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Nineteen fungi were tested for their ability to degrade aflatoxin

B1 (AFB1). An extracellular enzyme from the edible mushroomPleurotus ostreatus showed afaltoxin-degradation activitydetected by thin-layer chromatography (TLC). An enzymewith this activity was purified by two chromatographies onDEAE-Sepharose and Phenyl-Sepharose. The apparent mole-cular mass of the purified enzyme was estimated to be 90 kDaby SDS-PAGE. Optimum activities were found in the pH rangebetween 4.0 and 5.0 and at 25C. Also, degradation activity ofseveral dyes in the presence of H2O2 was tested, resulting in thedetection of bromophenol blue-decolorizing activity. Based onthese data, we suggest this enzyme is a novel enzyme withaflatoxin-degradation activity. Fluorescence measurementssuggest 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 metabolitesproduced 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; Nesbitt1962). These toxins are considered to play an importantrole in the high incidence of human hepatocellular carci-noma in certain areas of the world (Stern et al. 2001).

Because of the high toxicity to both, humans andanimals, trials to eliminate aflatoxin contaminationfrom food and feed have been carried out. Many appro-aches (Galvano et al. 2001) have been reported todegrade this toxin, however, there are no effectivemethods to prevent preharvest contamination, anddecontamination is also ineffective or not economi-cal.

In addition, many reports show the degradation ofaflatoxin by bacteria, yeasts and molds. However, thereis no practical application where biological degradationis efficiently used to reduce aflatoxins in foods (Doyleet al. 1982; Galvano et al. 2001).

On the other hand, studies on polycyclic aromatichydrocarbons (PAHs), such as dioxines, has attractedmuch attention because of their strong toxicity tohumans and animals. In the degradation of these com-pounds, the effectiveness by two main groups ofmicroorganisms, soil bacteria and white-rot fungi, hasbeen revealed. Among these, Pleurotus ostreatus hasbeen 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 aromaticorganic compounds as environmental pollutants im-portant to the dyestuff industries (Novotny et al. 1999,

2001; Vyas and Molitoris 1995). This ability has gener-ally been attributed to the lignin-degrading enzymesystem. The major enzymes in this system are mangan-ese-dependent peroxidase (MnP) and laccase (Vyas andMolitoris 1995).

In this paper, the screening of extracellular enzymesfor aflatoxin-degradation activity in white-rot andbrown-rot fungi, and the purification of an enzyme fromP. ostreatus possessing this activity are described.

0944-5013/03/158/03-237 $15.00/0 Microbiol. Res. 158 (2003) 3 237

Microbiol. Res. (2003) 158, 237242http://www.urbanfischer.de/journals/microbiolres

Purification and characterization of an aflatoxin degradation

enzyme fromPleurotus ostreatus

Marisa Motomura1, Tetsuo Toyomasu2, Keiko Mizuno1, Takao Shinozawa1,*

1 Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Gunma 376 8515, Japan2 The Mushroom Research Institute of Japan, Kiryu, Gunma 376-0051, Japan

Accepted: June 10, 2003

Abstract

Corresponding author:T. Shinozawae-mail: [email protected]


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Materials and methods

Fungi and aflatoxin. Nineteen fungi were obtained fromthe collection of Mori and Company Research Institute,Japan (Kiryu, Japan). These strains were maintained onslant tubes containing 1% glucose, 1% malt extract,0.4% yeast extract medium (GMY) with 1.5% agar at4C. Aflatoxin B1 (AFB1) was obtained from Sigma

Chemical Co. (Poole, UK).

Culture conditions. The strains, listed in Table 1, werecultured at 25C for 20 25 days in GMY medium withshaking.

Enzyme assay for aflatoxin-degradation activity. Afla-toxin B1 (final concentration, 5 g/ml) was incubated at25C for 1 h in a 500 l assay mixture containing 0.1 Msodium acetate buffer (pH 5.0) and 445 l of culturesupernatant or purified enzyme fraction. The reactionwas terminated by mixing with 500 l chloroform. Thelower chloroform layer, obtained by centrifugation at

1500 rpm for 15 min, was recovered and evaporated todryness at room temperature. The evaporated residuewas dissolved in 200 l chloroform and spotted on asilica gel plate (silica gel 60, Merck and Co, Inc., Rah-way, N. J.) for thin layer chromatography (TLC). Thecomponents were developed with a mixture of chloro-form-ethyl acetate-formic acid (6:3:1, vol/vol/vol), fol-lowed by visualization of the fluorescent spots on a UVtransilluminator. The aflatoxin-degradation activity wasalso estimated spectrophotometrically in the rangebetween 200 and 400 nm using a UV-visible spectro-photometer (Shimadzu UV-160A, Japan) in the absenceor presence of 0.1 mM H2O2. One unit of enzyme

activity is defined as the amount of enzyme that pro-duces a decrease of 0.01 absorbance units at 363 nm perminute.

Purification of the enzyme from the culture supernatantof P. ostreatus. One liter of culture supernatant wassupplemented with solid ammonium sulfate to 80%saturation under constant stirring. The solution wascentrifuged at 20000 g for 30 min and the precipitateswere dissolved in 50 mM sodium acetate buffer (pH 5.0)followed by overnight dialysis against buffer A (50 mMsodium acetate, pH 5.0, supplemented with 0.1 mMphenylmethanesulfonyl fluoride (PMSF)). The dialyzed

sample was applied to a DEAE-Sepharose column(Pharmacia Biotech, Uppsala, Sweden) pre-equilibratedwith buffer A. After washing with buffer A, the proteinswere eluted with buffer A containing 0.1 M NaCl. Theeluted fraction was supplemented with ammoniumsulfate to a final concentration of 1.0 M, and then ap-plied to a Phenyl-Sepharose column (Pharmacia)pre-equilibrated with buffer A containing 1.0 M ammo-nium sulfate. Proteins were eluted with a decreasing

linear gradient (1.0 to 0 M) of ammonium sulfate. Afterdialysis against buffer A, the aflatoxin-degradationactivity of each fraction was tested. Protein concen-trations were determined according to the Bradfordmethod (Bradford 1976) using bovine serum albuminas a standard.

Sodium dodecyl sulfate-polyacrylamide gel electro-

phoresis (SDS-PAGE). SDS-PAGE was performed in12.5% polyacrylamide gels according to the method ofLaemmli (1970). The separated proteins were stainedwith Coomassie Brilliant Blue R-250 (Fluka, Switzer-land), and their molecular weights were determined bycomparison with low range molecular weight markers(Pharmacia Biotech, Uppsala, Sweden).

Effect of pH and temperature on enzyme activity.The optimum temperature for enzyme activity wasdetermined in the range of 20 to 45C at 5C intervals.The pH optimum in the range of 4.0 to 10.0 was deter-mined using 50mM sodium acetate, sodium phosphateor 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 (WakoPure Chemicals, Osaka, Japan), Congo Red (Wako),Methylene Blue (Wako), Nile Blue (Sigma) and VictoriaBlue (Wako) were used as substrates. Decolorizingactivity was assayed at room temperature in a 500 lassay mixture of 0.1 M sodium acetate buffer (pH 5.0),50 M of each dye, and 375 l of the purified enzymefraction in the absence or presence of 0.1 mM H2O2.Decolorization was monitored spectrophotometricallyin the range of 400 to 700 nm using a UV-visiblespectrophotometer (Shimadzu UV-160A, Japan). Theactivity for each dye was also measured at the followingfixed 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 from19 mushroom strains and the supernatant from Pleu-rotus ostreatus showed aflatoxin-degradation activity(Table 1).

Purification of the enzyme was performed by twochromatographic steps. The protein solution, obtainedby ammonium sulfate precipitation of the culture super-natant followed by dialysis, was loaded on a DEAE-Sepharose column. In the next step, hydrophobic chro-matography on Phenyl-Sepharose was performed. Inboth steps, each fraction was monitored at 280 nm forprotein concentration, and activities were tested. Thepurification from the culture supernatant is summarized

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in Table 2. The enzyme was purified 148-fold with a50% yield. The apparent molecular mass of the purifiedenzyme was estimated to be 90 kDa by SDS-PAGE(Fig.1).

The effects of pH and temperature on enzymeactivity were studied. The aflatoxin-degradation activi-ties 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 aflatoxin-degradation activity by the purified enzyme in theabsence or presence of H2O2. When aflatoxin was treat-ed with the purified enzyme, its absorbance maximaat 265 and 363 nm decreased, showing the aflatoxin-degradation activity. In the presence of H2O2 the activitywas enhanced.

Aflatoxin-degradation activity was also confirmed bythin layer chromatography (TLC). Small amounts ofaflatoxin are detectable by TLC. Since aflatoxins fluo-resce under ultraviolet light, TLC is an easy and highly

parallel method and the development of the componentsis fast. When aflatoxin itself was developed, a spotdetected by UV-light appeared at position Rf 0.4(Fig. 5, lane 1). When aflatoxin was treated with theculture supernatant, the TLC plates showed a decrease

Microbiol. Res. 158 (2003) 3 239

Table 1. Screening of extracellular enzymes from mushroomculture supernatants for aflatoxin-degradation.

Mushroom Activity Mushroom Activity

Armillariella Lepista mellea nudeArmillariella Philiota (Armillaria) terrestristabescanClimacodon Pleurotus ++roseomaculatum ostreatusFomitopsis Polyporus pinicola arcularius

Ganoderma Pycnoporus applanatum coccineusGrifola Rigidoporus frondosa lineatusHygrocybo Sparassis flavesceus crispaHypsizigus Trametes +marmoreus versicolor Lentinula Volvariella edodes volvaceaLentinus lepideus

The activity was measured by thin layer chromatography. ()no activity; (+) weak activity; (++) strong activity

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

Fig. 2. Effect of pH on the aflatoxin-degradation enzymeactivity. The activity was analyzed at each pH in sodiumacetate buffer (), sodium phosphate buffer () or glycinesodium 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 95 36 0.38 1 100NH4(SO4)2precipitate 67 33 0.49 1.3 92DEAE-Sepharose 6 22 3.7 9.7 61Phenyl-Sepharose 0.32 18 56 148 50


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in fluorescence intensity of the aflatoxin spot. Further-more, the fluorescence intensities of aflatoxin spotstreated with enzymes purified by DEAE-Sepharoseor Phenyl-Sepharose were much weaker (Fig 5, lanes 4and 5).

It has been suggested that the multienzyme isolatedfromArmillariella tabescens detoxifies aflatoxin B1 byopening the difuran ring (Liu et al. 1998). It is known

that this opening does not change the fluorescence spec-trum of aflatoxin. However, cleavage of the lactone ringabolishes or decreases its fluorescence (Lee et al. 1981)This lactone structure is associated with the carcinogen-ic activity of the aflatoxin molecule (Bol and Smith1989). In our analyses by fluorescence spectra measure-ments (Fig. 4) and also by TLC (Fig. 5), treatment ofaflatoxin with purified enzyme resulted in a decreasein fluorescence intensity, suggesting the enzymatic clea-vage of the lactone ring. Additional understanding canbe obtained by characterizing the enzymatic products ofAFB1. For the large scale production of this enzyme,cloning and characterization of the gene are in progress.

240 Microbiol. Res. 158 (2003) 3

Fig. 4. Effect of H2O2 on the aflatoxin-degradation activity of the purified enzyme. The activity was analyzed by the absorban-ce change at 25C. (A) AFB1 as a control (B) enzyme assay of AFB1 without H2O2, (C) enzyme assay of AFB1 with the addi-tion of H2O2. In (B) and (C), 50 g of purified enzyme were used.

Fig. 3. Effect of temperature on the aflatoxin-degradationactivity. The activity was analyzed at each temperature in50 mM sodium acetate buffer (pH 5.0).


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

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