Saudi Journal of Biological Sciences (2011) 18, 293298

King Saud University

Saudi Journal of Biological Sciences

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Purication 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;
Purication;
Characterization;
Thermostability;
Substrate specicity

Abstract Peroxidase from date palm (Phoenix dactylifera L.) leaves was puried to homogeneity
and characterized biochemically. The enzyme purication included homogenization, extraction of
pigments followed by consecutive chromatographies on DEAE-Sepharose and Superdex 200. The
purication factor for puried 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 SDSPAGE. The enzyme was characterized for thermal and pH stability, and kinetic parameters were determined using guaiacol as substrate. The optimum activity was between pH 56. 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 efuents at low cost.
2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

* Corresponding author.
E-mail addresses: senaidy@ksu.edu.sa
mismael@ksu.edu.sa (M.A. Ismael).

(A.M.

Al-Senaidy),

1319-562X 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 specic function of plant peroxidases is still unclear
(Hiraga et al., 2001; Passardi et al., 2007; Cosio and Dunand,
2009). Peroxidases have been classied 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 specicity (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 specicity, 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, purication and biochemical characterization of peroxidase from date palm leaves.
2. Materials and methods
2.1. Enzyme extraction
Fresh mature leaets from date palm (Phoenix dactylifera L.)
were collected from King Saud University campus garden.
After limb and spines removal, leaets were washed thoroughly in distilled water and cut into small pieces. Leaet
pieces were frozen in liquid nitrogen and ground to a ne powder using a prechilled grinder for 24 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 ltered.
Extraction of the residue continued until the ltrate was free
from pigments. Residue obtained after the ltration 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 ltered
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 ultraltration through an Amicon lter 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 purication
The enzyme was puried by a combination of anion exchange
and size exclusion chromatography using an FPLC-System
(AKTA-purier GE Healthcare Bio-Sciences AB, Uppsala,
Sweden). The extract was applied on a DEAESepharose column (2.6 cm 10 cm) equilibrated in 10 mM TrisHCl, pH 8.0
at a ow rate of 1 ml/min. The column was washed with ve
bed volumes of equilibration buffer. Proteins were eluted with
a linear gradient of 00.5 M NaCl in equilibration buffer and
2 ml fractions were collected. Fractions showing peroxidase
activity were pooled and concentrated by ultraltration as
mentioned above. Concentrated enzyme solution was loaded
onto 1.6/60 gel ltration 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 UVvis spectrophotometer (Amersham Pharmacia, Biotech 2000). Substrate specicity and classication 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 xed 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 dened as amount of enzyme that
oxidizes 1 lmol/min of hydrogen donors under assay
conditions.
2.5. Activity staining and SDSpolyacrylamide gel
electrophoresis (SDSPAGE)
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
SDSPAGE in a Mini Protean III Electrophoresis Cell (BioRad), with 12% resolving and 4% stacking gel. Proteins were

Purication 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 inuence of pH on date palm peroxidase activity was
determined in the presence of buffers of wide pH range (pH
49) at a concentration of 100 mM. The following buffers were
used: Na-acetate buffer (pH 45.5); K-phosphate buffer (pH
67.5); and TrisHCl buffer (pH 89), 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 (3080 C) in a thermostatic water bath. The
thermal stability was estimated by incubating the enzyme in
the range of 5080 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

MichaelisMenten constants were determined by measuring
the initial rates of guaiacol oxidation at 25 C at different
H2O2 (0.013 mM) and guaiacol (0.16.0 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 puried 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 purication of date palm peroxidase.

Step

Total protein (mg)

Total activity (units)

Specic activity (U/mg)

Purication (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 purication of date palm peroxidase. Elution prole 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 purication protocol included concentration by ultraltration, anion exchange on DEAE-Sepharose, followed by a nal
purication on Superdex 200 gel ltration chromatography
to produce a homogeneous enzyme. A typical elution prole
of date palm leaves purication scheme is shown in Figs. 1
and 2. The scheme utilized resulted in almost 17-fold increase
in specic activity when compared to crude extract with a
recovery of 5.8% (Table 1). The purity of the enzyme was analyzed by SDSPAGE after the nal 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 puried date palm peroxidase, assayed at different pH ranging from pH 4 to 9, exhibited higher activity over a broad pH
range (pH 58), 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 4.08.0 (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) SDSPAGE (12%) of the puried peroxidase.

Lane 1, low molecular weight protein markers along with their
corresponding molecular masses. Lane 2, shows puried date palm
peroxidase. (B) Native PAGE prole 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
1030 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 6070 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 proles, 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 ltration prole 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

Purication and characterization of membrane-bound peroxidase from date palm leaves (Phoenix dactylifera L.)

Table 3 Substrate specicity of the puried 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

n.d.: not detected.

60

pH

80
60

0.20

40

20

Temperature optima (C)

Temperature stability (%)

40

0.12
0.08
0.04

20

0
3
6
9
1/[Guaiacol] (mM)

12

[Guaiacol] (mM)

30

40

50

60

70

80

90

Temperature (C)

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 (mol/minlmg)10 3

0
20

Table 2

0.16

-3

60

0.10

40

1/V (mol/minlmg)

Activity (%)

100

1/V(mol/minlmg)

v (mol/min/mg)103

B 120

20

-40

-20

0
0

0.08
0.06
0.04
0.02
0
20
40
1/[H2O2] (mM)
2

60

[H2O2] (mM)

Figure 5 Substrate saturation curve and LineweaverBurk plot

of date palm peroxidase activity in presence of different concentrations of (A) guaiacol, and (B) with H2O2 as xed substrate. The
LineweaverBurk 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 specicity

3.4. Kinetic studies
The activity of puried date palm leaves peroxidase toward selected hydrogen donors in the presence of H2O2 was compared
with that of guaiacol at xed concentrations (Table 3). The

The puried 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. MichaelisMenten
constants (Km) were determined from LineweaverBurk
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 afnity 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 specicity,
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.
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