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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
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).
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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
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).
++
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
239
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.
240
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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