0.31
6,167 ± 105 4,151 ± 75 5,427 ± 89 3,967 ± 48 2,747 ± 117
Vmax/Km (mol/ mmol · min) 606 1602 1467 885 216
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Acknowledgements The authors are grateful to the National Natural Science Foundation of China for funding (serial number 31071564).
References 1
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Bucheli, C.S.; Robinson, S.P. (1994): Contribution of Enzymic Browning to Color in Sugarcane Juice. Journal of Agricultural and Food Chemistry 42, 257–261 Spagna, G., et al. (2005): Characterization of a Tomato Polyphenol Oxidase and Its Role in Browning and Lycopene Content. Journal of Agricultural and Food Chemistry 53, 2032–2038 Fraignier, M.-P., et al. (1995): Biochemical and Immunochemical Characteristics of Polyphenol Oxidases from Different Fruits of Prunus. Journal of Agricultural and Food Chemistry 43, 2375–2380 Coombs, J.; Baldry, C.W. (1978): Formation of color in cane juice by enzyme-catalyzed reactions. Part I. Mechanism and chemical inhibition. Int. Sugar J. 80, 291–294 Goodacre, B.C.; Coombs, J. (1978): Formation of color in cane juice by enzyme-catalyzed reactions. Part II. Distribution of enzyme and color precursors. Int. Sugar J. 80, 323–327 Goodacre, B.C.; Hutson, J.; Coombs, J. (1980): Enzyme catalyzed formation of color in cane juice. Part III. Inactivation of o-diphenol oxidase by heating intact cane. Int. Sugar J. 82, 11–14 Paton, N.H. (1992): Sugar cane phenolics and first expressed juice color. Part II. Concentration of phenolics in sugar cane and color of first expressed juice. Int. Sugar J. 94, 157–160 Paton, N.H.; Duong, M. (1992): Sugar cane phenolics and first expressed juice color. Part III. Role of chlorogenic acid and flavonoids in enzymic browning of cane juice. Int. Sugar J. 94, 170–176 Qudsieh, H.Y.M., et al. (2002): Effect of Maturity on Chlorophyll, Tannin, Color, and Polyphenol Oxidase (PPO) Activity of Sugarcane Juice (Saccharum officinarum Var. Yellow Cane). Journal of Agricultural and Food Chemistry 50, 1615–1618 Bradford, M.M. (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254 Zhao, Z.G., et al. (2008): Simultaneous determination of ten major phenolic acids in sugarcane by a reversed phase HPLC method. Sugar Industry 133, 503–507 Zhu, L.C., et al. (2010): Isolation and purification of flavonoid glycosides from sugarcane (Saccharum officinarum L.) juice by high-speed countercurrent chromatography. Int. Sugar J. 112, 24–29 Zhao, Z.G., et al. (2009):Optimization of extraction of phenolic compounds from scum using response surface methodology. Int. Sugar J. 111, 13–19 Colombo, R.; Lancas, F.M.; Yariwake, J.H. (2006): Determination of flavonoids in cultivated sugarcane leaves, bagasse, juice and in transgenic sugarcane by liquid chromatography-UV detection. Journal of Chromatography A 1103, 118–124
15 Broothaerts, W., et al. (2000): Fast apple (Malus × domestica) and tobacco (Nicotiana tobacum) leaf polyphenol oxidase activity assay for screening transgenic plants. Journal of Agricultural and Food Chemistry 48, 5924– 5928 16 Feng, W., et al. (2007): Regeneration of denatured polyphenol oxidase in Toona sinensis (A. Juss.) Roam. Process Biochemistry 42, 1155–1159 17 Kumar, V.B.A.; Mohan, T.C.K.; Murugan, K. (2008): Purification and kinetic characterization of polyphenol oxidase from Barbados cherry (Malpighia glabra L.). Food Chemistry 110, 328–333 18 Chaisakdanugull, C.; Theerakulkait, C. (2009): Partial purification and characterisation of banana [Musa (AAA Group) ‘Gros Michel’] polyphenol oxidase. Int. J. of Food Science and Techn. 44, 840–846 19 Zamorano, J.-P., et al. (2009): Characterisation and tissue distribution of polyphenol oxidase of deepwater pink shrimp (Parapenaeus longirostris). Food Chemistry 112, 104–111 20 Perez-Gilabert, M.; Garcia Carmona, F. (2000): Characterization of catecholase and cresolase activities of eggplant polyphenol oxidase. Journal of Agricultural and Food Chemistry 48, 695–700 21 Gonzalez, E.M.; Ancos, B. de; Cano, M.P. (1999): Partial characterization of polyphenol oxidase activity in raspberry fruits. Journal of Agricultural and Food Chemistry 47, 4068–4072 22 Yang, Y.; Wang, Z. (2008): Some properties of polyphenol oxidase from lily. Int. J. of Food Science and Techn. 43, 102–107 23 Fortea, M.I., et al. (2009): Kinetic characterisation and thermal inactivation study of polyphenol oxidase and peroxidase from table grape (Crimson Seedless). Food Chemistry 113, 1008–1014 24 Yang, C.P., et al. (2000): Purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) pulp. Journal of Agricultural and Food Chemistry 48, 2732–2735 25 Aydemir, T. (2004): Partial purification and characterization of polyphenol oxidase from artichoke (Cynara scolymus L.) heads. Food Chemistry 87, 59–67 26 Aydemir, T.; Akkanlı, G. (2006): Partial purification and characterisation of polyphenol oxidase from celery root (Apium graveolens L.) and the investigation of the effects on the enzyme activity of some inhibitors. Int. J. of Food Science and Techn. 41, 1090–1098 27 Bursakov, S.A., et al. (1997): Enzymatic properties and effect of ionic strength on periplasmic nitrate reductase (NAP) from Desulfovibrio desulfuricans ATCC 27774. Biochemical and Biophysical Research Communications 239, 816–822 28 Ümit Ünal, M. (2007): Properties of polyphenol oxidase from Anamur banana (Musa cavendishii). Food Chemistry 100, 909–913 29 Perez-Gilabert, M., et al. (2001): Partial purification, characterization, and histochemical localization of fully latent desert truffle (Terfezia claveryi Chatin) polyphenol oxidase. Journal of Agricultural and Food Chemistry 49, 1922–1927 30 Nkya, E., et al. (2003): Purification and characterization of polyphenol oxidase from garland chrysanthemum (Chrysanthemum coronarium L.). Journal of Agricultural and Food Chemistry 51, 5467–5471 31 Wuyts, N.; De Waele, D.; Swennen, R. (2006): Extraction and partial characterization of polyphenol oxidase from banana (Musa acuminata Grande naine) roots. Plant Physiology and Biochemistry 44, 308–314 32 Rojas-Graü, M.A.; Soliva-Fortuny, R.; Martín-Belloso, O. (2008): Effect of Natural Antibrowning Agents on Color and Related Enzymes in FreshCut Fuji Apples as an Alternative to the Use of Ascorbic Acid. Journal of Food Science 73, S267–S272 33 Dog˘an, M.; Arslan, O.; Dog˘an, S. (2002): Substrate specificity, heat inactivation and inhibition of polyphenol oxidase from different aubergine cultivars. Int. J. of Food Science and Techn. 37, 415–423 34 Wititsuwannakul, D., et al. (2002): Polyphenol oxidases from latex of Hevea brasiliensis: purification and characterization. Phytochemistry 61, 115–121
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Technology/Technologie Purification partielle et caractérisation de la polyphénoloxydase de la canne à sucre (Saccharum officinarum L.) (Résumé) La polyphénol oxydase (PPO) de la canne à sucre a été extraite en utilisant 0.02 mol/L de tampon phosphate à pH 6.8 contenant 1.5 % de polyvinylpyrrolidone et 0.5 % de Triton X-100 et ensuite purifiée par fractionnement au sulfate d’ammonium 80 %, dialyse et chromatographie sur colonne de DEAEpearl 650M, Sephadex G-100. L’activité de la PPO a été purifiée 37.6 fois avec un rendement de 18.4 %. La PPO a montré une activité vis-à-vis du catéchol, de l’acide chlorogénique, du 4-méthylcatéchol, de l’acide caféique et de l’acide férulique, mais pas vis-à-vis de la l-tyyrosine. Les conditions optimales (valeur du pH et température) de la PPO ont été déterminées à l’aide des cinq substances. L’activité de la PPO est très thermostable entre 20 et 30 °C. Après chauffage pendant 10 min à 80 °C , 90 % de l’activité est perdue. Les valeurs de Km et de Vmax de la PPO ont été calculées pour chaque substrat, le meilleur substrat de la PPO étant l’acide chlorogénique. La PPO a été sensiblement inhibée par les ions métalliques (Cu 2+, Al3+ et Mg2+) à 1 et 10 mmole/L et fortement inhibée par NaHSO3 à 1 mmol/L.
Purificación parcial y caracterización de polifenoloxidasa de caña de azúcar (Saccharum officinarum L.) (Resumen) Se extrajo la polifenoloxidasa de caña de azúcar con 0,02 mol/L tampón fosfato (pH = 6,8) junto con 1,5 % polivenilpolipirrolidona y 0,5 % Triton X-100. La purificación parcial
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se llevó a cabo primero por un fraccionamiento con sulfato de amonio (80 %) y luego por cromatografía sobre DEAE-Toyopearl 650M y Sephadex G-100. Se purificó la polifenoloxidasa 37,6 veces obteniéndose un rendimiento de 18,4 %. La polifenoloxidasa mostró una actividad frente a catequina, ácido clorogénico, 4-metilcatequina, ácido cafeico y ácido ferúlico, pero no frente a L-tirosina. Se determinaron los valores óptimos del pH y de la temperatura de los cinco sustratos. La actividad de la polifenoloxidasa es termoestable entre 20 y 30 °C. Después del tratamiento térmico a 80 °C durante 10 min se pierde el 90 % de la actividad. Para cada sustrato se determinaron la constante de Michaelis Km y la velocidad máxima de rendimiento Vmax – el ácido clorogénico resultó ser el sustrato más apropiado. Iones de metal (Cu2+, Al3+ y Mg2+) en concentraciones de 1 a 10 mmol/L inhiben claramente la actividad de polifenoloxidasa; NaHSO3 y ácido ascórbico en una concentración de 1 mmol/L, en cambio, inhiben fuertemente la actividad de polifenoloxidasa.
Paper received on 24 January 2011 Authors’ addresses: Zhengang Zhao, Licai Zhu, Prof. Shujuan Yu*, College of Food Science and Technology, South China University of Technology, 381 Wushan Road, Guangzhou, 510640, P.R. China; Michael Saska, Audubon Sugar Institute, Louisiana State University Agricultural Center, St. Gabriel, LA, 70776,USA; * corresponding author: e-mail:
[email protected]
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