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Appl Environ Microbiol, June 1998, p. 2273-2274, Vol. 64, No. 6
Pulp and Paper Research Institute of Canada,
Pointe-Claire, Quebec, Canada H9R 3J9
Received 6 January 1998/Accepted 23 March 1998
Manganese peroxidase treatment lowered the kappa number of kraft
pulp and increased the alkali extractability of the residual lignin but
did not directly solubilize it. This indicates that MnP partially
oxidizes the lignin in the pulp but does not degrade it to soluble
fragments.
Manganese peroxidase (MnP) has been
implicated as an important enzyme in delignification of kraft pulps by
white rot fungi (6, 8, 12, 15). Treating kraft pulps with
MnP can lower their kappa numbers and increase their bleachability
(10, 13, 14). In the presence of the surfactant Tween 80, the enzyme can increase the brightness of hardwood kraft pulp
(9). This enzyme also releases methanol from methoxyl
groups on rings with free phenolic hydroxyls (15). The
bleaching effect is thought to be caused by residual lignin
oxidation by chelated Mn3+, generated by enzymatic
oxidation of Mn2+, but the reacting sites within the lignin
and the products of delignification have not yet been identified. To
better understand pulp bleaching by MnP, we have investigated its
action on softwood kraft pulp (SWKP) containing
14C-labelled residual lignin. Delignification of this pulp
by the white rot fungus Trametes versicolor has been
described previously (16). The fungus extensively
depolymerized and solubilized the residual lignin in the pulp and
mineralized 22% of it to CO2.
We show here that the decrease in kappa number caused by MnP treatment
is not associated with removal of lignin carbon from the pulp.
Pulps.
SWKP labelled with 14C in its
residual lignin (SWKP*2) was prepared by laboratory pulping of
lignin-labelled white spruce as previously described (16).
The labelled pulp was washed with 0.1 M NaOH and rinsed with water just
before use to remove radioactive materials that had leached from the
pulp during storage. Unlabelled pulp was prepared from white spruce in
the Paprican pilot plant.
Enzymes.
Glucose oxidase (Gox) was purchased from Sigma.
The activity of the Gox was assayed at pH 4.5, with peroxidase to
detect the production of H2O2 from oxidation of
glucose (1). MnP was produced from Trametes
versicolor 52J as previously described (13) and assayed
by spectrophotometric detection of Mn(III) malonate formation (15). Crude cellulase was purchased from Iogen Corp.,
Ottawa, Ontario, Canada.
Pulp treatments.
SWKP (0.5 g) was suspended in 50 ml of
5 mM malonate buffer, pH 4.5, containing 50 U of MnP,
0.2 mM MnSO4, and 10 mM glucose, in a 125-ml
Erlenmeyer flask. In experiments using labelled pulp, 1 × 105 dpm of SWKP*2 was added to each flask. Gox (1.76 U) was added to start the reaction, and the flasks were incubated on a
gyrotory shaker at 250 rpm for 24 h at 30°C. Control flasks
lacking either MnP or Gox were incubated in parallel with the treatment
flasks. After incubation, the flask contents were fractionated into
soluble, alkali-extractable, and cellulase lignin fractions as
previously described (16). Alkali extraction was carried out
at 10% consistency and 60°C for 60 min with 1.6% NaOH. Cellulase
lignin was the fraction soluble in 0.1 M NaOH at 37°C after digestion
of the pulp polysaccharides with crude cellulase.
Analytical methods.
Kappa numbers were determined by the
micro-kappa method (3). Klason lignin was measured by the
method of Effland (4), with estimation of acid-soluble
lignin from the A205 of the hydrolysis filtrates
(11).
Effect of MnP treatment on distribution of 14C from
lignin-labelled pulp.
Gox was used in these experiments to supply
H2O2 continuously at low concentration; MnP is
inactivated by H2O2 at millimolar concentrations (5). Treatment of the labelled pulp with the complete system of MnP plus Gox did not release significant amounts of
14CO2, nor did it solubilize the labelled
lignin; the level of soluble radioactivity after the treatment of the
pulp with both MnP and Gox was low, intermediate between those of the
two controls lacking one of the enzymes (Table
1). Treatment of the pulp with Gox alone
(no MnP control) consistently decreased the amount of soluble radioactivity; the basis for this effect was not determined. The enzyme
treatment did increase the amount of radioactivity extracted by alkali
from ca. 15% to ca. 25% of the total and decreased the amount of
label in the cellulase lignin fraction.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effects of Manganese Peroxidase on Residual Lignin
of Softwood Kraft Pulp
ABSTRACT
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TABLE 1.
Treatment of 14C-lignin-labelled SWKP with
MnP increases the alkali extractability of the label but not
its solubility
Effect of MnP treatment on Klason lignin content of pulp. Treatment with MnP plus Gox lowered the kappa number of the pulp by about 20%, but it did not change the Klason lignin content (Table 2). There were no significant differences among the controls lacking one or both of the enzymes. When the pulps were extracted with alkali, the kappa numbers of the treated and control pulps all decreased by about six units, but the pulp treated with the complete enzyme mixture lost significantly more Klason lignin (1.5% on pulp) than the control pulps did (0.5% on pulp).
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-carbonyls, which are easily degraded by alkaline
peroxide (7). MnP is known to oxidize lignin model compounds
predominantly to -carbonyl products (17, 18).
The partial oxidation of the lignin apparently did not cause conversion
to fragments small or hydrophilic enough to diffuse out of the fiber
walls. However, the enzyme treatment did increase the proportion of the
lignin that could be extracted by alkali. It seemed that the lignin
extracted by alkali included the partially oxidized lignin, because the
ratio of kappa number to Klason lignin in the extracted pulp was 6.5, similar to the ratios in the extracted control pulps. The
enhanced extractability of lignin after oxidation with MnP
parallels the effect of lignin oxidation by chlorine or chlorine
dioxide in conventional bleaching (2). The ability of
alkali to solubilize the oxidized lignins is attributed to ionization
of acidic groups and to decreased association of the lignin molecules;
there are also alkali-promoted chemical reactions that cleave oxidized
fragments and liberate ionizable groups in the lignin (2).
The inability of MnP to solubilize the residual lignin from kraft pulp
differs from the ability of intact cultures of Trametes versicolor to extensively solubilize this lignin (16).
MnP probably functions at an early stage in the degradation of lignin
by this fungus, leading to demethoxylation (15) and
increased alkali extractability (16). Other enzymes then
probably continue the degradation to release soluble lignin fragments.
Alternatively, the fungal mycelium might enhance the ability of MnP to
degrade lignin by secreting thiols (18) or other redox
mediators (12) or by trapping fragments produced
transiently.
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ACKNOWLEDGMENTS |
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We thank Michelle Ricard for technical assistance and Sylvie Renaud for providing MnP and for advice on treating pulp with it.
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FOOTNOTES |
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* Corresponding author. Mailing address: Paprican, 570 boul. St.-Jean, Pointe-Claire, Quebec, Canada H9R 3J9. Phone: (514) 630-4101 ext. 2244. Fax: (514) 630-4134. E-mail: reid{at}paprican.ca.
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REFERENCES |
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Appl Environ Microbiol, June 1998, p. 2273-2274, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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