Saudi Journal of Biological Sciences (2011) 18, 293–298
King Saud University
Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
Purification and characterization of membrane-bound peroxidase from date palm leaves (Phoenix dactylifera L.) Abdurrahman M. Al-Senaidy *, Mohammad A. Ismael Biochemistry Department, College of Science, King Saud University, P.O. Box 2454, Riyadh, Saudi Arabia Received 16 March 2011; revised 26 April 2011; accepted 27 April 2011
KEYWORDS Date palm leaves; Peroxidase; Purification; Characterization; Thermostability; Substrate specificity
Abstract Peroxidase from date palm (Phoenix dactylifera L.) leaves was purified to homogeneity and characterized biochemically. The enzyme purification included homogenization, extraction of pigments followed by consecutive chromatographies on DEAE-Sepharose and Superdex 200. The purification factor for purified date palm peroxidase was 17 with 5.8% yield. The purity was checked by SDS and native PAGE, which showed a single prominent band. The molecular weight of the enzyme was approximately 55 kDa as estimated by SDS–PAGE. The enzyme was characterized for thermal and pH stability, and kinetic parameters were determined using guaiacol as substrate. The optimum activity was between pH 5–6. The enzyme showed maximum activity at 55 C and was fairly stable up to 75 C, with 42% loss of activity. Date palm leaves peroxidase showed Km values of 0.77 and 0.045 mM for guaiacol and H2O2, respectively. These properties suggest that this enzyme could be a promising tool for applications in different analytical determinations as well as for treatment of industrial effluents at low cost. ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction
* Corresponding author. E-mail addresses:
[email protected] [email protected] (M.A. Ismael).
(A.M.
Al-Senaidy),
1319-562X ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of King Saud University. doi:10.1016/j.sjbs.2011.04.005
Production and hosting by Elsevier
Plant peroxidases (EC 1.11.1.7) are hemoproteins that catalyze the H2O2 dependent oxidation of a wide variety of substrates including phenolic compounds. Peroxidases are ubiquitous in nature and have been implicated in broad range of physiological functions in plants. Biosynthesis and degradation of lignin in cell walls, defense against pathogens and environmental and metal stress response are some of the proposed functions. However, the specific function of plant peroxidases is still unclear (Hiraga et al., 2001; Passardi et al., 2007; Cosio and Dunand, 2009). Peroxidases have been classified into three families, on the basis of amino acid homology and metal-binding capabilities. Class I, includes prokaryotic and plant intracellular enzymes from mitochondria and chloroplasts, such as ascorbate peroxidase and cytochrome c peroxidase; class II comprises
294 extracellular fungal peroxidases, such as manganese peroxidase and lignin-degrading peroxidase; and class III consists of secreted higher plant peroxidases (Passardi et al., 2005). Class III peroxidases both soluble and membrane-bound plant peroxidases are single-chain proteins that are often highly glycosylated. They exist in multiple forms of isoenzymes, with differences in function, molecular mass, heat stability, pH optimum, and substrate specificity (Sergio et al., 2007). Class III peroxidases from plant tissues were able to oxidize a wide range of phenolic compounds, such as guaiacol, pyrogallol, chlorogenic acid, catechin and catechol (Passardi et al., 2005). Because of broader catalytic activity, peroxidase from different sources can be used in wide range of clinical, food processing and industrial applications (Torres et al., 1997). Horseradish peroxidase (HRP) has been used as an important component of reagents for clinical diagnosis, laboratory experiments such as in ‘ELISA’ enzyme immunoassay kits, in removal of phenolics from industrial wastes and removal of peroxides from foodstuffs (Regalodo et al., 2004). Peroxidase has been detected, isolated, sequenced and characterized from a number of organisms including bacteria, fungi and higher plants (Passardi et al., 2007; Welinder, 1992). The structure, substrate specificity, and kinetic properties of various plant peroxidases are well known, particularly those from horseradish (Armoracia sp.). Date palm is widely grown in Saudi Arabia. Several publications have addressed the quantitative and qualitative aspects of date palm peroxidases (Baaziz et al., 1994; Cosio and Dunand, 2009). Baaziz (1989) reported that date palm leaves contained highly active soluble and ionically wall-bound peroxidases. The present investigation reports the isolation, purification and biochemical characterization of peroxidase from date palm leaves. 2. Materials and methods 2.1. Enzyme extraction Fresh mature leaflets from date palm (Phoenix dactylifera L.) were collected from King Saud University campus garden. After limb and spines removal, leaflets were washed thoroughly in distilled water and cut into small pieces. Leaflet pieces were frozen in liquid nitrogen and ground to a fine powder using a prechilled grinder for 2–4 min. Ground tissues were stored at 70 C until used. Fifty grams of ground tissue was suspended in chilled acetone ( 20 C), mixed thoroughly for 10 min and filtered. Extraction of the residue continued until the filtrate was free from pigments. Residue obtained after the filtration of acetone was kept to dryness for 30 min at room temperature. The dried residue was homogenized in a warring blender in 100 ml cold 100 mM sodium phosphate buffer, pH 6.0 containing 1% polyvinylpyrrolidone (PVP). The extract obtained was filtered through 4 layers of cheesecloth and centrifuged at 15,000g at 4 C for 20 min. The pellet and the solid material on the cheesecloth were suspended in 100 ml of 100 mM sodium phosphate pH 6.0, 100 mM sodium chloride and 0.1 mM PMSF. The suspension was then probe sonicated for 5 min and centrifuged at 15,000g for 10 min. The supernatant was concentrated by ultrafiltration through an Amicon filter with 10 kDa cut off and the retentate was dialyzed twice against 4 liters of 10 mM Tris HCl buffer (pH 8.0).
A.M. Al-Senaidy, M.A. Ismael 2.2. Protein purification The enzyme was purified by a combination of anion exchange and size exclusion chromatography using an FPLC-System (AKTA-purifier GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The extract was applied on a DEAE–Sepharose column (2.6 cm · 10 cm) equilibrated in 10 mM Tris–HCl, pH 8.0 at a flow rate of 1 ml/min. The column was washed with five bed volumes of equilibration buffer. Proteins were eluted with a linear gradient of 0–0.5 M NaCl in equilibration buffer and 2 ml fractions were collected. Fractions showing peroxidase activity were pooled and concentrated by ultrafiltration as mentioned above. Concentrated enzyme solution was loaded onto 1.6/60 gel filtration column (Superdex 200) equilibrated with 100 mM sodium phosphate buffer, pH 6.0 and eluted at 0.5 ml/min in the same buffer. The fractions showing activity were pooled and used as enzyme source for further studies. 2.3. Protein determination Quantitative protein determination was achieved according to the Bradford (1976) method, by measuring the optical density at 595 nm, with bovine serum albumin as standard. 2.4. Peroxidase activity assay Peroxidase activity was carried out spectrophotometrically using guaiacol as substrate (Lobarzewski et al., 1990). The increase in the absorption as a result of the formation of the oxidized product (tetraguaiacol) was measured at 470 nm. Reaction mixture contained 100 mM phosphate buffer (pH 6.0), 5 mM guaiacol, and 0.5 mM H2O2 at 25 C. The changes in absorbance were read for 3 min using a UV–vis spectrophotometer (Amersham Pharmacia, Biotech 2000). Substrate specificity and classification of date palm peroxidase enzyme was determined using different substrates with similar reaction mixture and assay conditions. All the substrates and H2O2 were at a fixed concentration of 0.5 mM. The rate of oxidation of o-dianisidine, was followed at 445 nm, (e445 = 30 mM 1 cm 1), guaiacol and catechol at 470 nm, (e470 = 26.6 mM 1 cm 1), ascorbic acid at 290 nm (e290 = 2.8 mM 1 cm 1) and pyrogallol at 430 nm (e430 = 2.47 mM 1 cm 1). One unit of enzymatic activity was defined as amount of enzyme that oxidizes 1 lmol/min of hydrogen donors under assay conditions. 2.5. Activity staining and SDS–polyacrylamide gel electrophoresis (SDS–PAGE) Activity staining of peroxidase was performed according to Laemmli (1970), on 8% nondenaturing polyacrylamide gel. Loading buffer without SDS and thiol reducing agent was used. A constant power supply of 10 mA was employed. After the run, peroxidase bands were detected by immersing the gel in a solution of 100 mM sodium phosphate buffer, pH 6.0 containing 1% guaiacol and 0.3% H2O2. Color development occurred within 10 min. Enzyme purity and molecular weight were analyzed by SDS–PAGE in a Mini Protean III Electrophoresis Cell (BioRad), with 12% resolving and 4% stacking gel. Proteins were
Purification and characterization of membrane-bound peroxidase from date palm leaves (Phoenix dactylifera L.) stained using the Coomassie Blue staining technique and molecular weight was estimated by comparison to molecular weight markers (Amersham Biosciences). 2.6. Effect of pH The influence of pH on date palm peroxidase activity was determined in the presence of buffers of wide pH range (pH 4–9) at a concentration of 100 mM. The following buffers were used: Na-acetate buffer (pH 4–5.5); K-phosphate buffer (pH 6–7.5); and Tris–HCl buffer (pH 8–9), respectively. The pH stability was investigated by measuring the remaining activity of the enzyme after it had been kept for 60 min in various pH conditions, and then the residual activities were tested under standard conditions. 2.7. Effect of temperature The effect of temperature on enzymatic activity was determined at pH 6.0 by incubating the reaction mixture at various temperatures (30–80 C) in a thermostatic water bath. The thermal stability was estimated by incubating the enzyme in the range of 50–80 C for 60 min prior to assay, the remaining activity was assayed at room temperature after brief cooling on ice. Residual activity was calculated as percent of the original activity in the unheated preparation. Table 1
295
2.8. Steady-state kinetics Michaelis–Menten constants were determined by measuring the initial rates of guaiacol oxidation at 25 C at different H2O2 (0.01–3 mM) and guaiacol (0.1–6.0 mM) concentrations. The apparent Km values were determined from Lineweaver– Burk plots at optimum pH and temperature conditions. 2.9. Effect of metal ions The effect of various metal ions on the enzyme activity was tested by addition of 10 mM chloride salts of Na+ and K+ for monovalent, Cu2+, Mg2+, Ca2+, Mn2+, Zn2+, Cd2+ for divalent and Al3+ for trivalent ions. The activity of the enzyme was determined after 20 min incubation under standard assay conditions in the presence and absence of the metal ion. Residual activity was calculated taking activity of control as 100%. 3. Results and discussion Peroxidase was purified from date palm leaves according to the protocol summarized in Table 1. Date palm leaves contain a high concentration of polyphenols, which cause browning of the crude extract during peroxidase extraction. Browning
Summary of the purification of date palm peroxidase.
Step
Total protein (mg)
Total activity (units)
Specific activity (U/mg)
Purification (fold)
Yield (%)
Crude extract Concentration DEAESepharose Superdex 200 GF
381 267 14.4 1.3
20,325 19,476 2683 1178
53.3 72.9 186.3 906.2
1.0 1.4 3.5 17
100 96 13.2 5.8
Figure 1 Chromatographic patterns observed during purification of date palm peroxidase. Elution profile obtained from ion exchange chromatography on DEAE-Sepharose CL-6B.
296
A.M. Al-Senaidy, M.A. Ismael
was avoided by acetone extraction of polyphenols and by treatment of the extract with 0.1% insoluble PVP. The purification protocol included concentration by ultrafiltration, anion exchange on DEAE-Sepharose, followed by a final purification on Superdex 200 gel filtration chromatography to produce a homogeneous enzyme. A typical elution profile of date palm leaves purification scheme is shown in Figs. 1 and 2. The scheme utilized resulted in almost 17-fold increase in specific activity when compared to crude extract with a recovery of 5.8% (Table 1). The purity of the enzyme was analyzed by SDS–PAGE after the final chromatographic step and a single band with a molecular weight of 55 kDa relative to standard molecular weight was observed on the gel (Fig. 3A). This molecular weight is in the range of peroxidases from various sources including horseradish, oil palm leaf (Sakharov et al., 2000), pepper fruit (Pomar et al., 1997), and artichoke leaves (Cardinali et al., 2007). The above-mentioned and peroxidase isoenzymes from various sources have different molecular weights ranging from 30,000 to 60,000 Dalton (Robinson, 1991). The activity staining of the enzyme, with guaiacol as substrate, showed a single band corresponding to the position of the peroxidase activity (Fig. 3B). 3.1. Optimum pH and temperature The purified date palm peroxidase, assayed at different pH ranging from pH 4 to 9, exhibited higher activity over a broad pH range (pH 5–8), with maximum activity around pH 5.5 (Fig. 4A). Other studies have reported similar results where, most peroxidases from different sources display optimum activity in the pH range between 4.5 and 6.5 (Leon et al., 2002; Deepa and Arumughan, 2002; Sakharov et al., 2001). Date palm peroxidase was stable in the pH range of 4.0–8.0 (Fig. 4A). The enzyme lost almost 80% of its activity at pH lower than pH 3.0 and higher than pH 9.0. The pH affects ionization state of side chain of enzyme amino acids. Loss of activity may be due to instability of the heme binding to the enzyme at low pH. Also at high pH, loss of activity may result from protein denaturation or ionic changes in the heme group (Adams, 1997). Date palm peroxidase was assayed at optimum pH in different temperatures, ranging between 25 and 80 C for 5 min. The enzyme remained active up to 80 C with maximum activity at
Figure 3 (A) SDS–PAGE (12%) of the purified peroxidase. Lane 1, low molecular weight protein markers along with their corresponding molecular masses. Lane 2, shows purified date palm peroxidase. (B) Native PAGE profile of date palm peroxidase localized using guaiacol.
55 C (Fig. 4B). Studies have reported a wide range of optimum temperature variability for peroxidase from different sources. Buckwheat seed peroxidase showed optimum temperature of 10–30 C (Suzuki et al., 2006) whereas, turnip and green asparagus, peroxidases have temperatures optima at 35 and 70 C, respectively (Motamed et al., 2009; Rodrigo et al., 1996). Thermostability of the date palm peroxidase was investigated by incubation of the enzyme at different temperatures for 60 min. The thermal stability in the form of residual enzyme activity was presented in Fig. 4B. The enzyme almost retained its original activity at 60–70 C for the whole incubation period. Inactivation increases with increasing temperature and time. However, the residual activity of date palm peroxidase was less than 15% at 80 C after 60 min incubation time. Results from both thermal activity and thermal stability profiles, indicated that date palm peroxidase is a heat resistant enzyme (Alokail and Ismael, 2005). Thermal stability of peroxidases is attributed to the presence of carbohydrate moiety in their structure. Prosthetic groups binding play an important role in peroxidase thermostability (Tigier et al., 1991). Inactivation of peroxidase at higher temperatures is due to the unfolding of the tertiary structure. It can be speculated that peroxidase enzyme preparation from date palm leaves can be used at higher temperatures ranging from 60 to 70 C in industrial and biotechnological applications. 3.2. Effect of metal ions
Figure 2 Gel filtration profile on Superdex 200 HR (1.6/60) of DEAE-Sepharose fraction.
The effect of metal ions on date palm peroxidase activity is presented in Table 2. The enzyme activity was enhanced by Ca2+, Mg2+, Mn2+, Zn2+ and Fe2+ ions with a residual activity of 121%, 114%, 116%, 107% and 133%, respectively. On the other hand, Cd2+ ions slightly inhibited the enzyme
Purification and characterization of membrane-bound peroxidase from date palm leaves (Phoenix dactylifera L.)
A
Table 3 Substrate specificity of the purified date palm peroxidase.
100
Relative activity (%)
297
80
pH optima pH stability
60 40
Substrate
Concentration (mM)
Relative activity (%)
Guaiacol o-Dianisidine Pyrogallol Catechol Ascorbic acid
0.5 0.5 0.5 0.5 0.5
100 323 65 23 n.d.*
*
20 0 3
4
5
6
7
8
n.d.: not detected.
A
9
60
pH
80 60
0.20
40
20
Temperature optima (°C) Temperature stability (%)
40
0.12 0.08 0.04
0
20
0
0 3 6 9 1/[Guaiacol] (mM)
2
4
12
6
[Guaiacol] (mM)
30
40
50
60
70
80
90
Temperature (°C)
B
Figure 4 (A) pH optima and pH stability of peroxidase from date palm leaves. (B) Temperature optima and temperature stability of peroxidase from date palm leaves. Effect of metal ions on date palm peroxidase activity.
Metal ion (10 mM)
Relative activity (%)
Control MgCl2 MnCl2 CaCl2 ZnCl2 CdCl2 FeCl3 CuCl2 AlCl3 NaCl KCl
100 ± 2.4 114 ± 5.2 116 ± 4.4 121 ± 2.6 107 ± 3.1 74 ± 5.6 133 ± 4.3 102 ± 2.3 98 ± 4.3 102 ± 2.6 104 ± 2.2
activity resulting in relative activity of 74%, whereas, no change in date palm leaves peroxidase activity was noticed with metals ions of Al3+, Cu2+ and monovalent ions Na+ and K+. Pandey and Dwivedi (2011) reported that divalent cations activated peroxidase up to 50 mM, while higher concentrations inhibited the enzyme in a concentration dependent manner.
v (µmol/minlmg)10 3
0 20
Table 2
0.16
-3
60
0.10
40
1/V (µmol/minlmg)
Activity (%)
100
1/V(µmol/minlmg)
v (µmol/min/mg)103
B 120
20
-40
-20
0 0
1
0.08 0.06 0.04 0.02 0 20 40 1/[H2O2] (mM) 2
60
3
4
[H2O2] (mM)
Figure 5 Substrate saturation curve and Lineweaver–Burk plot of date palm peroxidase activity in presence of different concentrations of (A) guaiacol, and (B) with H2O2 as fixed substrate. The Lineweaver–Burk plot (insets) was generated by plotting 1/velocity vs. 1/substrate.
enzyme exhibited a distinct preference for o-dianisidine, guaiacol, and pyrogallol as electron donors under similar conditions. On the other hand, date palm leaves showed no detectable activity toward ascorbic acid; suggesting that this enzyme is not ascorbate peroxidase (class I), but related to class III peroxidases.
3.3. Substrate specificity 3.4. Kinetic studies The activity of purified date palm leaves peroxidase toward selected hydrogen donors in the presence of H2O2 was compared with that of guaiacol at fixed concentrations (Table 3). The
The purified date palm peroxidase showed typical Michaelis– Menten kinetics for both guaiacol and H2O2. The substrate
298 saturation curve was obtained by interpolating the substrate concentrations against activity values. Michaelis–Menten constants (Km) were determined from Lineweaver–Burk plots. The enzyme had Km values of 0.77 and 0.045 mM for guaiacol (Fig. 5A) and H2O2 (Fig. 5B) substrates, respectively. The guaiacol Km value appears to be lower than those reported for the peroxidase from palm leaf (Deepa and Arumughan, 2002), green peas (Halpin et al., 1989) and turnip roots (Duarte-Vazquez et al., 2001). Low Km values suggest that the enzyme has a high apparent affinity toward guaiacol and H2O2 compared to other peroxidases previously reported. In conclusion, the properties of date palm peroxidase, such as high activity and good stability over a wide pH range, excellent thermostability, wide substrate specificity, and stability in the presence of high ionic metals concentration, demonstrate that date palm leaves peroxidase has good potential for industrial and medicinal applications. Therefore, we plan to clone and sequence the structural gene coding in this enzyme in order to develop a method for high level production of enzyme. References Adams, J.B., 1997. Regeneration and kinetics of peroxidase inactivation. Food Chem. 60, 201–206. Alokail, MS., Ismael, A., 2005. Thermostable characteristics of peroxidase from leaves of Arabian palm date (Phoenix dactylifera L.). Saudi J. Biol. Sci. 12, 25–32. Baaziz, M., 1989. The activity and preliminary characterization of peroxidases in leaves of thirteen cultivars of date palm (Phoenix dactylifera L.). Netc. Phytol. 111, 403–411. Baaziz, M., Aissam, F., Brakez, Z., Bendiab, K., El Hadrami, I., Cheikh, R., 1994. Electrophoretic patterns of acid soluble proteins and active isoforms of peroxidase and polyphenoloxidase typifying calli and somatic embryos of two reputed date palm cultivars in Morocco. Euphytica 76, 159–168. Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of dye binding. Anal. Biochem. 72, 248–254. Cardinali, A., Sergio, L., Di Venere, D., Linsalata, V., Fortunato, D., Conti, A., Lattanzio, V., 2007. Purification and characterization of a cationic peroxidase from artichoke leaves. J. Sci. Food Agric. 87, 1417–1423. Cosio, C., Dunand, C., 2009. Specific functions of individual class III peroxidase genes. J. Exp. Bot. 60, 391–408. Deepa, S.S., Arumughan, C., 2002. Purification and characterization of soluble peroxidase from oil palm (Elaeis quineensis Jacq.) leaf. Phytochemistry 61, 503–511. Duarte-Vazquez, M., Garcia-Almenda´ rez, B., Regalado, C., Whitaker, J., 2001. Purification and properties of neutral peroxidase isozymes from turnip (Brassica napus L. var) Purple top white globe roots. J. Agri. Food Chem. 49, 4450–4456. Halpin, B., Pressey, R., Jen, J., Mondy, N., 1989. Purification and characterization of peroxidase isoenzymes from green peas (Pisum sativum). J. Food Sci. 54, 644–649.
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