Substrate specificity and ionization potential in chloroperoxidase- catalyzed oxidation of diesel fuel

Article abstract of DOI:10.1021/es991270o

Straight-run diesel fuel containing 1.6% of sulfur was enzymatically oxidized with chloroperoxidase from Caldariomyces rumago. Most organosulfides and thiophenes were transformed to form sulfoxides and sulfones. The oxidized organosulfur compounds can be effectively removed by distillation. The resulting fraction after distillation contained only 0.27% sulfur, while the untreated straight-run diesel fuel after the same distillation process still showed 1.27% sulfur. To know the chemical nature of the products, nine organosulfur compounds and 12 polycyclic aromatic compounds (PACs) were transformed by chloroperoxidase in the presence of chloride and hydrogen peroxide. Organosulfur compounds were only oxidized to form sulfoxides and sulfones, and no chlorinated derivatives were detected, except for bithiophene. In contrast, PACs were exclusively chlorinated, and no oxidized derivatives could be found. No enzymatic activity was detected on PACs with an ionization potential higher than 8.52 eV, while in the lower region it was found that the higher the ionization potential of the PAC the lower the specific activity. On the other hand, the substrate ionization potential did not seem to influence chloroperoxidase activity in the oxidation of organosulfur compounds. All organosulfur compounds tested were oxidized by chloroperoxidase. From double-substrate experiments, it appears that organosulfur compounds are oxidized by both compound I and compound X enzyme intermediates, while PACs react only with the halogenating intermediate, compound X. Straight-run diesel fuel containing 1.6% of sulfur was enzymatically oxidized with chloroperoxidase from Caldariomyces fumago. Most organosulfides and thiophenes were transformed to form sulfoxides and sulfones. The oxidized organosulfur compounds can be effectively removed by distillation. The resulting fraction after distillation contained only 0.27% sulfur, while the untreated straight-run diesel fuel after the same distillation process still showed 1.27% sulfur. To know the chemical nature of the products, nine organosulfur compounds and 12 polycyclic aromatic compounds (PACs) were transformed by chloroperoxidase in the presence of chloride and hydrogen peroxide. Organosulfur compounds were only oxidized to form sulfoxides and sulfones, and no chlorinated derivatives were detected, except for bithiophene. In contrast, PACs were exclusively chlorinated, and no oxidized derivatives could be found. No enzymatic activity was detected on PACs with an ionization potential higher than 8.52 eV, while in the lower region it was found that the higher the ionization potential of the PAC the lower the specific activity. On the other hand, the substrate ionization potential did not seem to influence chloroperoxidase activity in the oxidation of organosulfur compounds. All organosulfur compounds tested were oxidized by chloroperoxidase. From double-substrate experiments, it appears that organosulfur compounds are oxidized by both compound I and compound X enzyme intermediates, while PACs react only with the halogenating intermediate, compound X.

Full text of DOI:10.1021/es991270o

Environ. Sci. Technol. 2000, 34, 2804-2809
in gasoline (2). The plan would cut the average sulfur level
in gasoline by 90%, from an average of about 330 ppm to 30
ppm, by 2004.
Substrate Specificity and Ionization
Potential in
Microbial desulfurization of fossil fuels has been under
active investigation for several decades and has been recently
reviewed (3-5). Research groups and companies worldwide
are developing the technology for fuel biodesulfurization,
the most successful being a unique refinery process using
bacteria to selectively remove sulfur from diesel. The patented
bacteria, Rhodococcus IGTS8, has been genetically engineered
to increase both activity and stability (6).
Chloroperoxidase-Catalyzed
Oxidation of Diesel Fuel
M A R C E L A A YA L A , * , †
N O R M A R . R O B L E D O , ‡
A G U S T I N L O P E Z - M U N G U I A , † A N D
R A F A E L V A Z Q U E Z - D U H A L T †
Recently, we have described an enzymatic method for
fuel desulfurization (7). The method includes the steps of
biocatalytic oxidation of organosulfur compounds contained
in straight-run diesel fuel by chloroperoxidase from Cal-
dariomyces fumago, followed by a distillation process in which
the oxidized compounds are removed. Chloroperoxidase
from Caldariomyces fumago (CPO) (EC 1.11.1.10) is a versatile
heme enzyme because of its catalytic diversity. CPO 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. This unusual combination of
enzymatic activities is the origin of a number of studies
involving CPO as a catalyst with potential applications,
including the petroleum industry. It has been demonstrated
that CPO is able to remove nickel and vanadium from
asphaltene fractions (8). CPO is also able to perform
interesting reactions, like the enantioselective epoxidation
of alkenes (9), oxidation of phenolic pollutants (10, 11),
oxygenation of sulfides (7, 12), oxidation of organophos-
phorus pesticides (13), and the determination of genotoxic
potential of pollutants (14), to give only a few examples.
However, the catalytic mechanism of CPO has not been
completely established, and the exact role of chloride and
the identity of the halogenating species remains a subject of
controversy (15-18).
The ability of fungal peroxidases to biotransform petro-
leum compounds, such as polyaromatic hydrocarbons
(PAHs), has been investigated before, specially lignin per-
oxidase (LiP) and manganese peroxidase (MnP) from Phan-
erochaete chrysosporium. These nonspecific extracellular
enzymes are believed to be involved in pollutant biotrans-
formation. Interestingly, the activity of LiP and MnP correlates
with the ionization potential (IP) of the PAHs. A threshold
IP value was found for each enzyme. LiP oxidizes PAHs with
IP e 7.55 eV as well as some heterocyclic compounds with
IP e 8 eV(19, 20), while MnP oxidizes PAHs with IPs as high
as 8.1 eV (21, 22). With this evidence it was possible to
distinguish whether a substrate was transformed via an
electron subtraction process.
Institute of Biotechnology, UNAM, Apartado Postal 510-3,
Cuernavaca, Morelos 62271, Mexico, and CEPROBI, IPN,
Yautepec, Morelos, Mexico
Straight-run diesel fuel containing 1.6% of sulfur was
enzymatically oxidized with chloroperoxidase from
Caldariomyces fumago. Most organosulfides and thiophenes
were transformed to form sulfoxides and sulfones. The
oxidized organosulfur compounds can be effectively removed
by distillation. The resulting fraction after distillation
contained only 0.27% sulfur, while the untreated straight-
run diesel fuel after the same distillation process still
showed 1.27% sulfur. To know the chemical nature of the
products, nine organosulfur compounds and 12 polycyclic
aromatic compounds (PACs) were transformed by
chloroperoxidase in the presence of chloride and hydrogen
peroxide. Organosulfur compounds were only oxidized to
form sulfoxides and sulfones, and no chlorinated derivatives
were detected, except for bithiophene. In contrast, PACs
were exclusively chlorinated, and no oxidized derivatives
could be found. No enzymatic activity was detected on
PACs with an ionization potential higher than 8.52 eV, while
in the lower region it was found that the higher the
ionization potential of the PAC the lower the specific
activity. On the other hand, the substrate ionization potential
did not seem to influence chloroperoxidase activity in
the oxidation of organosulfur compounds. All organosulfur
compounds tested were oxidized by chloroperoxidase.
From double-substrate experiments, it appears that
organosulfur compounds are oxidized by both compound I
and compound X enzyme intermediates, while PACs
react only with the halogenating intermediate, compound
X.
Considering that both organosulfur and PACs are con-
tained in diesel fuel, in the present work the enzymatic activity
of CPO toward a group of several organosulfur compounds
(thiophenes and organic sulfides) and PACs was determined.
The chemical nature of reaction products and the role of the
substrate ionization potential were analyzed.
Introduction
The environmental driver for diesel sulfur reduction is well-
established. Meeting sulfur regulations on petroleum prod-
ucts is driving up the cost of refining, because conventional
hydrodesulfurization becomes increasely expensive and less
efficient in handling sulfur removal as lower and lower sulfur
levels are reached (1). In the United States, there are plans
to greatly reduce motor-vehicle emissions and sulfur content
Experimental Section
Chem icals. Purified CPO from Caldariomyces fumago was
produced in a fructose medium and purified according to
Pickard (23); 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 compounds and aro-
matic thiophenes and sulfides were purchased from Aldrich
* Corresponding author phone: +52 73 291619; fax: +52 73
172388; e-mail: maa@ibt.unam.mx. Mailing address: Apartado Postal
510-3, Cuernavaca, Morelos 62271, Mexico.
† Institute of Biotechnology, UNAM.
‡ CEPROBI, IPN.
9
2 8 0 4 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 13, 2000
10.1021/es991270o CCC: $19.00
2000 Am erican Chem ical Society
Published on Web 05/31/2000

tometrically at 288 nm (MCD) and either 254 nm (thian-
threne) or 335 nm (pyrene).
TABLE 1. Sulfur Content of Straight-Run Diesel Fuel after
Enzymatic Oxidation with Chloroperoxidase from
Caldariomyces fumago Followed by a Distillation to
325 °C as Final Distillation Point
Kinetic Constants Determ ination. Reactions were per-
formed in 1 mL of 60 mM acetate buffer pH 3.0, 20 mM KCl
and either 15% for thianthrene or 20% acetonitrile for MCD
and pyrene. Reaction was started by addition of 1 mM H2O2.
The initial reaction rates were obtained by following the
decrease in absorbance at 254 nm for thianthrene (ꢀ )
35 mM-1 cm-1) and at 335 nm for pyrene (ꢀ ) 32.6 mM-1
cm-1).
distillation
enzymatic + distillation
a
TPH (%)
sulfur (%)
TPH (%)
sulfur (%)
distillate
residue
83
17
1.27
3.21
71
29
0.27
5.51
a
Total petroleum hydrocarbons.
Analytical Methods. Substrate concentration was mea-
sured in a Perkin Elmer (series 200) HPLC system, using a
C
18 Hypersyl 5 µm Hewlett-Packard column and eluted with
Chemical (Milwaukee, WI). HPLC-grade acetonitrile and
methylene chloride were purchased from Fisher Scientific
(Springfield, NJ).
an acetonitrile-water (70:30 v/ v) solvent mixture. Substrate
and products detection was carried out using a diode array
detector coupled to the HPLC system. The used wavelengths
for detection (λdet) are listed in Tables 3 and 4. Other UV
measurements were made in a Beckman Spectrophotometer
(DU 530). Product identification was performed in a Hewlett-
Packard GC (model 6890)-MS (model 5972) equipped with
a SPB-20 column (30m × 0.25 mm, Supelco). The GC system
was coupled to both a flame ionization detector (FID, general
detector) and a flame photometric detector (FPD, specific
sulfur detector). 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.
Reaction Conditions. Diesel fuel oxidations with chlo-
roperoxidase were carried as previously reported (7). Oxida-
tion reactions of individual organosulfur and aromatic
compounds were carried out in a 1-mL reaction mixture
containing 20 µM substrate and 15% acetonitrile in a 60 mM
acetate buffer, pH 3.0, with or without 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 started
by addition of 1 mM H2O2. Reaction rates were estimated by
monitoring the substrate peak in a HPLC system equipped
with a diode array detector. Enzyme activities were obtained
from the differences in peak area after 10 min of reaction,
transformed by a standard curve, and adjusted for protein
concentration. Reported values are the mean of three
replicates. Specific reaction rates are given as mol of substrate
Microdistillations were carried out according to the
standard test for boiling range distribution of petroleum
fractions by gas chromatography, ASTM D 2887-89. Organic
sulfur content on diesel fuel were determined by X-ray
fluorimetry in a Horiba X-ray fluorimeter. Total petroleum
hydrocarbons (TPH) were estimated by the USEPA 8015
method (modified).
converted per mol of enzyme per minute or simply in min-1
.
For products identification, 10-mL reactions were performed;
after 1 h, the mixture was acidified and extracted with
methylene chloride, and the extract was reduced under
nitrogen, before being analyzed by GC-MS.
Two-Substrate Reactions. Reaction mixtures contained
either 20 µM thianthrene or 30 µM pyrene and 100 µM
monochlorodimedone (MCD) in 15% acetonitrile in a 20 mM
KCl, 60 mM acetate buffer pH 3.0. The reaction was started
by addition of 0.25 mM H2O2 and monitored spectropho-
Results
Sraight-run diesel fuel, obtained from primary distillation
and containing 1.6% sulfur, was oxidized with chloroper-
oxidase in the presence of 20 mM KCl and 1 mM hydrogen
peroxide. The gas chromatographic analysis with both flame
TABLE 2. Mass Spectral Data of Productsa
substrate
product
mass spectral ions (m/z)
benzothiophene
benzothiophene sulfone
166 (42) [M+], 138 (9), 137 (100), 118 (15), 109 (48), 90 (14), 89 (16), 76 (15),
75 (15), 74 (14), 65 (9), 63 (13)
diphenyl sulfide
diphenyl sulfone
218 (27) [M+], 125 (100), 97 (26), 77 (53), 51 (47), 50 (16)
dibenzothiophene dibenzothiophene sulfone 216 (100) [M+], 187 (46), 160 (31), 150 (16), 139 (30)
thianthrene
5-thianthrene oxide
5,10-thianthrene dioxide
dichloroacenaphthene
trichloroacenaphthene
9,10-dichloroanthracene
dichlorobiphenylene
trichlorobiphenylene
dichlorofluorene
232 (16) [M+], 184 (100), 171 (15), 139 (14), 69 (14)
248 (77) [M+], 200 (86), 184 (84), 171 (100), 168 (23), 139 (30), 108 (24), 69 (36)
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)
248 (68), 246 (100) [M+], 176 (43), 87 (10)
acenaphthene
anthracene
biphenylene
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)
238 (25), 237 (7), 236 (40) [M+], 201 (31), 199 (18), 166 (63), 165 (100), 164 (17),
163 (24), 100 (11), 82 (35)
fluorene
phenanthrene
pyrene
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)
chloropyrene
dichloropyrene
chlorotriphenylene
238 (31), 236 (100) [M+], 200 (34), 100 (12)
272 (62), 270 (100) [M+], 235 (11), 200 (53), 135 (12), 100 (23)
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)
238(15), 236 (72) [M+], 234 (100), 201 (36), 164 (45), 157 (28), 155 (76), 142 (10),
119 (21), 93 (17), 82 (19), 79 (14), 69 (30)
triphenylene
bithiophene
dichlorobithiophene
trichlorobithiophene
tetrachlorobithiophene
272 (37), 271 (11), 270 (100) [M+], 233 (58), 198 (81), 191 (53), 189 (82), 163 (15),
154 (33), 135 (18), 119 (37), 103 (19), 93 (39), 81 (46), 79 (58), 69 (36), 58 (12)
308 (13), 306 (49), 304 (96) [M+], 302 (71), 267 (52), 232 (44), 223 (39), 197 (22),
188 (30), 162 (12), 153 (76), 117 (72), 98 (11), 93 (36), 81 (74), 79 (100), 69 (26)
a
Values in parentheses are relative abundances.
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TABLE 3. Specific Activity of CPO with Organosulfur
Compounds
specific
det (nm) IP (eV) activity (min-1
λ
)
1
2
3
4
5
6
7
8
9
thianthrene
254
300
248
232
226
254
238
256
240
7.80
7.83a
7.88
8.39
8.73
8.80
8.90
8.95
9.40
1310 (( 132)
2,2′-bithiophene
diphenyl sulfide
dibenzothiophene
benzothiophene
ethyl phenyl sulfide
benzenethiol
840 (( 8)
831 (( 32)
126 (( 9)
557 (( 42)
1725 (( 145)
116 (( 5)
2917 (( 58)
352 (( 10)
thioanisole
diphenyl disulfide
a
IP m easured by charge transfer (25).
TABLE 4. Specific Activity of CPO with Aromatic Compounds
specific
λ
det (nm) IP (eV) activity (min-1
)
1
2
3
4
5
6
7
8
9
azulene
9-m ethylanthracene
anthracene
biphenylene
2-m ethylanthracene
pyrene
acenaphtene
fluorene
270
254
250
248
248
236
226
260
236
250
256
220
250
280
260
7.43a 676 (( 34)
7.46
7.51
7.56a
7.70
7.72
7.73a
7.91
7.95a
8.07
8.10
8.18
8.64
8.77
9.43
758 (( 27)
134 (( 14)
10 (( 0.5)
107 (( 8)
53 (( 6)
FIGURE 1. Microdistillation of untreated and chloroperoxidase-
oxidized straight-run diesel fuel: FID, flame ionization detector
(general detector) and FPD, flame photometric detector (sulfur
selective detector).
65 (( 8)
thiophenes, organic sulfides, and thiols, and 15 aromatic
compounds were tested for chloroperoxidase transformation.
Table 2 shows the products identified by GC-MS. Products
from all the organosulfur compounds were their respective
sulfoxides and sulfones, except for biothiophene for which
chlorinated derivatives were detected. Sulfones are the final
product of CPO reactions; successive additions of both
enzyme and H2O2 to complete substrate modification did
not change the chemical nature of the products. In addition,
sulfone standard compounds were not substrate for CPO as
determined by GC and HPLC methods.
1.9 (( 0.13)
3 (( 0.2)
7 (( 0.1)
0.8 (( 0.09)
0.6 (( 0.01)
NRb
fluoranthene
10 phenanthrene
11 triphenylene
12 naphthalene
13 biphenyl
14 dibenzofuran
15 anthrone
NRb
NRb
a
b
IP m easured by photoelectron spectroscopy (25). NR: no reaction
detected.
ionization (FID) and flame photometric (FPD) detectors
showed that chloroperoxidase was able to oxidize most of
organosulfur compounds contained in the diesel fuel. The
oxidation was detected by the increase of boiling point
(retention time) of these compounds on the gas chromato-
gram monitored by the sulfur selective detector (FPD).
Microdistillation of both chloroperoxidase-oxidized and
untreated diesel fuels monitored by FID (general detection)
and FPD (sulfur selective detection) (Figure 1) shows that
the hydrocarbon distillation profile changes slightly after
enzymatic treatment. In contrast, the specific sulfur detector
(FPD) shows a significant change of the distillation profile,
in which most of organosulfur compounds were effectively
oxidized and their boiling points increased after enzymatic
treatment.
Oxidized sulfur compounds can be removed by a distil-
lation process (Table 1). After distillation, the sulfur content
in the enzymatically oxidized diesel fuel is only 0.27%, while
for the untreated fuel is 1.27%. The distillation of the straight-
run diesel fuel (1.6% sulfur) to a final distillation point of 325
°C produced a distillate containing 66% of the total sulfur,
while if the diesel fuel is previously oxidized with chloro-
peroxidase, the obtained distillate contained only 12% of the
total sulfur. Thus, by using an enzymatic oxidation with
chloroperoxidase coupled with a distillation process it is
possible to obtain a diesel fuel with six times lower sulfur
concentration than straight-run diesel fuel. Few hydrocar-
bons are also transformed during the enzymatic treatment,
and after distillation an additional 12% of them remain in
the residue (Table 1).
Aromatic hydrocarbons are also important constituents
of diesel fuel. It is well know that chloroperoxidase is able
to transform some PAHs (14, 24). Twelve of the 15 PACs
tested were transformed by CPO in the presence of 1 mM
H2O2 and 20 mM KCl, as monitored by HPLC. GC-MS analysis
of the reaction products showed that the substrates were
exclusively chlorinated during the reaction (Table 2). Fur-
thermore, in the absence of chloride there was not observable
reaction.
Tables 3 and 4 show the specific activity of CPO and IP
values for the organosulfur and PACs compounds assayed.
The ionization potentials (IP) taken are measured by electron
impact, except for azulene, biphenylene, fluoranthene, and
bithiophene (25). Figure 2 shows the correlation between IP
values and specific activity for PACs and organosulfur
compounds.
To determine the effect of the presence of a good substrate
for halogenation, such as monochlorodimedone (MCD),
thianthrene oxidation and pyrene halogenation reactions
were performed in the presence of 0.1 mM MCD (Figure 3).
Under these conditions, thianthrene was initially oxidized
to form a sulfoxide with a significantly low rate (Table 3).
Once MCD was exhausted, thianthrene oxidation rate became
similar to that found in the absence of MCD (Figure 3a). In
the case of pyrene, halogenation did not start until all MCD
was halogenated, suggesting a strong affinity of MCD for the
enzyme (Figure 3b). Under our experimental conditions, the
specific activity of halogenation of MCD is 3480 min-1. The
specific reaction rate is only slightly affected in the presence
of pyrene (3200 min-1). On the other hand, the presence of
thianthrene decreases the specific reaction rate for haloge-
nation of MCD (2830 min-1), while the initial rate for
To know the chemical nature of the products from the
enzymatic reaction, nine organosulfur compounds, including
9
2 8 0 6 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 13, 2000

TABLE 5. Kinetic Constants for Thianthrene Oxidation and
Pyrene Halogenation
substrate
kcat (s-1
)
KM (µM)
kcat/KM (µM-1 s-1
)
MCD
thianthrene
pyrene
94
64
37
1.4
1.5
32
67
44
1.2
as can be seen from the catalytic efficiencies kcat/ KM. Though
both the affinity and the catalytic constant are higher for
thianthrene, the main effect comes from the affinity of the
enzyme for the substrate, which is 1 order of magnitude lower
for pyrene.
Discussion
Enzymatic oxidation of diesel fuel allows the organosulfur
compounds to be separated by a single distillation process.
Chloroperoxidase from C. fumago is a very active enzyme
able to perform transformation of complex oil fractions, such
as diesel (7) and asphaltenes (8). Chloroperoxidase shows
three different catalytic activities: halogenase, peroxidase,
and catalase (26-28). In addition, some reports have claimed
that chloroperoxidase catalyzes two-electron reactions (per-
oxygenase), which could be considered a kind of monooxy-
genase activity (29-32). Nevertheless, when organosulfur
compounds such as thiophenes and organosulfides are
substrates, mainly sulfoxides are formed by the peroxidase
activity (Table 2 and Figure 1). All nine organosulfur
compounds tested were oxidized by chloroperoxidase, even
when the reaction system contained 20 mM KCl (Table 3),
except for 2,2′-bithiophene from which halogenated deriva-
tives were detected. These results are in agreement with
previous work reporting that sulfoxides are produced from
chloroperoxidase activity (30, 32, 33).
FIGURE 2. Influence of the substrate ionization potential on the
specific activity of CPO. Substrates are organosulfur compounds
(b) and polycyclic aromatic compounds (O). Numbers in superior
and inferior panels correspond to those in Tables 3 and 4,
respectively.
On the other hand, polycyclic aromatic compounds (PACs)
are halogenated (Table 2). Other peroxidases, such as lignin
peroxidase (19, 20) and manganese peroxidase (21, 22) and
even hemoproteins with peroxidase activity (24, 34), produce
mainly quinones from PAHs oxidation. Specific activity of
chloroperoxidase on PACs halogenation shows a clear
correlation with the substrate ionization potential (Figure
2). Because ionization potential could be defined as the energy
involved in taking out one electron from the substrate
molecule, this correlation suggests a one-electron mechanism
with a free radical-mediated reaction. Only PACs with
ionization potential lower than 8.52 eV were halogenated
(Table 4). In general, the lower the ionization potential of the
PAC, the higher the specific activity of the chloroperoxidase
for that substrate (Figure 2). The ionization potential value
of 8.52 eV appears to be a threshold, as none of the
compounds tested having higher ionization potentials were
transformed by chloroperoxidase. This threshold value is
significantly higher than those reported for other peroxidases.
Lignin peroxidase is able to oxidize PAHs and form quinones
up to a PAHs ionization potential of 8.0 eV (20), and
manganese peroxidase from P. chrysosporium shows a
threshold value for PAHs substrates of 8.1 eV (22). Interest-
ingly, no clear correlation could be found between the
ionization potential and the specific activity for organosulfur
compounds (Figure 2). In fact, we were not able to found a
single organosulfur compound, thiophene or sulfide, which
is not transformed by chloroperoxidase.
FIGURE 3. Competition between (A) MCD and thianthrene and (B)
MCD and pyrene.
thianthrene oxidation is 300 min-1, until MCD is exhausted.
The addition of the MCD halogenation and thianthrene
oxidation rates results in a value close to that obtained with
MCD alone.
Kinetic constants for pyrene halogenation and thianthrene
oxidation were determined (Table 5). Chloroperoxidase is a
more efficient catalyst in the reaction of oxidation of
thianthrene than in the reaction of halogenation of pyrene,
A possible production of chlorinated derivatives from
PAHs by CPO reactions is an undesirable side of the process.
However, as shown in Tables 3 and 4 (enzyme activity on
single substrates) and in Table 5 (affinity constants, KM, for
thianthrene and pyrene), organosulfur compounds are better
substrates and therefore can compete favorably with PAHs.
This means that in a mixture containing both types of
9
VOL. 34, NO. 13, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 2 8 0 7

FIGURE 4. Proposed catalytic cycle of chloroperoxidase.
compounds, the sulfur compounds would be preferentially
oxidized by CPO. Nevertheless, the reaction conditions and
the biocatalyst preparation should be designed to minimize
the halogenation reactions because of the environmental
implications of chlorinated aromatic compounds.
1 order of magnitude lower. Under these conditions the
available halogenating active sites are readily saturated by
MCD; pyrene, which is unable to compete for compound X,
is transformed only until MCD is exhausted.
Chloroperoxidase from C. fumago catalyzes the oxidation
of most of organosulfur compound found in straight-run
diesel fuel. This oxidation allows the desulfurization of diesel
fuel by distillation. Sulfoxides and sulfones are the main
products from CPO reaction on organosulfur compounds,
while halogenated aromatic compounds are the only prod-
ucts from PACs reactions. Furthermore, PACs halogenation
by chloroperoxidase seems to be dependent on the substrate
ionization potential. In general, PACs with an ionization
potential of < 8.52 eV were halogenated. Our results support
a free radical mechanism for enzymatic halogenation and a
catalytic cycle in which compound X [ClO(FeIII)porphyrin]
could be responsible for both substrate halogenation and
oxidation in a chlorine-dependent process.
So far, the catalytic cycle of chloroperoxidase is not
completely elucidated (15, 16, 18, 35). The proposed mech-
anism (Figure 4) includes a first activation step, in which
hydrogen peroxide transforms the (FeIII)porphyrin group
(native state) to oxo(FeIV)porphyrin radical cation (compound
I). Then compound I can follow two ways: the oxidation of
a substrate molecule to form an oxo(FeIV)porphyrin without
the associated porphyrin π-radical cation (compound II) or
the reaction with a chlorine ion to form a ClO(FeIII)porphyrin
group, called compound X, which is the only responsible for
the enzymatic reaction of halogenation. In addition, this
compound X seems also to be able to perform oxidation
reactions liberating a chlorine ion. After both reactions,
compound X returns to the native (FeIII)porphyrin state. From
this proposed mechanism, it seems that the organosulfur
compounds are able to react with both compound I and
compound X, while PACs are only reactive to compound X.
This is in agreement with our results, as when a high affinity
halogenation substrate (MCD) is present in the medium, a
slow thianthrene oxidation is found (Figure 3a). The oxidation
rate is lower because most of compound I is rapidly
transformed to compound X, due to rapid compound X
turnover by MCD reaction. Thus the observed thianthrene
oxidation is mainly mediated by compound X. When MCD
is exhausted, thianthrene competes more favorably with the
chlorine ions for compound I, its transformation involving
both forms: compound I and compound X. This competition
between a halogenation substrate (MCD) and a peroxidase
substrate (catechol) has been previously reported (35). In
this case, MCD quantitatively replaces catechol as a substrate
for part of the enzymatic reaction. In contrast, and as
expected, chloroperoxidase is not able to react with pyrene
when MCD is present in the medium (Figure 3b), a situation
that can be explained by the significant differences between
the catalytic efficiencies of MCD (kcat/ KM ) 55 µM-1 s-1) and
pyrene (kcat/ KM ) 1.2 µM-1 s-1). The main effect comes from
the different affinity of chloroperoxidase for the substrates,
whereas for MCD KM ) 1.2 µM, and for pyrene KM ) 32 µM,
The broad specificity and high activity of chloroperoxidase
encourage further investigation in the use of this enzyme as
an efficient catalyst in a desulfurization process, including
an enzymatic treatment followed by a fractional distillation
step. Sulfur removal from a very complex mixture, such as
petroleum fractions, is far from being accomplished. Con-
ventional hydrodesulfurization becomes expensive and less
efficient as lower and lower sulfur levels are reached. The
biotechnological process could be applied after a conven-
tional desulfurization process in order to reach these new
regulatory low-levels for sulfur content in fuels. At the
moment, the use of chloride as an activator in this process
seems unavoidable, since in this hydrophobic medium,
chloroperoxidase presents very low activity and chloride
greatly improves the reaction rate. Unfortunately, the pres-
ence of halogens would yield some environmentally unde-
sirable products. Our research is currently focused on the
protein engineering of chloroperoxidase in order to reduce
its halogenase activity, maintaining or increasing the per-
oxidase activity. In addition, different approaches to improve
the stability of chloroperoxidase are under research, such as
genetic engineering and cross-linking of enzyme crystals.
The stabilization of enzymes in non-conventional low-water
content medium is a priority for the successful development
of industrial enzymatic processes.
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Acknowledgments
This work was supported by DGPA-UNAM grant IN 220598
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Received for review November 10, 1999. Revised manuscript
received March 16, 2000. Accepted April 5, 2000.
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