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Applied and Environmental Microbiology, October 2001, p. 4588-4593, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4588-4593.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Conversion of Milled Pine Wood by Manganese
Peroxidase from Phlebia radiata
Martin
Hofrichter,*
Taina
Lundell, and
Annele
Hatakka
Division of Microbiology, Department of
Applied Chemistry and Microbiology, University of Helsinki,
FIN-00014 Helsinki, Finland
Received 20 March 2001/Accepted 9 July 2001
 |
ABSTRACT |
Purified manganese peroxidase (MnP) from the white-rot
basidiomycete Phlebia radiata was found to convert in
vitro milled pine wood (MPW) suspended in an aqueous reaction solution
containing Tween 20, Mn2+, Mn-chelating organic acid
(malonate), and a hydrogen peroxide-generating system (glucose-glucose
oxidase). The enzymatic attack resulted in the polymerization of
lower-molecular-mass, soluble wood components and in the partial
depolymerization of the insoluble bulk of pine wood, as demonstrated by
high-performance size exclusion chromatography (HPSEC). The surfactant
Tween 80 containing unsaturated fatty acid redsidues promoted the
disintegration of bulk MPW. HPSEC showed that the depolymerization
yielded preferentially lignocellulose fragments with a predominant
molecular mass of ca. 0.5 kDa. MnP from P. radiata
(MnP3) turned out to be a stable enzyme remaining active for 2 days even at 37°C with vigorous stirring, and 65 and 35% of the
activity applied was retained in Tween 20 and Tween 80 reaction
mixtures, respectively. In the course of reactions, major part of the
Mn-chelator malonate was decomposed (85 to 87%), resulting in an
increase of pH from 4.4 to >6.5. An aromatic nonphenolic lignin
structure (
-O-4 dimer), which is normally not attacked by MnP, was
oxidizible in the presence of pine wood meal. This finding indicates
that certain wood components may promote the degradative activities of
MnP in a way similar to that promoted by Tween 80, unsaturated fatty
acids, or thiols.
| |
INTRODUCTION |
Biodegradation of lignin by
basidiomycetous white-rot fungi is, at least partly, brought about by
extracellular lignin and manganese peroxidases (12, 26).
MnP discovered in Phanerochaete chrysosporium in 1984 (28) is obviously produced exclusively by wood and
soil-litter-decomposing basidiomycetes (14). Evidence has
been provided that the enzyme is involved in the biodegradation of
lignin (14, 36, 38, 40) and other recalcitrant substances such as humic acids (25) or organopollutants (1, 5,
15). There are a number of efficient delignifying fungi that
secrete MnP as the only extracellular peroxidase, including
Lentinula (Lentinus) edodes (30), Panus
tigrinus (35), Ceriporiopsis subvermispora
(32), Phanerochaete sordida (41),
Dichomitus squalens (39), and Pleurotus
ostreatus (7); some other ligninolytic fungi produce
MnP, along with lignin peroxidase (e.g., Phanerochaete chrysosporium, Phlebia radiata, Trametes
versicolor, and Nematoloma frowardii) (12, 14,
19, 26). MnP works via chelated Mn3+,
which acts as a small diffusible oxidant (redox mediator) attacking preferentially phenolic lignin structures (8, 9). The
oxidative strength of the MnP-Mn2+/3+ system is
enhanced in the presence of certain cooxidants as glutathione or
unsaturated lipids, which enable it to oxidize also recalcitrant nonphenolic lignin moieties with higher redox potential (4, 22,
23).
MnP was found to depolymerize and even mineralize part of polymeric
lignin in cell-free systems (20, 29, 44). Most of these
investigations, however, were carried out by using isolated dispersed,
natural, or synthetic lignins (DHP) as substrates, and so far only a
few studies have been published in which solid lignocelluloses (e.g.,
pulp or straw) were treated with MnP (18, 27, 40). The
present study therefore focuses on the action of purified MnP enzyme
from the white-rot fungus P. radiata on solid pine wood that
was merely milled prior to use. In this context, the effect of lipid
surfactants such as Tween 20 and Tween 80 was examined.
| |
MATERIALS AND METHODS |
Organism and enzyme preparation.
The corticoid white-rot
fungus P. radiata 79 (ATCC 64658) used for the production of
MnP was isolated and characterized as described earlier (10,
37). The fungus was cultured in a nitrogen-limited succinate-buffered medium (pH 4.5) containing 200 µM
Mn2+ in a 20-liter bioreactor. MnP from P. radiata (MnP3; pI 3.6) was purified from the culture liquid of 8- to 10-day-old cultures as described previously (37).
MPW.
Milled pine wood (MPW) applied as substrate for MnP was
prepared from a mixture of air-dried sap and heart wood of Scots pine (Pinus silvestris) by using a rotating jar ball mill
according to the method of Lundquist (34) except that no
organic solvent or water was added during the milling process. Thus,
the obtained powder (MPW1) contained all wood components including the
extractives. Part of the MPW1 was further extracted in 5-mg portions
with 1 ml of 0.5% Tween 20 or Tween 80 in 50 mM sodium malonate (also containing 2 mM MnCl2) with vigorous shaking for
2 days. After centrifugation, the supernatant containing the
extractable wood constituents (MPWX, mostly aromatic compounds) was
separated from the insoluble wood pellet (MPW2). The latter was washed
twice with ethyl acetate and water prior to use. Dry weight
measurements showed that about 20% (19.9% ± 2.3% = 99.5 ± 11.5 µg ml
1, n = 5) of the
MPW could be dissolved in both Tween 20 and Tween 80 under these
conditions. Most experiments were carried out with MPW1; MPW2 and MPWX
were used as additional substrates to distinguish between
depolymerizing and polymerizing MnP activities.
HPSEC.
High-performance size exclusion chromatography
(HPSEC) was used for the determination of the molecular mass
distribution of lignocellulose fragments formed by MnP (18,
20). The high-performance liquid chromatography (HPLC) system
(HP 1090 Liquid Chromatograph; Hewlett-Packard, Waldbronn, Germany)
equipped with a diode array detector was fitted with a HEMA-Bio linear
column (8 by 300 mm, 10 µm; Polymer Standard Service, Mainz,
Germany). The mobile phase consisted of 20% acetonitrile and 80% of
an aqueous solution of 0.5% NaNO3 and 0.2%
K2HPO4; the pH was adjusted
to 10.0 by the addition of NaOH. Sodium polystyrene sulfonates (1.3 to
168 kDa, Polymer Standard Service) and biphenyl dicarboxylic acid
(0.246 kDa) served as molecular mass standards (16).
Determination of organic acids.
The concentrations of
malonate and other organic acids (oxalate, tartrate, and malate) acting
as Mn chelator and buffer substances in the reaction system were
determined after 48 h by using the HPLC system mentioned above but
fitted with a Aqua-C18 column (4.6 by 250 mm, 5 µm; Phenomex, St. Torrance, Calif.). Phosphoric acid (10 mM) served
as eluent under isocratic conditions, and authentic standards of the
organic acids served as references (detection wavelength, 210 nm)
(17).
Enzyme assay.
MnP activity and Mn3+
concentration were measured at 420 nm by monitoring the oxidation of
ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid);
420 = 36 mM1
cm1] as described previously (17,
20).
Enzymatic reactions.
The basic reaction mixture contained in
a total of 1 ml of 50 mM sodium malonate (pH 4.5), 2 mM
MnCl2, 0.5% Tween 20 or 80, 10 mM glucose, 0.1 U
of glucose oxidase (from Aspergillus niger; Sigma-Aldrich)
ml1, and 2 U of MnP3 from P. radiata
(40 nM) ml1. MPW1 (5 mg
ml1) or MPW2 (ca. 4 mg
ml1) served as solid target substrates. The
soluble MPWX fraction (1 ml) was supplemented with the respective
reactants and was, like MPW1 and MPW2, incubated in 10-ml reaction
tubes closed with parafilm with vigorous stirring at 37°C for 48 h. Samples (10 µl) for the measurement of MnP activity and
Mn3+ concentration were collected immediately
after the reaction was started, as well as in 0.5- to 10- h intervals;
larger samples (50 µl) for HPSEC analyses were obtained after 24 and
48 h. Considering the amount of extractable wood components (~1
mg of lignin plus other aromatics ml1) and the
change in the extinction coefficient due to oxidation of this material,
we calculated roughly how much aromatic material was realeased by MnP
from solid wood (MPW1, MPW2) by comparing the area below the respective
HPSEC elution profiles.
In order to check the stability of alternative chelators for the MnP
system, MPW1 was incubated in the reaction mixture mentioned above
(with Tween 20) but replacing malonate by equivalent amounts of
oxalate, malate, or tartrate. After a reaction of 48 h, the samples were analyzed by HPSEC, as well as for their organic acid concentration, pH, and remaining MnP activity.
Oxidation of a nonphenolic LMC.
The aromatic dimer
1-(3,4-dimethoxyphenyl)-2(2-methoxyphenoxy)propane-1,3-diol
(11) was used to examine the influence of MPW1 on the
oxidation of recalcitrant nonphenolic -O-4 lignin structures by MnP.
The conditions were identical to those described above except that the
Tween 20-containing reaction solution (total of 1 ml) was additionally
supplemented with 100 µM lignin model compound (LMC). The keto-form
of LMC [keto-LMC = 1-(3,4-dimethoxyphenyl)-2(2-methoxyphenoxy)-propane-1-one (11)], veratryl alcohol, veratryl aldehyde, veratric
acid, and guaiacol (2-methoxyphenol) served as authentic references of
possible oxidation products. HPLC was used for quantitative analysis
after 24 h by using the equipment described above, including the
Aqua-C18 column (Phenomex). Separations were run
at constant 40°C by using a stepwise gradient of 0 to 45%
acetonitrile (0% [0 min], 0% [10 min], 40% [20 min], 45% [25
min], 0% [30 min], and 0% [35 min]) in 0.05% phosphoric acid
for 35 min with a constant flow rate of 0.75 ml
min1. Eluted substances were detected at 275 and 310 nm.
| |
RESULTS |
MnP activity during MPW treatment.
Time courses of MnP
activity during the treatment of MPW1 are shown in Fig.
1 and were very similar regardless of
whether the reaction mixture contained Tween 20 or Tween 80. The
activity of MnP increased in the very beginning (from 2 to 2.4 U
ml1), which was due to the presence of Tween 20 or Tween 80 that somehow enhanced the enzyme activity (probably by
improving the accessibility of ABTS). After 1 h, MnP activity
started to decline, reaching temporary minima of 0.55 and 0.43 U
ml1 after 18 h for Tween 20 and Tween 80 containing samples, respectively. Simultaneous with the decrease of MnP
activity, Mn3+ appeared in the reaction solution
and reached its maximum concentration of 200 µM after 18 h.
Within the next 6 h, the Mn3+ level dropped
noticeably (to 70 µM), which was accompanied by a partial recovery of
MnP activity, particularly in the Tween 20-containing samples (from
0.55 to 1.3 U ml1; and from 0.43 to 0.7 U
ml1 in Tween 80 samples). Afterward, the MnP
activity remained nearly constant until the end of the experiment.
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FIG. 1.
Activity of MnP, concentration of Mn3+, and
pH during the in vitro treatment of MPW1 with MnP. The upper graph
refers to Tween 20-containing samples; the lower one refers to Tween
80-containing samples. Symbols:  , actual MnP activity;  ,
Mn3+ concentration;  , pH.
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|
In the course of the experiment, the pH of the reaction solutions
increased from ca. 4.5 to 6.5 to 6.8 (Fig.
2). HPLC analyses
revealed
that the pH increase was caused by a substantial decomposition
of
malonate during MnP catalysis (Table
1).
More than 80% of
malonate disappeared and was very probably converted
into carbon
dioxide and only to a small extent into oxalate.
Alternative organic
acids acting as buffer substances and chelators for
manganese
were also decomposed by the MnP system, although to a
noticeably
lesser extent than was the malonate (Table
2). Only in the case
of oxalate was a
similar drastic increase in pH as with malonate
observed.
However, ca. 60% of oxalate remained intact after 48
h. On the
other hand, the organic acids tested were not as efficient
as malonate
in promoting the attack on MPW1 by MnP (see below).
HPSEC analyses.
Figure 2
summarizes the HPSEC elution profiles of the water-soluble products
formed as the result of the MPW treatment with MnP. Although we do not
know anything about the molecular weight of native lignin in the
untreated bulk of wood, it is obvious that both polymerizing and
depolymerizing reactions took place and that these reactions were
dependent on the type of Tween used.
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FIG. 2.
HPSEC elution profiles of soluble products from
different MPW preparations treated with MnP3 in the presence of
malonate for 48 h. The upper row refers to Tween 20-containing
samples; the lower row to Tween 80-containing samples. Key: dotted
lines, controls without MnP after 48 h; thin lines, MnP-containing
samples after 24 h; bold lines, MnP-containing samples after
48 h. (A) MPW2 plus Tween 20. (B) MPW2 plus Tween 80. (C) MPWX
plus Tween 20. (D) MPWX plus Tween 80. (E) MPW1 plus Tween 20. (F) MPW1
plus Tween 80.
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|
MPW2 (free of soluble wood components) was only slightly attacked by
MnP in the presence of Tween 20, resulting in the formation
of moderate
amounts (ca. 300 µg ml
1) of
low-molecular-mass fragments (~0.5 kDa and smaller; Fig.
2A). Tween 80 stimulated noticeably the
depolymerization process,
resulting in an increase of water-soluble
fragments, among which
were also higher-molecular-mass products (>5
kDa; Fig.
2B), and
we estimated that ca. 800 µg of lignin fragments
and other aromatics
ml
1 was released.
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FIG. 3.
Oxidation of a nonphenolic LMC by MnP in the presence of
MPW (MPW1) for 24 h. Reversed-phase HPLC analysis was performed at
275 nm by using a water-acetonitrile gradient. (A) Standards (100 µM)
of veratryl alcohol (peak 1), veratric acid (peak 2), guaiacol (peak
3), veratryl aldehyde (peak 4), LMC (peak 5), and keto-LMC (peak 6).
(B) Complete reaction mixture plus LMC plus MPW1, but without MnP. (C)
Complete reaction mixture plus LMC plus MnP, but without MPW1. (D)
Complete reaction mixture plus LMC plus MnP plus MPW1.
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|
The conversion of MPWX obviously resulted in the polymerization of
soluble wood constituents to high-molecular-mass products
(~50 kDa),
although individual differences were ascertained for
Tween 20- and
Tween 80-containing samples (Fig.
2C and D). High-molecular-mass
products (~50 kDa) were predominantly formed in the presence of
Tween
20 (Fig.
2C), whereas the elution profiles of Tween 80-containing
samples also included more of the lower-molecular-mass fractions
(~0.5 and 5 kDa; Fig.
2D). Elution profiles of the MPW1 products
show
characteristics of both MPW2 and MPWX (Fig.
2E and F). Depolymerization
was particularly pronounced in the presence of Tween 80, where
the
low-molecular-mass fractions dominated in the end over the
high-molecular-mass ones. On the other hand, estimation of total
aromatic fragments indicates that in the presence of both Tween
20 and
Tween 80, similar amounts were released (600 and 700 µg
ml
1,
respectively).
The replacement of malonate as a chelator for
Mn
3+ did not lead to an increased
depolymerization of MPW1. On the contrary, the
respective HPSEC
profiles for tartrate-, malate-, and oxalate-containing
samples
demonstrated that malonate was the most efficient chelator
of the
MnP-Mn
2+/3+ system (data not shown). Whereas the
formation of high-molecular-mass
fragments was not influenced or even
enhanced, the resulting amounts
of low-molecular-mass substances were
in all three cases lower.
Furthermore, the remaining MnP activities
after MPW1 treatment
show that oxalate, malate, and tartrate were not
as efficient
as malonate in maintaining MnP activity (Table
2).
Oxidation of LMC.
MnP3 from P. radiata was not able
to attack a nonphenolic dimer (LMC) in the presence of Tween 20. Within
48 h of incubation, only a negligible part of LMC (<3%) was
converted, and no indication for the formation of the keto-form of LMC
was found (Fig. 3C). However, in the presence of MPW1 (5 mg
ml1), a significant amount of LMC (ca. 35% = 35 µM) disappeared in the reaction solution accompanied by the nearly
equivalent formation of the corresponding keto-form of LMC (31.9 µM,
arrow in Fig. 3D); other oxidation products, such as monomeric
fragments (e.g., veratric acid or veratryl aldehyde), were not observed
(Fig. 3A and D). The keto-form of LMC was also detected when MPWX was
used instead of MPW1, although oxidation was less pronounced (data not
shown). In addition, Fig. 3 shows that MnP treatment reduced the amount
of soluble lower-molecular-mass constituents of MPW1, which formed a
broad peak absorbing at 275 nm in the HPLC elution profile of MnP-free
controls (Fig. 3B). A number of sharp, distinct peaks sitting up on
this broad peak and with characteristic aromatic UV spectra disappeared
completely from the reaction solution as a result of MnP treatment
(Fig. 3D). It can be concluded that these soluble aromatic monomers and
oligomers were preferentially polymerized by the MnP system, which
matches well the results of the HPSEC analyses. Last, but not least,
there was also an indication for the depolymerizing activity of MnP.
Thus, the elution profile of treated MPW1 was altered toward
more-hydrophilic products eluting very early from the reversed-phase
column (Fig. 3B and D).
| |
DISCUSSION |
MnP3 from P. radiata is able to convert MPW suspended
in an appropriate reaction solution in vitro. The enzymatic attack
results in the polymerization of lower-molecular-mass, soluble wood
components and in the partial depolymerization of the insoluble bulk of
pinewood. Tween 80, acting as both a surfactant and cooxidant for the
MnP system (23), promotes the disintegration of insoluble
MPW into smaller, soluble lignocellulose fragments. MnP3 was found to
be a rather stable enzyme that remained active for at least 2 days under the reaction conditions applied. An aromatic nonphenolic lignin
structure (-O-4), which is normally not attacked by MnP but is
cleaved by LiP (33), can be oxidized in the presence of
pinewood meal, indicating the support of MnP activities by certain wood components.
The ability of MnP from different fungi to depolymerize isolated
lignins partly has been demonstrated in vitro within some studies.
Among the lignins tested were synthetic preparations (19, 20,
44), chlorolignin (29) and natural lignins from spruce or pine (4, 13), Hevea spp.
(6), and wheat straw (18). Our results also
show that MnP not only attacks isolated lignins in aqueous reaction
systems but also attacks more-complex solid lignocellulose such as MPW.
There are only a few reports on the conversion of entire
lignocelluloses (wood, straw, or pulp) by oxidative fungal enzymes. Softwood kraft pulp was treated with MnP from Trametes
versicolor to oxidize residual lignin (38, 40). In
contrast to our present results, MnP was not able to "solubilize"
lignin directly in previous studies, i.e., no water-soluble lignin
fragments were found in the reaction mixtures after MnP treatment
(38, 40). The reason for this might be the use of a low
malonate concentration (5 mM) and the lack of a surfactant such as
Tween. On the other hand, it has been shown that the MnP treatment
lowered the kappa number of kraft pulp and increased the alkali
extractability of residual lignin (38), indicating that
MnP partially oxidized the lignin in pulp under the conditions applied.
Similar results were obtained with hardwood kraft pulp and wheat straw
meal when MnP from Phanerochaete sordida (27)
and the abiotic model system MnO2-oxalate,
respectively, were used (31). The authors of the former
study noticed, similar to our observations, a positive effect of Tween
80 toward the oxidation of lignin by MnP and the bleaching of pulp.
Unlike isolated disperged lignins such as DHP, the disintegration of
nonextracted solid lignocellulose (MPW1) was accompanied in our present
study (at least in the early stages of attack) by substantial formation
of high-molecular-mass, water-soluble fragments resulting from the
obvious polymerization of low-molecular-mass wood components. The
substantial polymerization of aromatic monomers such as guaiacol
(2-methoxyphenol) and 2,6-dimethoxyphenol to insoluble macromolecules
has recently been described for an MnP from Bjerkandera
adusta (21). The fact that the polymers formed by MnP
in our experiments did not precipitate suggests that their highly
hydrophilic nature is based on a high grade of initial oxidation.
It is interesting that the Mn3+ concentration
seems to regulate the MnP activity; the higher the
Mn3+ concentration was during the course of MPW
conversion, the lower was the actual activity of MnP, and vice versa.
It is still unclear, however, just what is responsible for this
endproduct regulation, but some kind of feedback control on the enzyme
level cannot be ruled out. Some indication for the regulation of MnP
activity by Mn3+ was already found in a previous
study (45). The activity of crude MnP from
Clitocybula dusenii declined considerably when Mn3+ pyrophosphate was added to the assay mixture.
The decomposition of malonate in the course of MnP catalysis is a
phenomenon that we already observed in an earlier study in the absence
of any secondary substrate (17). This radical process,
which also applies to oxalate and other organic acids, limits the
efficiency of MnP catalysis in cell-free batch systems, since the
concomitant degradation of the required chelator and buffer agent leads
to disproportionation of Mn3+ and to an
unfavorable increase of pH. To overcome this problem in future
experiments, malonate could be re-added continuously, holding the pH constant.
The MnP system obviously has an effect on certain lignin structures in
pine wood, but only the "cooperation" with cooxidants as the
unsaturated fatty acid residues in Tween 80 may enable the substantial
disintegration of intact lignin. Reactive peroxyl radicals
derived from unsaturated lipids (24) might be responsible for the enhanced pine wood conversion. Whether the lipid substances in
question are produced by the fungus (3), are present in the wood, or originate from both sources has to be proven in each particular case. In this context, our finding that the presence of MPW1
enables MnP to oxidize a nonphenolic lignin model dimer is remarkable.
It can be concluded that certain substances either contained in MPW or
formed from MPW by MnP somehow promote the oxidation of
more-recalcitrant lignin structures. The nature of these wood
constituents is still unknown. As already mentioned, lipids comprising
unsaturated fatty acids (22, 23, 24), but also thiols
(4, 43) or aromatic mediators, which have been shown to
expand laccase activities in vitro (2, 42), should be
taken into consideration.
Our future studies will focus on the action of MnP on other types of
wood (e.g., other softwoods such as spruce or larch and also hardwood),
on wood constituents acting as natural cooxidants (redox mediators),
and on synergistic effects of other lignin-modifying enzymes (lignin
peroxidase and laccase) on the MnP-catalyzed conversion of wood.
| |
ACKNOWLEDGMENT |
This work was supported finacially by the European Union (project
"Fungal metalloenzymes oxidizing aromatic compounds of industrial interest," QLK3-1999-00590).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Applied Chemistry and Microbiology, P.O. Box 56, Biocenter 1, Viikinkaari 9, FIN-00014 Helsinki, Finland. Phone: 358-9-191-59321. Fax: 358-9-191-59322. E-mail:
martin.hofrichter{at}helsinki.fi.
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Applied and Environmental Microbiology, October 2001, p. 4588-4593, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4588-4593.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
