Biocatalytic chlorination of aromatic hydrocarbons by chloroperoxidase of caldariomyces fumago

Article abstract of DOI:10.1016/S0031-9422(01)00326-0

Chloroperoxidase from Caldariomyces fumago was able to chlorinate 17 of 20 aromatic hydrocarbons assayed in the presence of hydrogen peroxide and chloride ions. Reaction rates varied from 0.6 min^-1 for naphthalene to 758 min^-1 for 9-methylanthracene. Mono-, di- and tri-chlorinated compounds were obtained from the chloroperoxidase-mediated reaction on aromatic compounds. Dichloroacenaphthene, trichloroacenaphthene 9,10-dichloroanthracene, chloropyrene, dichloropyrene, dichlorobiphenylene and trichlorobiphenylene were identified by mass spectral analyses as products from acenaphthene, anthracene, pyrene and biophenylene respectively. Polycyclic aromatic hydrocarbons with 5 and 6 aromatic rings were also substrates for the chloroperoxidase reaction. The importance of the microbial chlorination of aromatic pollutants and its potential environmental impact are discussed.

Full text of DOI:10.1016/S0031-9422(01)00326-0

Phytochemistry 58 (2001) 929–933
www.elsevier.com/locate/phytochem
Biocatalytic chlorination of aromatic hydrocarbons by
chloroperoxidase of Caldariomyces fumago
Rafael Vazquez-Duhalta,*, Marcela Ayalaa, Facundo J. Marquez-Rochab
aBiotechnology Institute UNAM, AP 510-3, Cuernavaca, Morelos 62271, Mexico
bMarine Bioprocess Enginering Laboratory, CICESE, Baja California, 22860, Mexico
Received 19 March 2001; received in revised form 5 July 2001
Abstract
Chloroperoxidase from Caldariomyces fumago was able to chlorinate 17 of 20 aromatic hydrocarbons assayed in the presence of
hydrogen peroxide and chloride ions. Reaction rates varied from 0.6 minÀ1 for naphthalene to 758 minÀ1 for 9-methylanthracene.
Mono-, di- and tri-chlorinated compounds were obtained from the chloroperoxidase-mediated reaction on aromatic compounds.
Dichloroacenaphthene, trichloroacenaphthene, 9,10-dichloroanthracene, chloropyrene, dichloropyrene, dichlorobiphenylene and
trichlorobiphenylene were identified by mass spectral analyses as products from acenaphthene, anthracene, pyrene and biopheny-
lene respectively. Polycyclic aromatic hydrocarbons with 5 and 6 aromatic rings were also substrates for the chloroperoxidase
reaction. The importance of the microbial chlorination of aromatic pollutants and its potential environmental impact are discussed.
# 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Fungal chloroperoxidase; Enzymatic chlorination; Polycyclic aromatic hydrocarbons
1. Introduction
In spite of the microbial capacity to produce haloge-
nated compounds (Neidleman, 1975; de Jong and Field,
1997), information is scarce on the microbial production
of toxic organochlorine compounds. In addition to de
novo synthesis of chlorinated compounds, microorgan-
isms transform some non-halogenated xenobiotics into
organochlorine compounds with a possible increase in
their toxicity. In this work, we show the capacity of
chloroperoxidase from the fungus Caldariomyces
fumago to chlorinate aromatic hydrocarbons, including
polycyclic aromatic hydrocarbons (PAHs). PAHs are
widely dispersed in the environment, and they are con-
sidered to be a potential health risk because of their
possible carcinogenic and mutagenic activities. Chloro-
peroxidase (CPO) is a 42, 000 Da extracellular heme glyco-
enzyme containing ferriprotoporphyrin IX as the
prosthetic group (Sundaramoorthy et al., 1995). CPO
exhibits a broad spectrum of chemical reactivities, it is a
peroxide-dependent chlorinating enzyme and it also cata-
lyzes peroxidase-, catalase- and cytochrome P450-type
reactions of dehydrogenation, H2O2 decomposition and
oxygen insertion, respectively (Yi et al., 1999). CPO is only
one of a variety of halogenase enzymes that can be found in
nature, other enzymes such as vanadium and non-heme
halogenases (van Pee et al., 2000; Vollenbroek et al., 1995)
are also potentially able to chlorinate organic pollutants.
Organochlorine industrial compounds, chlorinated
pesticides and polychlorinated biphenyls (PCBs) are
considered among the most important pollutant xeno-
biotics. Their toxic effects have been extensively studied
(Evangelista de Duffard and Duffard, 1996; Giesy and
Kannan 1998; Tilson and Kodavanti, 1998). Because of
their potential public health risk, some organochlorine
compounds, such as PCBs, have been banned in western
countries, but many are still manufactured and used as
pesticides, plasticizers, paint and printing-ink compo-
nents, adhesives, flame retardants, hydraulic and heat
transfer fluids, refrigerants, solvents, additives for cut-
ting oils, and textile auxiliaries. Thus, contamination
with organochlorine compounds still occurs and this is
of great public concern due to potential toxicity to
humans and wildlife. Microbiological studies have been
almost entirely focused on the degradation of these
toxic compounds (Robinson, 1998; Wiegel and Wu,
2000; Chaudhry and Chapalamadugu, 1991).
* Corresponding author at: Instituto de Biotecnologıa UNAM,
Aparto Postal 510-3, Cuernavaca, Morelos 62250, Mexico. Tel.: +52-
5622-7655; fax: +52-7317-2388.
E-mail address: vazqduh@ibt.unam.mx (R. Vazquez-Duhalt).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00326-0

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930
2. Results and discussion
methylanthracene. Interestingly, recalcitrant and carci-
nogenic 5- and 6-aromatic rings PAHs were substrates
for chloroperoxidase in the presence of hydrogen per-
oxide and chloride ions. Chloroperoxidase is able to
perform a broad range of reactions, like the enantio-
selective epoxidation of alkenes (Zaks and Dodds,
1995), oxidation of phenolic pollutants (Aitken et al.,
1994; Carmichael et al., 1983), oxygenation of sulfides
(Colonna et al., 1990), oxidation of organophosphorus
pesticides (Hernandez et al., 1998), and the PAH–DNA
adduct formation (Marquez-Rocha et al., 1997), to give
only a few examples. In addition, CPO is able to oxidize
very complex molecules, such as asphaltenes (Fedorak
et al., 1993) and complex mixtures such as petroleum
distillates (Ayala et al., 1998). Thus this halogenating
enzyme is the most versatile enzymatic hemoprotein.
Other halogenases are also able to perform halogenations,
mainly chlorination, on a variety of substrates (Neidle-
man, 1975; Vollenbroek et al., 1995).
The chemical nature of the reaction products was
determined by gas chromatography–mass spectrometry
(GC–MS). The mass spectra of the products from the
enzymatic reaction with acenaphthene, anthracene,
biphenylene, fluorene, phenanthrene, pyrene, and tri-
phenylene are shown in Table 2. Monochlorinated,
dichlorinated and trichlorinated compounds were found
(Fig. 1), and they showed major ions at m/z=[M+]-35,
m/z=[M+]-70, and m/z=[M+]-105, respectively. No
oxygen incorporation was detected in any of the pro-
ducts from the CPO-mediated reactions. In addition, no
reaction could be detected with any PAH tested under
peroxidase activity conditions (pH 5.0 and in the
absence of chlorine ions). Significant information is
available on the toxicity of chlorinated aromatic com-
pounds, such as polychlorophenols (International
Chloroperoxidase was able to utilize 17 of the 20
aromatic hydrocarbons assayed as substrate (Table 1).
Only biphenyl, and the oxygen-containing dibenzofuran
and anthrone, were not substrates for CPO under our
reaction conditions. The specific activity values were
from 0.6 minÀ1 for naphthalene to 758 minÀ1 for 9-
Table 1
Specific activity of chloroperoxidase from Caldariomyces fumago
against aromatic compounds
Aromatic compound
Specific activity
(minÀ1
)
9-Methylanthracene
Azulene
Anthracene
758 (Æ27)
676 (Æ34)
134 (Æ14)
107 (Æ8)
87 (Æ8)
84 (Æ6)
81 (Æ7)
65 (Æ8)
53 (Æ6)
45 (Æ7)
25 (Æ10)
10 (Æ0.5)
7 (Æ0.1)
3 (Æ0.2)
1.9 (Æ0.13)
0.8 (Æ0.09)
0.6 (Æ0.01)
NRa
2-Methylanthracene
7,12-Dimethylbenzanthracene
Benzo[a]pyrene
7-Methylbenzo[a]pyrene
Acenaphthene
Pyrene
Benzo[ghi]perylene
Perylene
Biphenylene
Phenanthrene
Fluoranthene
Fluorene
Triphenylene
Naphthalene
Biphenyl
Dibenzofuran
Anthrone
NRa
NRa
a
NR, no reaction detected.
Table 2
Mass spectral data of the products from the chloroperoxidase-mediated reaction on aromatic compounds
Substrate
Product
Mass spectral ions (m/z)a,b
Acenaphthene
Dichloroacenaphthene
Trichloroacenaphthene
224 (39), 222 (64) [M+], 187 (100), 152 (95), 93 (17), 75 (24).
258 (57), 256 (81) (M+], 221 (66), 186 (100), 150 (50), 110 (27), 98 (18), 75 (23).
Anthracene
Biphenylene
9,10-Dichloroanthracene
Dichlorobiphenylene
Trichlorobiphenylene
248 (68), 246 (100) [M+], 176 (43), 87 (10).
222 (64), 220 (100) [M+], 185 (17), 150 (45), 75 (11).
258 (30), 256 (93), 254 (100) [M+], 219 (13), 184 (49), 149 (14), 74 (10).
Fluorene
Dichlorofluorene
238 (25), 237 (7), 236 (40) [M+], 201 (31), 199 (18), 166 (63), 165 (100), 164 (17),
163 (24), 100 (11), 82 (35).
Phenanthrene
Chlorophenanthrene
214 (32), 213 (16) [M+], 212 (100), 177 (20), 176 (55), 175 (11), 174 (10), 151 (14),
150 (14), 106 (17), 88 (33), 87 (11), 75 (13).
Pyrene
Chloropyrene
Dichloropyrene
238 (31), 236 (100) [M+], 200 (34), 100 (12).
272 (62), 270 (100) [M+], 235 (11), 200 (53), 135 (12), 100 (23).
Triphenylene
Chlorotriphenylene
265 (7), 264 (34), 263 (21) [M+], 262 (100), 227 (14), 226 (63), 225 (16), 224 (23),
200 (11), 132 (9), 131 (20), 113 (56), 112 (43), 100 (21), 99 (12), 87 (9).
a
Values in parentheses are relative abundances.
[M+] molecular ion.
b

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931
Fig 1. Chlorinated products from the enzymatic halogenation of aromatic compounds by chloroperoxidase in the presence of hydrogen peroxide
and chloride ions.
Agency for Research on Cancer, 1999) and PCBs
(Robinson, 1998; Wiegel and Wu, 2000), and to a less
extent, on the toxicity of chlorinated derivatives of
polycyclic aromatic hydrocarbons. Chlorinated PAHs
have been tested for mutagenic activity by the Ames test
on Salmonella typhimurium (Colmsjo et al., 1984; John-
sen et al., 1989). Chlorinated fluorene, flouranthene and
benzo(a)pyrene acted as strong mutagens both in the
presence and in the absence of metabolic activation,
while only benzo(a)pyrene showed mutagenic activity as
parent hydrocarbon. Mono- and di-chloropyrene iso-
mers showed from 40 to 4000 times higher mutagenic
activity than 1-nitropyrene and pyrenoquinones, respec-
tively. On the other hand, a mixture of chlorinated
chrysene isomers was considerably more potent than the
parent hydrocarbon in terms of embryolethality and
cytochrome P450 induction (7-ethoxyresorufin-O-de-
ethylase and aryl hydrocarbon hydroxylase) (Gustafsson
et al., 1994). The chlorinated chrysene caused anoma-
lies, including edema and beak defects, similar to those
reported after treatment of chick embryos with coplanar
PCBs. These effects of the chlorinated mixture were
mainly accounted for by 6-chlorochrysene and 6,12-
dichlorochrysene. Chloronaphthalene was 5000-times

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R. Vazquez-Duhalt et al. / Phytochemistry 58 (2001) 929–933
932
more potent than naphthalene for the inhibition of
mitochondrial respiration in molar basis (Beach and
Harmon, 1992). Monochloronaphthalene represent a
risk for human health (Tsunenar et al., 1982) and for
aquatic organisms (Ward et al., 1981).
mM acetate buffer, pH 3.0, with 20 mM KCl at room
temperature. From 0.4 pmol to 0.2 nmol of the purified
enzyme were used in the mixtures. Reactions were star-
ted by addition of 1 mM H2O2 and monitored by HPLC.
Reaction rates were measured from the differences in
peak area after 10 min and referred to the purified pro-
tein concentration for specific activity calculations.
Reported values are the mean of three replicates. Specific
reaction rates are given as mol of substrate converted
per mol of enzyme per minute or simply in minÀ1. Reac-
tions for 5 and 6 aromatic rings PAHs 7,12-dimethylbenz-
anthracene, benzo[a]pyrene, 7-methylbenzo[a]pyrene,
benzo[ghi]perylene and perylene were also monitored by
fluorescence spectrum, with an excitation at 300 nm, in
a Luminescence spectroꢀmeter, Perkin-Elmer, Model LS
50, during 2 min at 25 C. The CPO activity was mea-
sured as the disappearance of the respective maximum
emission peak for each PAH over a two minute reaction.
3. Conclusions
Chloroperoxidase from the imperfect fungus Caldar-
iomyces fumago is the most versatile enzyme in the
hemoprotein family (Yi et al., 1999). CPO performs
halogenase, peroxidase, catalase and cytochrome P450-
like reactions. However, under our reaction conditions
and with PAHs as substrates, CPO only acts as halo-
genase and no oxygenated products could be detected.
In contrast, peroxidase activity on aromatic compounds
produces mainly quinones, such as in the case of lignin
peroxidase (Hammel et al., 1986; Vazquez-Duhalt et al.,
1994) and manganese peroxidase (Bogan et al., 1996).
Caldariomyces fumago has been isolated from damp
sites and also has been reported as a marine fungus
(Dawson and Sono, 1987; Colonna et al., 1999). Chloro-
peroxidase, as other halogenases, is an extracellular
enzyme that can react with a variety of substrates in the
microbial environment. This enzyme is able to catalyze
PAH-DNA adduct formation in vitro (Marquez-Rocha
et al., 1997), suggesting the production of genotoxic
aromatic intermediates. The present work shows that
the transformation of aromatic pollutants into chlori-
nated derivatives by microbial enzymes may occur in
polluted sites. This biocatalytic process should be con-
sidered because the toxicity and environmental impact
of aromatic compounds may be increased.
4.3. Analytical methods
Substrate concentration was measured in a Perkin-
Elmer (series 200) HPLC system, using a C18 Hypersyl 5
mm Hewlett-Packard column eluted with an acetoni-
trile–water (70:30 v/v) solvent mixture. Substrate and
product detection was carried out using a diode array
detector coupled to the HPLC system. Product identifi-
cation was performed in a Hewlett-Packard GC (model
6890)-MS (model 5972) equipped with a SPB-20 column
(30 mÂ0.25 mm, Supelco). The temperature program
ꢀ
started at 100 C for 2 min; the temperature was raised
to 290 ꢀC at a rate of 8 ꢀC/min and kept at 290 ꢀC for 10
min.
Acknowledgements
4. Experimental
This work was supported by The National Council of
Science and Technology of Mexico (CONACYT grant
33611-U) and by The Mexican Oil Institute (grant FIES
98-110-VI).
4.1. Chemicals
Purified CPO [EC 1.11.1.10] from C. fumago 89362
(Commonwealth Mycological Institute, Kew, Surrey,
UK) was produced in a fructose medium and purified
according to Pickard et al. (1991); all preparations used
in this study had an Rz=1.36, which corresponds to
95% purity. Hydrogen peroxide and buffer salts were
obtained from J.T. Baker (Phillisburg, NJ). Polycyclic
aromatic hydrocarbons were purchased from Aldrich
Chemical (Milwaukee, WI). HPLC-grade acetonitrile
and methylene chloride were purchased from Fisher
Scientific (Springfield, NJ).
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