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Applied and Environmental Microbiology, March 1999, p. 923-928, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Mechanism of Peroxidase Inactivation in Liquid
Cultures of the Ligninolytic Fungus Pleurotus
pulmonarius
Brigitte
Böckle,
María Jesús
Martínez,
Francisco
Guillén, and
Ángel T.
Martínez*
Centro de Investigaciones Biológicas,
Consejo Superior de Investigaciones Científicas, E-28006
Madrid, Spain
Received 20 July 1998/Accepted 2 December 1998
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ABSTRACT |
It has recently been reported that Pleurotus
pulmonarius secretes a versatile peroxidase that oxidizes
Mn2+, as well as different phenolic and nonphenolic
aromatic compounds; this enzyme has also been detected in other
Pleurotus species and in Bjerkandera species.
During culture production of the enzyme, the activity of the main peak
was as high as 1,000 U/liter (measured on the basis of the
Mn3+-tartrate formation) but this peak was very ephemeral
due to enzyme instability (up to 80% of the activity was lost within
15 h). In culture filtrates inactivation was even faster; all
peroxidase activity was lost within a few hours. Using different
inhibitor compounds, we found that proteases were not responsible for
the decrease in peroxidase activity. Peroxidase instability coincided with an increase in the H2O2 concentration,
which reached 200 µM when filtrates were incubated for several hours.
It also coincided with the onset of biosynthesis of anisylic compounds
and a decrease in the pH of the culture. Anisyl alcohol is the natural
substrate of the enzyme aryl-alcohol oxidase, the main source of
extracellular H2O2 in Pleurotus
cultures, and addition of anisyl alcohol to filtrates containing stable
peroxidase activity resulted in rapid inactivation. A decrease in the
culture pH could also dramatically affect the stability of the P. pulmonarius peroxidase, as shown by using pH values ranging from
6 to 3.25, which resulted in an increase in the level of inactivation
by 10 µM H2O2 from 5 to 80% after 1 h.
Moreover, stabilization of the enzyme was observed after addition of
catalase, Mn2+, or some phenols or after dialysis of the
culture filtrate. We concluded that extracellular
H2O2 produced by the fungus during oxidation of
aromatic metabolites is responsible for inactivation of the peroxidase
and that the enzyme can protect itself in the presence of different
reducing substrates.
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INTRODUCTION |
Lignin degradation by basidiomycetes
belonging to the genus Pleurotus is being investigated
because of the industrial potential of some of these fungi for
selectively removing lignin from wheat straw (26, 32). It
has been shown by using in vivo 14C labeling that
Mn2+ stimulates lignin mineralization by Pleurotus
pulmonarius under solid-state fermentation (SSF) conditions
(5). This suggests that Mn3+ is involved;
Mn3+ can be generated directly by the
Mn2+-oxidizing peroxidases secreted by ligninolytic fungi
(22, 38) or indirectly by other ligninolytic enzymes
(36). In the presence of chelators secreted by fungi
(30), the Mn3+ formed could be responsible for
the attack on lignin "at a distance" by the fungal mycelium. This
degradation pattern, which is characteristic of extensive fungal
delignification of wood in nature (3), has been found in
straw treated with Pleurotus species (32).
Seven extracellular Mn2+-oxidizing peroxidases have been
purified from liquid and SSF cultures of Pleurotus eryngii
and P. pulmonarius and characterized (33). Two
additional peroxidases have been obtained from liquid cultures of
Pleurotus ostreatus (2, 42). All of these enzymes
efficiently oxidize Mn2+ to Mn3+ and therefore
have been described as Mn peroxidases. However, six of them, including
the enzymes from P. pulmonarius, exhibit activity on
phenolic and nonphenolic aromatic substrates and dyes, and the optimum
pH for Mn-independent reactions is around 3 (24, 33, 34).
Because of this they can be considered representatives of a new type of
peroxidase with catalytic properties intermediate between the catalytic
properties of Phanerochaete chrysosporium Mn-dependent
peroxidases (MnP), which require Mn2+ to complete the
catalytic cycle, and the catalytic properties of lignin peroxidase
(LiP) (29). Molecular characterization of the new
peroxidases isolated from P. eryngii has revealed that on
the basis of amino acid sequence and molecular architecture these
enzymes are more similar to P. chrysosporium LiP than to MnP
(41) and that they have an Mn-binding site which accounts for their ability to oxidize Mn2+ (25).
Peroxidases with similar catalytic properties have been found recently
in Bjerkandera adusta (23) and
Bjerkandera sp. (35).
P. pulmonarius is a strongly ligninolytic fungus, as
revealed by the greater wheat lignin mineralization by this organism than by P. chrysosporium or other Pleurotus
species (5). High levels of the new peroxidase described
above are produced by this fungus in peptone-containing media. However,
the main activity peak is very ephemeral. Efficient production and
purification of this enzyme from liquid cultures are hampered by the
very rapid decline in peroxidase activity that occurs. A similar
phenomenon has been described for production of enzymes by other
ligninolytic fungi, and proteinases have been found to be largely
responsible for this decline in enzyme activity (13, 40). In
the present work we investigated the causes of peroxidase instability
in P. pulmonarius cultures and ways to protect the enzyme
against inactivation.
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MATERIALS AND METHODS |
Fungi and culture conditions.
P. pulmonarius CBS
507.85 (= IJFM A578) was grown in 2% glucose-0.2% yeast
extract-0.5% peptone medium containing 1 g of
H2PO4 per liter and 0.5 g of
MgSO4 per liter (pH 5.5) (28). A homogenized 7-day-old culture in the same medium was used as the inoculum (4%,
vol/vol), and incubation was carried out at 28°C and 200 rpm. Samples
were collected after different incubation periods, filtered, and
analyzed directly or stored at 
80°C.
Enzymatic activities.
P. pulmonarius peroxidase
activity was generally estimated on the basis of the formation of an
Mn3+-tartrate complex (
238, 6,500 M
1 cm1) during oxidation of 0.1 mM
MnSO4 in 0.1 M sodium tartrate (pH 5) containing 0.1 mM
H2O2. In experiments which included EDTA or
compounds with high levels of UV absorbance, the peroxidase activity
was estimated by measuring the direct oxidation of 2.5 mM
2,6-dimethoxyphenol (469, 27,500 M1
cm1) in 0.1 M sodium tartrate (pH 3) containing 0.1 mM
H2O2 (34). Aryl-alcohol oxidase
(AAO) activity was determined by measuring the amount of veratraldehyde
(310, 9,300 M1 cm1) formed
from 5 mM veratryl alcohol in 0.1 M phosphate buffer (pH 6). Laccase
activity was measured by using 10 mM 2,6-dimethoxyphenol in 0.1 M
sodium tartrate (pH 5). One unit of activity was defined as the amount
of enzyme that transformed 1 µmol of substrate per min.
Proteinase studies.
Proteinase activity was measured by
using 0.25% azocasein in 50 mM phosphate buffer (pH 6). After
incubation for 30 min at 30°C, azocasein was precipitated with
trichloroacetic acid, and the activity in the supernatant was
determined by measuring the absorbance at 440 nm. One unit of
proteinase activity was defined as the amount of enzyme that produced a
change of 1 absorbance unit per min. Additional information concerning
proteinase activity was obtained by adding 0.2% insoluble azo
dye-impregnated collagen (azocoll) (Sigma) to whole cultures or
filtrates and monitoring the release of soluble red-dyed peptides (at
520 nm) during 24 h of incubation at 28°C. Finally, different
compounds were tested as proteinase inhibitors by using azocasein as
the substrate; the compounds tested included phenylmethylsulfonyl
fluoride (PMSF) (0.1 mM), 4-(2-aminoethyl)-benzenesulfonyl fluoride
(AEBSF) (1 mg/ml), EDTA (1 to 5 mM), leupeptin (1 µM), and pepstatin
(1 µM).
Estimation of hydrogen peroxide concentration.
The
H2O2 concentration was estimated by using
horseradish peroxidase (HRP) and phenol red as the substrate
(39). Samples were incubated with 0.01% phenol red in 0.2 M
phosphate buffer (pH 6) containing 2.5 U of HRP per ml in a 950-µl
(total volume) reaction mixture. The reaction was stopped by adding 50 µl of 3 M NaOH. The filtrates were not heated (to inactivate the
enzymes) before the H2O2 contents were
estimated because heating resulted in lower concentrations, and
incubation was limited to 30 s in order to prevent slow oxidation
of phenol red by other components of the culture filtrate.
Analysis of aromatic metabolites.
Ten-milliliter samples of
culture filtrates were adjusted to acid pH values and were extracted
twice with 25 ml of diethyl ether. The water was removed with anhydrous
Na2SO4, the ether was evaporated under an
N2 stream, and the compounds that were extracted were
resuspended in 12.5 µl of pyridine (containing 4 µg of
ethylvanillin as an internal standard) and derivatized with 20 µl of
bis(trimethylsilyl)trifluoroacetamide for 10 min at 50°C. The
trimethylsilyl derivatives were analyzed by gas chromatography-mass spectrometry by using a type SPB1 column (30 m by 0.25 mm [inside diameter]) which was programmed so that the temperature increased from
100 to 280°C at a rate of 4°C min1, and they were
identified by comparing their mass spectra with mass spectra obtained
from the National Bureau of Standards library or mass spectra of
standard compounds. Yields were calculated from the peak areas of the
different compounds and the internal standard and were corrected with
the response factors.
Enzyme purification.
The P. pulmonarius
peroxidase was purified from culture filtrates after 64 h of
incubation in the medium described above. The purification process
included (i) ultrafiltration and dialysis (cutoff, 5 kDa) with 10 mM
sodium tartrate (pH 4.5), (ii) Q-cartridge (Bio-Rad) chromatography (to
remove laccase and pigment), (iii) Sephacryl S-200 chromatography (to
remove AAO), and (iv) Mono-Q chromatography (pH 5) to complete the
purification (34).
Evaluation of peroxidase stability.
Peroxidase instability
during fungal growth was estimated by incubating culture and culture
filtrate samples for several hours at room temperature and then
measuring the remaining activity and H2O2
concentration. The effects of different incubation conditions and
additives were also determined (the compounds and concentrations used
are shown in Tables 1 and
2). Finally, we investigated inactivation
of purified peroxidase (700 U/liter) by H2O2 at
concentrations ranging from 1.4 to 70 µM at different pH values by
using 0.1 M tartrate (pH 3.25 to 5) and 0.1 M phosphate (pH 6).
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TABLE 1.
Effects of different compounds and enzymes as stabilizers
of P. pulmonarius peroxidase: experiments performed with
filtrates from 75-h cultures exhibiting unstable peroxidase activity
(estimated on the basis of Mn3+-tartrate formation)
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TABLE 2.
H2O2 production and inactivation
of P. pulmonarius peroxidase by aromatic compounds and
protection by Mn2+ and enzymes: experiments performed with
filtrates from 64-h cultures exhibiting initially stable peroxidase
activity (estimated on the basis of
Mn3+-tartrate formation)
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RESULTS |
Production and instability of P. pulmonarius peroxidase
in liquid cultures.
The time courses of extracellular peroxidase,
AAO, and laccase activities in a representative culture of P. pulmonarius are shown in Fig. 1. The
culture pH values are also shown, and the minimum pH occurred around
days 4 and 5. The peroxidase activity exhibited an ephemeral peak
around day 3, and there were only a few hours of maximal activity.
Samples were collected from P. pulmonarius cultures after
different growth periods, and enzymatic activities were monitored in
filtrates for several hours. Figure 2
shows the main peroxidase peak together with the time courses of
activity in filtrates. The enzyme became unstable just before the
maximum level of activity was reached in the culture. The greatest
instability in the culture filtrates (45% of the activity was lost
after 1 h) was observed after 81 h of growth. In the same
filtrates laccase and AAO activities did not change appreciably. In
order to determine the cause of the sudden inactivation of peroxidase
and to identify conditions that prevented enzyme loss during
purification, samples were taken before and after the maximum level of
peroxidase activity was observed (corresponding to filtrates with
unstable and stable activities shown in Tables 1 and 2, respectively),
and enzyme inactivation was investigated under different conditions.
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FIG. 1.
Time course of extracellular peroxidase activity
estimated on the basis of Mn3+-tartrate formation ( ),
laccase ( ), AAO ( ), and proteinase ( ) activities, and pH
(.....) during 13 days of growth of P. pulmonarius in
glucose-yeast extract-peptone medium. The data are data from a typical
culture.
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FIG. 2.
Time course of extracellular peroxidase activity
estimated on the basis of Mn3+-tartrate formation () and
AAO activities () in P. pulmonarius cultures during the
main peak of peroxidase activity (Fig. 1). The courses of peroxidase
activity in culture filtrate samples (taken at different time points
and incubated at room temperature) are indicated by arrows. The
corresponding peroxidase instability, expressed as the percentage of
activity lost 1 h after filtering (), is also shown.
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Factors involved in peroxidase instability.
First, we
considered whether extracellular fungal proteinases could be
responsible for the enzyme inactivation observed. As shown in Fig. 1,
the maximal level of extracellular proteinase activity found in
P. pulmonarius cultures was 25 U/liter, and no degradation
of azocoll was observed with whole cultures or filtrates. When
proteinase inhibitors were used, we observed that most of the activity
probably corresponded to serine-type proteinases, since it was
inhibited by PMSF and AEBSF, and exhibited neutral or alkaline optimum
pH values. Moreover, the effects of the proteinase inhibitors PMSF,
AEBSF, EDTA, leupeptin, and pepstatin were tested with an unstable
filtrate in which 50% of the peroxidase was inactivated after 2 h, but none of these inhibitors stabilized the enzyme (Table 1).
Second, peroxidase stabilization by different compounds, including some
enzyme substrates, was tested by using the unstable
filtrate described
above (Table
1). Addition of Mn
2+ resulted in appreciable
stabilization at concentrations greater
than 10 µM. Among the other
divalent cations tested, only Fe
2+ stabilized the enzyme to
some extent. Addition of the phenolic
substrates
p-cresol
and 1,6-dichlorophenol also resulted in appreciable
stabilization.
However, none of the compounds mentioned above
was able to reactivate
the peroxidase once the activity in the
filtrates had been lost. When
purified enzyme was added to preincubated
filtrates that had lost
activity, rapid inactivation of the new
enzyme was observed, suggesting
that an inactivating factor was
present. Because dialysis of the
culture filtrate (against 10
mM phosphate buffer at the same pH as the
culture pH) resulted
in peroxidase stabilization, we concluded that a
low-molecular-weight
compound was involved in
inactivation.
Third, by using scavengers of active oxygen species, we found that
neither the superoxide anion radical (O
2
)
nor the hydroxyl radical (OH
) was involved, since
inactivation was not modified by the presence
of superoxide dismutase
or mannitol (Table
1). However, addition
of catalase resulted in enzyme
stabilization, which indicated
that H
2O
2 could
be the agent causing inactivation of the enzyme.
Moreover, direct
addition of H
2O
2 or generation of
H
2O
2 in cultures
and filtrates from
methoxybenzylic alcohols (see below) led to
peroxidase inactivation. As
described above for Mn
2+ and phenolic substrates, catalase
and/or superoxide dismutase
(200 U/ml) did not restore the activity of
inactivated
peroxidase.
Finally, based on the fact that the decrease in pH observed in cultures
paralleled the decrease in the peroxidase peak (Fig.
1), the influence
of pH on inactivation by H
2O
2 was studied (Fig.
3). Purified enzyme was incubated for
1 h with different H
2O
2 concentrations at
different pH values in the absence of reducing
substrates. The amount
of enzyme used (700 U/liter) corresponded
to 0.14 µM, as calculated
from a specific activity of 115 U/mg
and a molecular mass of 43 kDa
(
5), which was similar to the
concentration found in
cultures. At pH 6 the enzyme was quite
stable in the presence of
H
2O
2 concentrations up to 15 µM (which
corresponded to more than 100 enzyme equivalents). However, at
lower pH
values the stability decreased, and the same
H
2O
2 concentrations
resulted in 70 to 80%
peroxidase inactivation at pH values ranging
from 3.75 to 3.25.
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FIG. 3.
Effect of pH on peroxidase inactivation by
H2O2. Purified enzyme from P. pulmonarius (700 U/liter, as estimated on the basis of
Mn3+-tartrate formation) was incubated at room temperature
with different initial concentrations of H2O2
at different pH values (from top to bottom, pH 6, 5, 4, 3.75, and
3.25). The activities remaining after 1 h are shown.
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Production of H2O2.
The
H2O2 levels found in fresh filtrates obtained
from P. pulmonarius cultures are shown in Fig.
4. The onset of
H2O2 production and biosynthesis of anisylic
compounds coincided with the onset of peroxidase instability (Fig. 2).
The total amounts of anisylic compounds (Fig. 4) were estimated by gas
chromatography-mass spectrometry, including 75 to 95% anisaldehyde and
5 to 25% anisic acid. Both anisaldehyde and anisic acid can
participate in H2O2 generation via redox
cycling involving cell-bound reducing activities on aromatic aldehydes
and acids and AAO extracellular oxidation of the alcohols formed
(17, 19).
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FIG. 4.
H2O2 concentrations in fresh
filtrates from P. pulmonarius cultures () and after
incubation of the culture filtrates for 3 h at room temperature
(). The concentrations of anisylic compounds in fresh culture
filtrates () are also shown.
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As shown in Fig.
5,
H
2O
2 in filtrates containing unstable
peroxidase activity (obtained after 75 h of cultivation) was
produced
constantly, and the enzyme was inactivated simultaneously. The
fact that addition of catalase (and filtrate dialysis) protected
the
peroxidase against inactivation supported the hypothesis that
H
2O
2 was involved. Similar protection was
obtained by adding Mn
2+, although the
H
2O
2 levels were only partially lowered. In the
case of filtrates containing stable peroxidase (obtained after
64 h of growth), addition of 1 mM veratryl or anisyl alcohol resulted
in
enzyme inactivation that paralleled H
2O
2
production (Table
2). As determined for naturally unstable peroxidase,
simultaneous
addition of catalase or Mn
2+ prevented
inactivation. The effect of Mn
2+ depended on the
concentration; significant protection was obtained
with 0.1 mM
Mn
2+. Addition of tartrate as a chelator partially
decreased peroxidase
stabilization by Mn
2+.
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FIG. 5.
Peroxidase activity estimated on the basis of
Mn3+-tartrate formation (A) and
H2O2 concentration (B) during incubation of
culture filtrates from 75-h cultures of P. pulmonarius at
room temperature () and effects of continuous dialysis (),
addition of catalase (100 U/ml) (), and addition of 0.1 mM
Mn2+ ( ).
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DISCUSSION |
The action of extracellular proteases has been described by
Dosoretz et al. (14) as a major factor that decreases
peroxidase levels in cultures of P. chrysosporium, and
similar findings have been reported for other ligninolytic fungi
(7, 44). In P. chrysosporium two types of
proteases were described, and these two types were related to decreases
in LiP and MnP levels in both liquid cultures (4, 9, 13) and
SSF cultures (10). Therefore, we initially considered
whether proteases could be responsible for the rapid decreases in
peroxidase activity observed in cultures of P. pulmonarius
(33). However, the enzyme was not stabilized by protease
inhibitors, including PMSF, which efficiently protects P. chrysosporium peroxidases (12, 13). As discussed below, our results indicate that H2O2 is a major
factor in inactivation of the P. pulmonarius peroxidase in cultures.
H2O2 and aromatic metabolites.
H2O2 is required for lignin degradation, as
described by Faison and Kirk (15), but it is a strong
oxidant that has deleterious effects on living cells. The
H2O2 involved in lignin degradation is probably
turned over rapidly, and the excess is decomposed by
mycelium-associated catalase. This would explain the larger amounts of
H2O2 found in culture filtrates than in whole
cultures, where the mycelium prevents undesirably high concentrations.
In
Pleurotus and
Bjerkandera species
H
2O
2 is produced by AAO (
11,
17,
20), in contrast to
P. chrysosporium, in which
H
2O
2 is generated by glyoxal oxidase
(
27). Nevertheless, the
H
2O
2 levels
found in cultures of
P. pulmonarius were similar to
the
H
2O
2 levels reported for
P. chrysosporium (
45). The present
study revealed that
P. pulmonarius is a good AAO producer, and
the levels
obtained were the highest levels reported for this
enzyme. AAO exhibits
high levels of activity with anisylic alcohol
and other primary
aromatic alcohols and low levels of activity
with some aromatic
aldehydes (
18). It has been reported that
the basidiomycetes
are able to synthesize a variety of phenolic
and nonphenolic aromatic
compounds, such as vanillin (
16), veratryl
alcohol
(
43), and anisaldehyde (
20). In addition, during
growth
on natural substrates simple aromatic compounds are released as
a result of lignin depolymerization (
8). We suggest that the
cyclic H
2O
2-producing system proposed by
Guillén and Evans (
17)
for
P. eryngii,
involving extracellular AAO and mycelium-associated
dehydrogenases
acting on anisylic and other aromatic compounds,
is responsible for
H
2O
2 generation in
P. pulmonarius
cultures.
The balance among the aromatic alcohols, aldehydes, and acids
depends on the efficiency of the oxidizing and reducing systems
(
17,
20), and due to the high level of AAO activity in
P. pulmonarius, anisaldehyde predominates in cultures of
this organism.
It is necessary to point out that production of anisylic
compounds
in
P. pulmonarius cultures starts a few hours
before the time
when the maximal levels of H
2O
2
are observed, which coincides
with the time when the greatest
peroxidase instability is
observed.
In culture filtrates, the redox cycle is interrupted since the reducing
counterpart, the mycelium, is absent. Any traces of
anisyl alcohol are
quickly oxidized. Since AAO exhibits broad
substrate specificity
(
17), we postulated that the slow generation
of
H
2O
2 in filtrates was maintained by AAO
activity with other
aromatic substrates. These substrates could include
phenolic or
nonphenolic aromatic alcohols present in cultures of
P. pulmonarius and related species (
20). As shown
by Guillén and Evans (
17),
the concentrations of some
benzylic alcohols (substrates of AAO)
are relatively high in the
presence of
Pleurotus mycelium because
of the balance
between oxidizing and reducing enzymatic activities
(whereas mainly
anisaldehyde is found during incubation with anisylic
compounds). Other
mechanisms could also generate H
2O
2; for
example,
we confirmed that Mn-mediated production of
H
2O
2 occurred after
glyoxylate was added to
culture filtrates of
P. pulmonarius (data
not shown).
However, this type of reaction (
31) may not have
been
involved in peroxidase inactivation because the presence
of
Mn
2+ would have protected the
enzyme.
Peroxidase inactivation and protection.
Inactivation of
P. chrysosporium LiP and MnP by H2O2
has been investigated previously. Odier and Artaud (37)
reported that LiP is more sensitive to an excess of
H2O2 (compound III formation by 25 equivalents
of H2O2) than MnP (250 H2O2 equivalents required) and HRP (500 H2O2 equivalents required) are, but differences
in the reaction pH values (usually pH 3 for LiP, pH 4.5 for MnP, and
around pH 6 for HRP) should be taken into account. The P. pulmonarius peroxidase is inactivated by
H2O2, and the susceptibility of this enzyme
varies with pH. In this way, addition of 250 equivalents of
H2O2 (corresponding to 35 µM
H2O2 under our experimental conditions) resulted in more than 90% inactivation of this peroxidase at pH 3, around 60% inactivation at pH 4.5, and only 30% inactivation at pH 6. This effect of pH on peroxidase stability is important in order to
understand the inactivation of the enzyme in P. pulmonarius cultures, in which a sudden decrease in pH is observed near the peak of
peroxidase activity. Compared with P. chrysosporium
peroxidases, the P. pulmonarius enzyme appears to be more
resistant to an excess of H2O2 than MnP is at
pH 4.5, and it exhibited resistance similar to that of LiP at lower pH
values. It is interesting that the maximal peroxidase instability found
in cultures was nearly the same as the maximal peroxidase instability
observed after we incubated purified peroxidase under
H2O2 concentration conditions (ca. 15 µM) and
pH conditions (ca. pH 4.5) similar to those found in the unstable cultures.
The experiments designed to stabilize
P. pulmonarius
peroxidase provided information about the way in which inactivation is
caused. The enzyme was stabilized by reducing substrates, such
as
phenols and Mn
2+. Stabilization of HRP in the presence of
H
2O
2 by phenols has
been described by Halliwell
(
21), and veratryl alcohol acts
as a LiP stabilizer in
P. chrysosporium cultures (
6,
45).
Although
veratryl alcohol can be oxidized by
P. pulmonarius
peroxidase,
addition of this compound to cultures resulted in rapid
peroxidase
inactivation because it is a better substrate of AAO
(
18) than
of peroxidase (
33) (and strongly
promotes H
2O
2 generation).
Addition of
Mn
2+ efficiently protected the
P. pulmonarius
peroxidase against inactivation,
but only a partial decrease in the
H
2O
2 concentration was observed.
This suggests
that H
2O
2 generation continues in the presence
of
Mn
2+ and that this compound acts as a reductant of the
oxidized enzyme
which prevents formation of compound III and peroxidase
bleaching,
as described previously for LiP (
46). Protection
of the enzyme
by low Mn
2+ concentrations implies that
Mn
3+ is recycled, probably by oxidation of
H
2O
2 to O
2(
1),
whereas the lower level of protection provided by phenols
could be
related to difficulties in product recycling. During
peroxidase
purification from
Pleurotus liquid cultures, addition
of
about 100 µM MnSO
4 before separation of the mycelium
stabilizes
the peroxidase during the critical steps, ultrafiltration
and
dialysis. Peroxidase inactivation by the mechanism described here
does not seem to occur during fungal growth on lignocellulosic
substrates because the vanillin and syringaldehyde (and corresponding
acids) released during oxidative depolymerization of lignin probably
act as electron donors for
Pleurotus peroxidase. This
conclusion
is supported by the results of Camarero et al.
(
5), who showed
that extended peroxidase production occurred
from weeks 1 to 4,
when
P. pulmonarius was grown on wheat
straw under SSF
conditions.
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ACKNOWLEDGMENTS |
We thank A. Prieto for the gas chromatography-mass spectrometry
analyses and C. Rodríguez for skillful technical assistance.
B.B. thanks the HCM Programme and the FAIR Programme of the European
Union for two postdoctoral fellowships supporting her stay at the
Centro de Investigaciones Biológicas (Madrid). This work was
supported by project BIO96-393 (Evaluation of Enzymatic and
Radical-Mediated Mechanisms in Lignin Degradation by Fungi from the
Genera Pleurotus and Phanerochaete) of the
Spanish Biotechnology Program and by project AIR2-CT93-1219 (Biological
Delignification in Paper Manufacture) of the European Union.
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FOOTNOTES |
*
Corresponding author. Mailing address: Centro de
Investigaciones Biológicas (CIB), Consejo Superior de
Investigaciones Científicas (CSIC), Velázquez 144, E-28006 Madrid, Spain. Phone: 34915611800. Fax: 34915627518. E-mail:
cibm149{at}fresno.csic.es.
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Applied and Environmental Microbiology, March 1999, p. 923-928, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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[Abstract]
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Abstract
Introduction
