Transformation of diphenyl ethers by Trametes versicolor and characterization of ring cleavage products

Article abstract of DOI:10.1023/A:1008384019897

The white-rot fungi Trametes versicolor SBUG 1050, DSM 11269 and DSM 11309 are able to oxidize diphenyl ether and its halogenated derivatives 4- bromo- and 4-chlorodiphenyl ether. The products formed from diphenyl ether were 2- and 4-hydroxydiphenyl ether. Both 4-bromo- and 4-chlorodiphenyl ether were transformed to the corresponding products hydroxylated at the non- halogenated ring. Additionally, ring-cleavage products were detected by high performance liquid chromatography and characterized by gas chromatography/mass spectrometry and proton nuclear magnetic resonance spectroscopy. Unhalogenated diphenyl ether was degraded to 2-hydroxy-4- phenoxymuconic acid and 6-carboxy-4-phenoxy-2-pyrone. Brominated derivatives of both these compounds were formed from 4-bromodiphenyl ether, and 4- chlorodiphenyl ether was transformed in the same way to the analogous chlorinated ring cleavage products. Additionally, 4-bromo- and 4-chlorophenol were detected as intermediates from 4-bromo- and 4-chlorodiphenyl ether, respectively. In the presence of the cytochrome-P450 inhibitor 1- aminobenzotriazole, no metabolites were formed by cells of Trametes versicolor from the diphenyl ethers investigated. Cell-free supernatants of whole cultures with high laccase and manganese peroxidase activities were not able to transform the unhydroxylated diphenyl ethers used. The white-rot fungi Trametes versicolor SBUG 1050, DSM 11269 and DSM 11309 are able to oxidize diphenyl ether and its halogenated derivatives 4-bromo- and 4-chlorodiphenyl ether. The products formed from diphenyl ether were 2- and 4-hydroxydiphenyl ether. Both 4-bromo- and 4-chlorodiphenyl ether were transformed to the corresponding products hydroxylated at the non-halogenated ring. Additionally, ring-cleavage products were detected by high performance liquid chromatography and characterized by gas chromatography/mass spectrometry and proton nuclear magnetic resonance spectroscopy. Unhalogenated diphenyl ether was degraded to 2-hydroxy-4-phenoxymuconic acid and 6-carboxy-4-phenoxy-2-pyrone. Brominated derivatives of both these compounds were formed from 4-bromodiphenyl ether, and 4-chlorodiphenyl ether was transformed in the same way to the analogous chlorinated ring cleavage products. Additionally, 4-bromo- and 4-chlorophenol were detected as intermediates from 4-bromo- and 4-chlorodiphenyl ether, respectively. In the presence of the cytochrome-P450 inhibitor 1-aminobenzotriazole, no metabolites were formed by cells of Trametes versicolor from the diphenyl ethers investigated. Cell-free supernatants of whole cultures with high laccase and manganese peroxidase activities were not able to transform the unhydroxylated diphenyl ethers used.

Full text of DOI:10.1023/A:1008384019897

Biodegradation 10: 279–286, 1999.
1999 Kluwer Academic Publishers. Printed in the Netherlands.
2
79
©
Transformation of diphenyl ethers by Trametes versicolor and
characterization of ring cleavage products
Kai Hundt, Ulrike Jonas, Elke Hammer & Frieder Schauer
Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universität, Greifswald D-17487, Germany
e-mail: hundt@microbio1.biologie.uni-greifswald.de
Accepted 8 July 1999
Key words: Diphenyl ether, ligninolytic enzymes, metabolites, oxidation, Trametes versicolor, white-rot fungi
Abstract
The white-rot fungi Trametes versicolor SBUG 1050, DSM 11269 and DSM 11309 are able to oxidize diphenyl
ether and its halogenated derivatives 4-bromo- and 4-chlorodiphenyl ether. The products formed from diphenyl
ether were 2- and 4-hydroxydiphenyl ether. Both 4-bromo- and 4-chlorodiphenyl ether were transformed to the
corresponding products hydroxylated at the non-halogenated ring. Additionally, ring-cleavage products were de-
tected by high perfomance liquid chromatography and characterized by gas chromatography/mass spectrometry
and proton nuclear magnetic resonance spectroscopy. Unhalogenated diphenyl ether was degraded to 2-hydroxy-
4
-phenoxymuconic acid and 6-carboxy-4-phenoxy-2-pyrone. Brominated derivatives of both these compounds
were formed from 4-bromodiphenyl ether, and 4-chlorodiphenyl ether was transformed in the same way to the
analogous chlorinated ring cleavage products. Additionally, 4-bromo- and 4-chlorophenol were detected as in-
termediates from 4-bromo- and 4-chlorodiphenyl ether, respectively. In the presence of the cytochrome-P450
inhibitor 1-aminobenzotriazole, no metabolites were formed by cells of Trametes versicolor from the diphenyl
ethers investigated. Cell-free supernatants of whole cultures with high laccase and manganese peroxidase activities
were not able to transform the unhydroxylated diphenyl ethers used.
Introduction
et al. 1995). There are also naturally occuring halogen-
ated diphenyl ether derivatives, which are synthesized
mainly by fungi and marine organisms (Elyakov et
al. 1991; Unson et al. 1994, Takahashi et al. 1992;
Takahashi et al. 1993).
White-rot fungi have been investigated extensively
for their ability to transform xenobiotics. In this study
the biotransformation of diphenyl ether and its halo-
genated derivatives 4-bromo- and 4-chlorodiphenyl
ether as model compounds for polyhalogenated di-
phenyl ethers was investigated using strains of the
white-rot fungus Trametes versicolor. The abilities
of the ligninolytic enzyme system to degrade these
compounds were considered.
In recent years, halogenated diphenyl ethers have
become increasingly important environmental pollut-
ants. Chlorinated diphenyl ethers are formed as by-
products during the synthesis of chlorinated phenols
and phenoxy acids (Ahlborg & Thunberg 1980). Fur-
thermore, brominated diphenyl ethers are used ex-
tensively as flame retardants in electronic equipment.
Since their first commercial introduction in the 1970s,
the concentrations of brominated diphenyl ethers in
the environment have risen sharply. High concentra-
tions were detected in sea sediments, sewage sludge
and animal tissue (Pijnenburg et al. 1995). Up to
now, most research has dealt with degradation of di-
phenyl ethers by bacteria (Takase et al. 1986, Pfeifer
et al. 1989, 1993; Schmidt et al. 1992, 1993). Only a
few reports exist on the transformation of these com-
pounds by fungi (Seiglé-Murandi et al. 1991; Schauer
In the investigation of pollutant degradation by
white-rot fungi the role of ligninolytic enzymes in-
cluding manganese peroxidases, lignin peroxidases
and laccases is a question of considerable interest. In
contrast to what is known about some polycyclic aro-

2
80
matic hydrocarbons (Bumpus 1989; Hammel 1992;
Bezalel et al. 1996; Collins et al. 1996; Kotterman
et al. 1998) little or no information exists concerning
their abilities for the degradation of biarylic com-
pounds like polychlorinated biphenyls, diphenyl eth-
ers or dibenzo-p-dioxins (Bumpus et al. 1985, Valli et
al. 1992). Obviously, in addition to the action of extra-
cellular ligninolytic enzymes, intracellular processes
also seem to be of importance for the biotransforma-
tion of these compounds by white rot fungi (Dietrich
et al. 1995, Takada et al. 1996).
from cultures without addition of xylidine. Samples
of 2 ml of supernatant with a manganese peroxidase
−1
activity of 0.1 units ml and a laccase acitivity of
−1
0.05 units ml was put in vials together with 3 ml
sodium tartrate buffer (0.05 M, pH 5), 10 mM MnSO4
◦
and 1 mM H2O2. The vials were incubated at 30 C
for 72 hours and then treated in the same way as in the
experiments done with whole cells.
For investigating the degradation of diphenyl ether
by laccase in the presence of the mediator ABTS (2,2-
azino-bi-(3-ethyl-benzthiazolin-sulfonate)) partially
purified laccase, obtained as described by Jonas et al.
(
1998), was used. Diphenyl ether diluted in dimethyl-
formamide was added to a final concentration of 3 mM
in sodium acetate buffer (0.1 M) containing 0.1 units
Material and methods
−1
ml
of laccase activity. The assay was incubated
Microorganisms, growth and culture conditions
for 72 h. Afterwards, the aqueous supernatants were
analyzed by HPLC as described in the text.
The cytochrome-P450 inhibitor 1-aminobenzotri-
azole was added to a final concentration of 0.25
mM directly to the cultures at the beginning of the
incubation.
The strain Trametes versicolor SBUG-M 1050 was
from the Culture Collection of the University of Gre-
ifswald, Germany. The strains Trametes versicolor
DSM 11269 and Trametes versicolor DSM 11309
used were purchased from the German Culture Col-
lection of Microorganisms and Cell Cultures (DSMZ,
Braunschweig).
Enzyme assays
For cultivation, a nitrogen-rich (8mM) medium
NRM) was used. Its composition was: glucose 5 g,
Laccase activity was determined spectrophotometric-
ally at 420 nm with ABTS as substrate (Bourbonnais
(
KH2PO4 0.5 g, L-asparagine 0.52 g, yeast extract 0.5
g, KCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4 0.01 g,
deionized water 950 ml. 50 ml of a mineral salt solu-
tion were added containing Mn(CH3COO)2·4H2O 8
mg ZnSO4·7H2O 2 mg, Ca(NO3)2·4H2O 50 mg,
CuSO4·5H2O 3mg. The pH of the NRM-medium was
&
Paice 1990) using the method described by Jonas et
al. (1998). One unit was defined as 1 µmol of ABTS
oxidized per min.
Manganese peroxidase activity was measured ac-
cording to the method of Akamatsu & Shimada (1996)
with a reaction mixture containing MnSO4 (3.3 mM),
H2O2 (0.25 mM), sodium tartrate buffer (50 mM, pH
4
.5. The diphenyl ethers to be investigated were pipet-
ted as a solution in acetone (0.25 mM w/v) into 100
ml Erlenmeyer flasks. The solvent was evaporated at
room temperature for 12 h. Afterwards, the flasks were
filled with 40 ml NRM containing 0.05% (w/v) Tween
4
.5) and a sufficient amount of supernatant. The in-
crease in absorbance at 264 nm due to the formation
−1
−1
)
of Mn(III)-ions was measured (ꢀ264 = 6 mM cm
and one unit of manganese peroxidase was defined as
8
0 and inoculated with 3 ml of homogenized my-
3+
1
µmol of Mn formed per min.
celium (IKA Ultra-Turrax, Janke & Kunkel, Staufen,
Germany) of a pre-culture which had been grown in
NRM for 10 days under static conditions. After inocu-
Lignin peroxidase was measured by following the
oxidation of veratryl alcohol to veratrylaldehyde as
described by Kirk et al. (1990).
◦
lation the cultures were incubated at 30 C on a rotary
shaker at 160 rpm.
Analytical methods
Laccase activity of the pre-culture was induced by
adding 20 mM xylidine. For studying the ability of
laccase to transform diphenyl ethers, 2 ml samples
of culture supernatants with a laccase activity of 2,5
Analysis of metabolites was done by HPLC for
aqueous culture supernatants. RP (Reverse phase)-
Analytical HPLC was carried out onto a Hewlett-
Packard 1050 Series pump system and a 1040 M
Series I Diode array detector. Data analysis was done
with a Hewlett Packard HPLC Chemstation (Hewlett
Packard, Waldbronn, Germany). A solvent system
−1
units ml and no detectable manganese peroxidase
activity were put into vials and 3 ml sodium acetate
buffer (0.1 M, pH 5) was added. Supernatants with
high manganese peroxidase activities were obtained

2
81
of methanol and phosphoric acid (0.1% w/v) was
used with a linear gradient from 30% methanol and
7
0% phosphoric acid to 100% methanol (flow rate: 1
ml/min) within 16 min.
Purification of metabolites was performed on a
Merck-Hitachi HPLC system equipped with a Model
L 6200 A Intelligent Pump, a Rheodyne 7161 injection
valve with a 100µl-loop and a Model L-4250 absorb-
ance detector operating at 254 nm. A solvent system
of methanol and acetic acid (0.1%) was used with the
same conditions described for the analytical HPLC
system. For both HPLC systems a RP 18 column (Li-
chro Cart 125-4, 5 µm, endcapped, Merck, Darmstadt,
Germany) was used.
Figure 1. Formation of products from 4-bromodiphenyl ether in
cultures of Trametes versicolor SBUG-M 1050.
For gas chromatographic analysis whole cultures
were extracted without a previous centrifugation step.
It was necessary to extract the cells also because of the
adsorption of the metabolites investigated to the fungal
mycelia. After 7 days of incubation, the cultures were
extracted with ethyl acetate at pH 7 and once again
after acidification of the aqueous residue to pH 2. The
resulting organic phases were dried over anhydrous
sodium sulfate and evaporated with a vacuum evap-
sulfonate)) was obtained from Boehringer (Ingelheim,
Germany). Tween 80 was obtained from Serva Com-
pany Ltd. (Heidelberg, Germany). All solvents were
of a purity suitable for chromatography.
Results
All strains of Trametes versicolor investigated formed
the same pattern of metabolites from diphenyl ether,
◦
orator at 50 C. Finally, the extracts were evaporated
to dryness under a stream of nitrogen.
4
-chloro- and 4-bromodiphenyl ether. Thus, the abil-
Gas chromatography/mass spectrometry (GC/MS)
was done using a Fisons GC 8000 gaschromatograph
ity of Trametes versicolor to transform diphenyl ethers
is not strain-specific. For more detailed studies of the
transformation products the strain SBUG-M 1050 was
used.
By comparing their HPLC and GC-MS-data with
those of authentic standards, two products formed
from diphenyl ether were identified as 2- and 4-
hydroxydiphenyl ether in supernatants as well as
in neutral extracts. It was calculated that at least
(
Fisons, Mainz-Kastel, Germany) equipped with a
DB-5-MS fused silica capillary column (J & W Sci-
entific, Folsom, California) and coupled with a Fisons
MD 800 EI mass selective detector. Measurements
were performed under the following conditions using
◦
helium as carrier gas: injector temperature 240 C,
◦
◦
◦
temperature program: 80 C to 300 C at 8 C/min,
◦
1
0 min 300 C.
2
0% of the concentration of diphenyl ether ad-
Non-volatile compounds were converted to their
ded to the cultures was converted to the mono-
hydroxylated products 2- and 4-hydroxydiphenyl
ether. The halogenated diphenyl ethers investig-
ated were metabolized to the corresponding mono-
methylated derivatives (De Boer & Backer 1956) us-
ing a methylation apparatus with diazomethane as
methylating agent (Aldrich, Steinheim, Germany).
1
0
Proton nuclear magnetic resonance ( H-NMR-)
hydroxylated products 2-hydroxy-4 -bromo- and 4-
0
0
spectroscopy was carried out using a Bruker ARX
hydroxy-4 -bromo-diphenyl ether or 2-hydroxy-4 -
chloro- and 4-hydroxy-4 -chlorodiphenyl ether. Dur-
0
3
00 MHz spectrometer (Bruker, Karlsruhe, Germany),
deuterated methanol as solvent and tetramethyl silane
as reference.
ing further incubation, the monohydroxylated di-
phenyl ethers disappeared again from the supernatants
(
Figure 1) and were not detectable by HPLC after
Chemicals
seven days, but low quantities could be identified in
the ethylacetate extracts.
Diphenyl ether (>98%), 4-chloro- (99%) and
Furthermore, low pH ethylacetate extracts of cul-
tures exposed to the investigated diphenyl ethers were
analyzed by GC-MS. Two additional metabolites A
and B from each diphenyl ether used were found in
4
-bromodiphenyl ether (>99%) as well as 1-
aminobenzotriazole were purchased from Ald-
rich. ABTS (2,2-azino-bi-(3-ethyl-benzthiazolin-

2
82
the low pH extracts. For these intermediates, GC-MS
was successful only after methylation. After methyl-
ation, product A, which was formed from diphenyl
ether, had a molecular ion peak at m/z 246 and re-
+
+
markable fragments at m/z 218 (M -CO), 187 (M -
+
COOCH3), 159 (M -COOCH3-CO), 93 (C6H5O), 77
(
C6H5), and 59 (COOCH3). These data are consist-
ent with 6-carboxy-4-phenoxy-2-pyrone, the lactone
of 2-hydroxy-4-phenoxymuconic acid. This product
was already characterized as metabolite formed from
diphenyl ether by Trichosporon spec. SBUG 752
(
Schauer et al. 1995).
Product B formed from nonhalogenated diphenyl
ether had after methylation a molecular ion peak at m/z
92 (Figure 2A). The base peak of m/z 233 resulted
2
in the loss of COOCH3 from the molecular ion. This
and the occurrence of a m/z 59 fragment indicated the
formation of a carboxylic acid as an intermediate. The
1
H-NMR spectrum (Figure 2B) of the non-methylated
product B showed 7 proton signals, five of them be-
ing quite similar to those of the non-substituted parent
compound diphenyl ether or 6-carboxy-4-phenoxy-2-
pyrone (Schauer et al. 1995). The spin-spin coupled
signals at δ = 5.31 (d, 1) and δ = 6.90 (d, 1) indic-
ated two aliphatic methine protons. The low coupling
constant J3,5 of 2.3 Hz point to a long-range coup-
Figure
2. Mass
spectrum
of
methylated
2-hydroxy-
1
1
ling of these protons. Because of the mass, H-NMR
4-phenoxymuconic acid (A) and H-NMR spectrum of the
non-methylated derivative (B) formed from diphenyl ether by
Trametes versicolor.
spectral data and the fact that both products A and B
can be chemically transformed to each other, product
B can be assumed to be the ring cleavage product
2
-hydroxy-4-phenoxy muconic acid.
Two further intermediates (C and D) resulting from
aromatic ring cleavage were also obtained from 4-
bromodiphenyl ether. Like for diphenyl ether, they
could be detected by GC-MS only after methylation.
Due to their characteristic mass spectra resulting from
bromine and chlorine substituents, products from 4-
bromo- and 4-chlorodiphenylether were easy to detect
by GC-MS.
Product C formed from 4-bromodiphenylether had
after methylation a molecular ion peak at m/z 324
+
and remarkable fragment ions at 296 (M -CO) 265
(
+
M -COOCH3), 172 (C6H5OBr) 155 (C6H4Br) and
5
9 (COOCH3, Figure 3). Because of the similarities
in the behaviour of the compound in the analytical
procedures and the fragmentation pattern of the mass
spectrum to that of the lactone formed from unsubsti-
tuted diphenyl ether it was concluded that it was 6-
Figure
3. Mass
spectrum
of
methylated 6-carboxy-
the lactone of
0
4
4
4
-(4 -bromophenoxy)-2-pyrone,
-(4 -bromophenoxy)-2-hydroxymuconic acid formed from
-bromodiphenyl ether by Trametes versicolor.
0
0
carboxy-4-(4 -bromophenoxy)-2-pyrone, the lactone
0
of 4-(4 -bromophenoxy)-2-hydroxymuconic acid.

2
83
by cleavage of the non-halogenated ring. After GC-
MS analysis of the extracts also 4-bromophenol was
found as metabolite from 4-bromodiphenyl ether as
well as 4-chlorophenol from 4-chlorodiphenyl ether.
During incubation with 4-bromodiphenyl ether
in the presence of the cytochrome-P450-inhibitor 1-
aminobenzotriazole no metabolites were formed al-
though the fungus was able to grow in the presence
of 0.25 mM of 1-aminobenzotriazole.
Trametes versicolor secreted laccase and man-
ganese peroxidase but no lignin peroxidase into the
supernatant. Laccase activity was enhanced distinctly
in the presence of diphenyl ether, 4-bromo- and 4-
chlorodiphenyl ether. Nevertheless, supernatants of
cultures of Trametes versicolor showing a manganese
−
1
peroxidase activitity (0.1 units ml ) and a laccase
−1
activity (2,5 units ml ) were not able to transform
any of the diphenyl ethers used. No products were
formed during an incubation period of 72 hours. Even
in the presence of the laccase mediator ABTS no
products were formed from diphenyl ether by laccase
as described for anthracene by Johannes et al. (1996).
0
Figure 4. Mass spectrum of methylated 4-(4 -bromo-
1
phenoxy)-2-hydroxymuconic acid (A) and H-NMR spectrum of
the non-methylated derivative (B) formed from 4-bromodiphenyl
ether by Trametes versicolor.
Discussion
The mass spectrum of product D showed a molecu-
lar ion at m/z 370 and a base peak of m/z 311 indicat-
ing the loss of COOCH3. Further important fragment
The white-rot fungus Trametes versicolor is able
to transform diphenyl ether and its monohalogen-
ated derivatives 4-bromo- and 4-chlorodiphenyl ether.
After hydroxylation reactions a ring-cleavage step oc-
curs which leads to a phenoxymuconic acid deriv-
ative and its corresponding lactone (Figure 5). The
structure of the degradation products suggests that
Trametes versicolor metabolizes diphenyl ethers in
the same way as the yeast Trichosporon spec. SBUG
752 (Schauer et al. 1995). This yeast strain, which
does not express any ligninolytic enzymes, oxid-
izes diphenyl ether via hydroxylation up to the di-
hydroxylated and presumably to the trihydroxylated
product (Figure 5). The latter is finally cleaved by an
ortho-fission of the aromatic ring. Like Trichosporon
spec. SBUG 752 Trametes versicolor degrades di-
phenyl ether to 6-carboxy-4-phenoxy-2-pyrone, the
lactone of 2-hydroxy-4-phenoxymuconic acid. In the
experiments presented here additionally 2-hydroxy-4-
phenoxymuconic acid, which was hitherto not detec-
ted as an intermediate, was also found in extracts of
cultures incubated with diphenyl ether.
+
+
ions were detected at m/z 339 (M -CH3O), 255 (M -
+
C3O3HCH3), 199 (M -C6H5O) and 172 (C6H5OBr,
1
Figure 4A). The signals in the H-NMR spectrum
(
4
Figure 4B) were like those measured for 2-hydroxy-
-phenoxymuconic acid, except that the signals of
aromatic protons were those of a para-substituted aro-
matic ring (Figure 4B). From these results it was
0
concluded that product D was 4-(4 -bromophenoxy)-
2
-hydroxymuconic acid, the brominated ring-cleavage
product analogous to that formed from unbrominated
diphenyl ether. This product was stable to acidic con-
ditions (0.5 mg of product in 1 ml of 25% HCl was
shaken at room temperature for 24 h) but unstable on
alkaline hydrolysis (0.5 mg of product in 1 ml of 40%
KOH was shaken at room temperature for 24 h), which
resulted in the formation of 4-bromophenol. These
findings are consistent with those obtained from 2-
phenoxymuconic acid as a similar bacterial metabolite
from diphenyl ether by Takase et al. (1986).
From 4-chlorodiphenyl ether the products 4-
Diphenyl ethers halogenated in the para-position
were transformed to the corresponding ring cleavage
products by cleavage of the non-halogenated ring. The
0
(
4 -chlorophenoxy)-2-hydroxymuconic acid and 6-
0
carboxy-4-(4 -chlorophenoxy)-2-pyrone were formed

2
84
able to transform 4-bromodiphenyl ether either. These
results may suggest that at least the initial hydroxyla-
tion steps of diphenyl ether transformation may be
performed by intracellular, nonligninolytic enzyme
systems under the given conditions. This notion is sup-
ported by the fact that biarylic ether compounds like
diphenyl ethers in general have high oxidation poten-
tials. Hammel et al. (1986) investigated the oxidation
abilities of lignin peroxidase for polycyclic aromatic
hydrocarbons and found that only PAHs with an ion-
ization potential lower than 7.55 eV were modified.
The ionization potential for diphenyl ether was de-
termined to be 8.09 eV (Lide 1995) and the values for
its halogenated derivatives might be even higher. By
the Mn(II)/Mn(III)-reaction catalyzed by manganese
peroxidase PAHs with a ionization potential of 7.7
eV or lower are oxidized (Cavalieri & Rogan 1985).
Laccases degrade only compounds with even lower
ionization potentials (De Jong et al. 1994).
In some cases authors could show that in the pres-
ence of specific mediators, aromatic compounds with
higher oxidation potentials can be oxidized by lac-
cases (Collins et al. 1996; Johannes et al. 1996) and
manganese peroxidases (Scheibner et al. 1997), too.
Collins et al. (1996) incubated PAHs with laccases
from Trametes versicolor and the laccase mediator
ABTS. However, they found that even in the presence
of this mediator compound only PAH with an ioniza-
tion potential lower than 7.45 eV were oxidized. Thus,
it seems understandable that also diphenyl ether with
its relatively high ionization potenial is not attacked
under the conditions we used.
Figure 5. Proposed pathway for the degradation of diphenyl ethers
by Trametes versicolor.
fact that only the non-halogenated ring of 4-bromo-
and 4-chlorodiphenyl ether is cleaved by Trametes
versicolor may support the hypothesis that a tri-
hydroxylated compound with hydroxyl groups lying
next to each other is required as precursor of the ring
cleavage product. If the aromatic ring of the diphenyl
ether is halogenated in the para-(4-)-position, three
hydroxyl groups cannot be placed next to each other
without dehalogenation. However, for more detailed
studies concerning this problem, investigations with
diphenyl ethers halogenated in ortho-position are ne-
cessary. The route of diphenyl ether degradation as
found for a yeast (Schauer et al. 1995) and presented in
this paper seems to be similar both in nonligninolytic
as well as in white-rot fungi and may be therefore
performed by nonligninolytic enzymes.
Yadav et al. (1995) observed extensive degrada-
tion of Arochlor 1242 and 1254 by Phanerochaete
chrysosporium both in non-ligninolytic as well as
in ligninolytic defined medium. Kr cˆ mar & Ulrich
(
1998) observed no degradation of lower-chlorinated
and higher-chlorinated biphenyl (PCB) congeners by
Phanerochaete chrysosporium when incubated in a
nitrogen-limited medium. Nevertheless, they observed
a significant PCB degradation in a non-limited me-
dium. All the results we obtained point to a transform-
ation catalyzed by nonligninolytic enzymes.
Phenol and its halogenated derivatives 4-bromo-
and 4-chlorophenol are formed from the diphenyl eth-
ers used by Trametes versicolor. This fact may suggest
that further degradation of the diphenyl ethers hap-
pens after ring cleavage. However, from the results
presented it is not yet clear whether the formation
of the halophenols occurs by further degradation of
the detected cleavage products or by direct enzymatic
Trichosporon spec. SBUG 752 oxidizes non-
halogenated diphenyl ether efficiently. However, from
4
-chlorodiphenyl ether only much smaller amounts
of monohydroxylated products were formed (Hen-
ning 1993). In contrast to these results, cells of the
investigated strains of Trametes versicolor formed
ring-cleavage products both from diphenyl ether,
4
-bromo and 4-chlorodiphenyl ether. No products
were formed from 4-bromodiphenyl ether in the
presence of the strong cytochrome P-450 inhibitor
1
-aminobenzotriazole. Supernatants containing high
manganese peroxidase and laccase activities were not

2
85
scission of the ether bridge between the two intact
aromatic rings.
The results suggest that in Trametes versicolor not
only a polymerization of hydroxylated biarylic ether
compounds by laccases (Jonas et al. 1998) occurs, but
also further transformation up to ring cleavage.
Hammel KE (1992) Oxidation of aromatic pollutants by lignin-
degrading fungi and their extracellular peroxidases. In: Metal
ions in biological systems (pp 41–60). John Wiley & Sons, New
York
Hammel KE, Kalynaraman B & Kirk, TE (1986) Oxidation of
polycyclic aromatic hydrocarbons and dibenzo[p]dioxins by
Phanerochaete chrysosporium ligninase. J. Biol. Chem. 261:
16948–16952
Henning K (1993) Oxidation von Diphenylether durch die basidio-
mycetale Hefe Trichosporon beigelii. Ph.D. thesis University of
Greifswald, Germany
Acknowledgments
Johannes C, Majcherczyk A & Hüttermann A (1996) Degradation
of anthracene by laccase of Trametes versicolor in the presence
of different mediator compounds. Appl. Microbiol. Biotechnol.
We would like to thank M. Kindermann, S. Siegert and
B. Witt for performing H-NMR spectroscopy. Thanks
1
46: 313–317
also to R. Jack for revising the manuscript. This work
was supported by a grant of the Stiftung Stipendien-
fonds des Verbandes der chemischen Industrie (VCI)
Deutschlands e. V. to K.H.
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