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Microbiol. Res.

(2003) 158, 237242


http://www.urbanfischer.de/journals/microbiolres

Purification and characterization of an aflatoxin degradation


enzyme from Pleurotus ostreatus
Marisa Motomura1, Tetsuo Toyomasu2, Keiko Mizuno1, Takao Shinozawa1,*
1
2

Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Gunma 3768515, Japan
The Mushroom Research Institute of Japan, Kiryu, Gunma 376-0051, Japan

Accepted: June 10, 2003

Abstract
Nineteen fungi were tested for their ability to degrade aflatoxin
B1 (AFB1). An extracellular enzyme from the edible mushroom
Pleurotus ostreatus showed afaltoxin-degradation activity
detected by thin-layer chromatography (TLC). An enzyme
with this activity was purified by two chromatographies on
DEAE-Sepharose and Phenyl-Sepharose. The apparent molecular mass of the purified enzyme was estimated to be 90 kDa
by SDS-PAGE. Optimum activities were found in the pH range
between 4.0 and 5.0 and at 25C. Also, degradation activity of
several dyes in the presence of H2O2 was tested, resulting in the
detection of bromophenol blue-decolorizing activity. Based on
these data, we suggest this enzyme is a novel enzyme with
aflatoxin-degradation activity. Fluorescence measurements
suggest that the enzyme cleaves the lactone ring of aflatoxin.
Key words: aflatoxin degradation Pleurotus ostreatus
enzyme purification lactone cleavage

Introduction
Aflatoxins are toxic and carcinogenic metabolites
produced by molds, especially Aspergillus parasiticus,
Aspergillus flavus, Aspergillus nomius and Aspergillus
tamarii, commonly found in crops such as corn, cotton,
peanuts and tree nuts (Hesseltine et al. 1966 ; Nesbitt
1962). These toxins are considered to play an important
role in the high incidence of human hepatocellular carcinoma in certain areas of the world (Stern et al. 2001).

Corresponding author: T. Shinozawa


e-mail: shinozawa@bce.gunma-u.ac.jp
0944-5013/03/158/03-237

$15.00/0

Because of the high toxicity to both, humans and


animals, trials to eliminate aflatoxin contamination
from food and feed have been carried out. Many approaches (Galvano et al. 2001) have been reported to
degrade this toxin, however, there are no effective
methods to prevent preharvest contamination, and
decontamination is also ineffective or not economical.
In addition, many reports show the degradation of
aflatoxin by bacteria, yeasts and molds. However, there
is no practical application where biological degradation
is efficiently used to reduce aflatoxins in foods (Doyle
et al. 1982; Galvano et al. 2001).
On the other hand, studies on polycyclic aromatic
hydrocarbons (PAHs), such as dioxines, has attracted
much attention because of their strong toxicity to
humans and animals. In the degradation of these compounds, the effectiveness by two main groups of
microorganisms, soil bacteria and white-rot fungi, has
been revealed. Among these, Pleurotus ostreatus has
been shown to have a high biodegradation activity
(Baldrian et al. 2000).
P. ostreatus has been reported to have the ability, also,
to degrade and decolorize various dyes and aromatic
organic compounds as environmental pollutants important to the dyestuff industries (Novotny et al. 1999,
2001; Vyas and Molitoris 1995). This ability has generally been attributed to the lignin-degrading enzyme
system. The major enzymes in this system are manganese-dependent peroxidase (MnP) and laccase (Vyas and
Molitoris 1995).
In this paper, the screening of extracellular enzymes
for aflatoxin-degradation activity in white-rot and
brown-rot fungi, and the purification of an enzyme from
P. ostreatus possessing this activity are described.
Microbiol. Res. 158 (2003) 3

237

Materials and methods


Fungi and aflatoxin. Nineteen fungi were obtained from
the collection of Mori and Company Research Institute,
Japan (Kiryu, Japan). These strains were maintained on
slant tubes containing 1% glucose, 1% malt extract,
0.4% yeast extract medium (GMY) with 1.5% agar at
4C. Aflatoxin B1 (AFB1) was obtained from Sigma
Chemical Co. (Poole, UK).
Culture conditions. The strains, listed in Table 1, were
cultured at 25 C for 20 25 days in GMY medium with
shaking.
Enzyme assay for aflatoxin-degradation activity. Aflatoxin B1 (final concentration, 5 g/ml) was incubated at
25 C for 1 h in a 500 l assay mixture containing 0.1 M
sodium acetate buffer (pH 5.0) and 445 l of culture
supernatant or purified enzyme fraction. The reaction
was terminated by mixing with 500 l chloroform. The
lower chloroform layer, obtained by centrifugation at
1 500 rpm for 15 min, was recovered and evaporated to
dryness at room temperature. The evaporated residue
was dissolved in 200 l chloroform and spotted on a
silica gel plate (silica gel 60, Merck and Co, Inc., Rahway, N. J.) for thin layer chromatography (TLC). The
components were developed with a mixture of chloroform-ethyl acetate-formic acid (6:3:1, vol/vol/vol), followed by visualization of the fluorescent spots on a UV
transilluminator. The aflatoxin-degradation activity was
also estimated spectrophotometrically in the range
between 200 and 400 nm using a UV-visible spectrophotometer (Shimadzu UV-160A, Japan) in the absence
or presence of 0.1 mM H2O2. One unit of enzyme
activity is defined as the amount of enzyme that produces a decrease of 0.01 absorbance units at 363 nm per
minute.
Purification of the enzyme from the culture supernatant
of P. ostreatus. One liter of culture supernatant was
supplemented with solid ammonium sulfate to 80%
saturation under constant stirring. The solution was
centrifuged at 20000 g for 30 min and the precipitates
were dissolved in 50 mM sodium acetate buffer (pH 5.0)
followed by overnight dialysis against buffer A (50 mM
sodium acetate, pH 5.0, supplemented with 0.1 mM
phenylmethanesulfonyl fluoride (PMSF)). The dialyzed
sample was applied to a DEAE-Sepharose column
(Pharmacia Biotech, Uppsala, Sweden) pre-equilibrated
with buffer A. After washing with buffer A, the proteins
were eluted with buffer A containing 0.1 M NaCl. The
eluted fraction was supplemented with ammonium
sulfate to a final concentration of 1.0 M, and then applied to a Phenyl-Sepharose column (Pharmacia)
pre-equilibrated with buffer A containing 1.0 M ammonium sulfate. Proteins were eluted with a decreasing
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Microbiol. Res. 158 (2003) 3

linear gradient (1.0 to 0 M) of ammonium sulfate. After


dialysis against buffer A, the aflatoxin-degradation
activity of each fraction was tested. Protein concentrations were determined according to the Bradford
method (Bradford 1976) using bovine serum albumin
as a standard.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was performed in
12.5% polyacrylamide gels according to the method of
Laemmli (1970). The separated proteins were stained
with Coomassie Brilliant Blue R-250 (Fluka, Switzerland), and their molecular weights were determined by
comparison with low range molecular weight markers
(Pharmacia Biotech, Uppsala, Sweden).
Effect of pH and temperature on enzyme activity.
The optimum temperature for enzyme activity was
determined in the range of 20 to 45C at 5C intervals.
The pH optimum in the range of 4.0 to 10.0 was determined using 50mM sodium acetate, sodium phosphate
or glycine sodium hydroxide buffers at pH 4.0 to 6.0,
6.0 to 8.0, or 8.0 to 10.0, respectively.
Dye decolorizing activity. Bromophenol Blue (Wako
Pure Chemicals, Osaka, Japan), Congo Red (Wako),
Methylene Blue (Wako), Nile Blue (Sigma) and Victoria
Blue (Wako) were used as substrates. Decolorizing
activity was assayed at room temperature in a 500 l
assay mixture of 0.1 M sodium acetate buffer (pH 5.0),
50 M of each dye, and 375 l of the purified enzyme
fraction in the absence or presence of 0.1 mM H2O2.
Decolorization was monitored spectrophotometrically
in the range of 400 to 700 nm using a UV-visible
spectrophotometer (Shimadzu UV-160A, Japan). The
activity for each dye was also measured at the following
fixed wavelengths, 591 nm (Bromophenol blue),
488 nm (Congo Red), 668 nm (Methylene Blue),
638 nm (Nile Blue) or 619 nm (Victoria Blue).

Results and discussion


AFB1 was treated with culture supernatants from
19 mushroom strains and the supernatant from Pleurotus ostreatus showed aflatoxin-degradation activity
(Table 1).
Purification of the enzyme was performed by two
chromatographic steps. The protein solution, obtained
by ammonium sulfate precipitation of the culture supernatant followed by dialysis, was loaded on a DEAESepharose column. In the next step, hydrophobic chromatography on Phenyl-Sepharose was performed. In
both steps, each fraction was monitored at 280 nm for
protein concentration, and activities were tested. The
purification from the culture supernatant is summarized

in Table 2. The enzyme was purified 148-fold with a


50% yield. The apparent molecular mass of the purified
enzyme was estimated to be 90 kDa by SDS-PAGE
(Fig. 1).
The effects of pH and temperature on enzyme
activity were studied. The aflatoxin-degradation activities showed pH optima in the range of 4.0 to 5.0 (Fig. 2)
and an optimum temperature of 25C (Fig. 3).
Fig. 4 shows the UV spectrum of the aflatoxindegradation activity by the purified enzyme in the
absence or presence of H2O2. When aflatoxin was treated with the purified enzyme, its absorbance maxima
at 265 and 363 nm decreased, showing the aflatoxindegradation activity. In the presence of H2O2 the activity
was enhanced.
Aflatoxin-degradation activity was also confirmed by
thin layer chromatography (TLC). Small amounts of
aflatoxin are detectable by TLC. Since aflatoxins fluoresce under ultraviolet light, TLC is an easy and highly

Table 1. Screening of extracellular enzymes from mushroom


culture supernatants for aflatoxin-degradation.
Mushroom

Activity

Mushroom

Activity

Armillariella
mellea
Armillariella
(Armillaria)
tabescan
Climacodon
roseomaculatum
Fomitopsis
pinicola
Ganoderma
applanatum
Grifola
frondosa
Hygrocybo
flavesceus
Hypsizigus
marmoreus
Lentinula
edodes
Lentinus
lepideus

Lepista
nude
Philiota
terrestris

Pleurotus
ostreatus
Polyporus
arcularius
Pycnoporus
coccineus
Rigidoporus
lineatus
Sparassis
crispa
Trametes
versicolor
Volvariella
volvacea

parallel method and the development of the components


is fast. When aflatoxin itself was developed, a spot
detected by UV-light appeared at position Rf 0.4
(Fig. 5, lane 1). When aflatoxin was treated with the
culture supernatant, the TLC plates showed a decrease

Fig. 1. SDS-PAGE analysis of proteins during the purification of the aflatoxin-degradation enzyme. Lane 1, culture
supernatant of P. ostreatus (10 g/lane); lane 2, peak fraction
with activity from DEAE-Sepharose chromatography (8 g/
lane); lane 3, peak friction with activity from Phenyl-Sepharose
chromatography (2 g/lane). Molecular size standards (Pharmacia LKB) were phosphorylase b (94 kda), albumin
(67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa).

++

The activity was measured by thin layer chromatography. ()


no activity; (+) weak activity; (++) strong activity

Fig. 2. Effect of pH on the aflatoxin-degradation enzyme


activity. The activity was analyzed at each pH in sodium
acetate buffer (), sodium phosphate buffer () or glycine
sodium hydroxide buffer ().

Table 2. Purification of an aflatoxin-degradation enzyme from Pleurotus ostreatus culture supernatant.


Fraction

Total protein (mg)

Total activity (U)

Sp. act. (U/mg)

Purification (fold)

Yield (%)

Culture Supernatant
NH4(SO4)2precipitate
DEAE-Sepharose
Phenyl-Sepharose

95
67
6
0.32

36
33
22
18

0.38
0.49
3.7
56

1
1.3
9.7
148

100
92
61
50

Microbiol. Res. 158 (2003) 3

239

Fig. 3. Effect of temperature on the aflatoxin-degradation


activity. The activity was analyzed at each temperature in
50 mM sodium acetate buffer (pH 5.0).

in fluorescence intensity of the aflatoxin spot. Furthermore, the fluorescence intensities of aflatoxin spots
treated with enzymes purified by DEAE-Sepharose
or Phenyl-Sepharose were much weaker (Fig 5, lanes 4
and 5).
It has been suggested that the multienzyme isolated
from Armillariella tabescens detoxifies aflatoxin B1 by
opening the difuran ring (Liu et al. 1998). It is known
that this opening does not change the fluorescence spectrum of aflatoxin. However, cleavage of the lactone ring
abolishes or decreases its fluorescence (Lee et al. 1981)
This lactone structure is associated with the carcinogenic activity of the aflatoxin molecule (Bol and Smith
1989). In our analyses by fluorescence spectra measurements (Fig. 4) and also by TLC (Fig. 5), treatment of
aflatoxin with purified enzyme resulted in a decrease
in fluorescence intensity, suggesting the enzymatic cleavage of the lactone ring. Additional understanding can
be obtained by characterizing the enzymatic products of
AFB1. For the large scale production of this enzyme,
cloning and characterization of the gene are in progress.

Fig. 4. Effect of H2O2 on the aflatoxin-degradation activity of the purified enzyme. The activity was analyzed by the absorbance change at 25C. (A) AFB1 as a control (B) enzyme assay of AFB1 without H2O2, (C) enzyme assay of AFB1 with the addition of H2O2. In (B) and (C), 50 g of purified enzyme were used.
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Microbiol. Res. 158 (2003) 3

Fig. 5. TLC analysis of the aflatoxin-degradation activity.


Lane 1, AFB1 only; lane 2, AFB1 treated with assay medium;
lane 3, AFB1 treated with 105 g of P. ostreatus culture supernatant; lane 4, AFB1 treated with 87 g of enzyme purified by
DEAE-Sepharose chromatography; lane 5, AFB1 treated with
50 g of enzyme purified by Phenyl-Sepharose chromatography. The TLC plate was photographed under long-wave UV
light.

In the analysis of dye-degradation by the enzyme, decolorizing activity enhanced by H2O2 for bromophenolblue was detected (Fig. 6). High capacities to degrade
different dyes have been reported for P. ostreatus and
Irpex lacteus (Novotny et al. 2001). Furthermore, an
extracellular peroxidase (MW 71Kda) with H2O2
depedent decolorizing activity for remazol brilliant blue
R (RBBR) has been purified from P. ostreatus (Shin
et al. 1997). For our enzyme, although H2O2 enhances
both its aflatoxin-degradation activity and bromophenol
blue-decolorizing activity, the dependencies of these
activities on H2O2 are not strict (Figs. 4 and 6) and the
molecular mass (MW 90 kda) is different from that of
the above peroxidase. Therefore, our enzyme can be
concluded to be different from that peroxidase.
Pleurotus ostreatus is a non-toxic, edible and very
popular fungus. It exhibits higher organopollutant
biodegradtion activity than other white rot fungi (Baldrian et al. 2000). It has been reported that the biodegradation of pollutants such as polyaromatic hydrocarbons and chlorophenols by Pleurotus ostreatus involves
the ligninolytic enzyme system, laccase (MW 56 kDa)
and manganese peroxidase (MW 37 kDa) (Baldrian
et al. 2000; Kubatova et al. 2001). However, the role of
ligninolytic enzymes in these degradations is not really
clear.

Fig. 6. Spectrophotometric analysis of Bromophenol blue-decolorization activity. A, incubated in the abscence of enzyme and
H2O2 for 1 min (1) and 30 min (2); B, incubated with 50 g of purified enzyme and 0 M H 2O2 for 1 min (1) and 30 min (2);
C, incubated in the absence of enzyme and in the presence of 100M H 2O2 for 1 min (1) and 30 min (2); D, incubated with
50 g of purified enzyme and 100 M H 2O2 for 1 min (1) and 30 min (2).
Microbiol. Res. 158 (2003) 3

241

This paper describes an enzyme with aflatoxindegradation activity. Since the enzyme was purified
from an edible mushroom, P. ostreatus, its application to
degradation of aflatoxin in foods and feeds is promising.
For this, it is important to investigate the role of this
aflatoxin-degradation enzyme and also the best conditions for its activity.

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