THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 6, Issue of February 25, pp.

3636-3643,1991
0 1991 by The American Society for Biochemistry and Molecular Biology, Inc Printed in U.S.A.

of Its Role in Viuo*

Lignin Peroxidase: Toward a Clarification
(Received for publication, July 19,1990)

Simo SarkanenS, Ramon A. Razals, Thomas Piccariellos, Etsuo Yamamotoa,and Norman G . LewissT
From the §Departments of Wood Science and Biochemistry, Virginia Polytechnic Instituteand State University,
Blacksburg, Virginia 24061 and the $Department of Forest Products, University of Minnesota, St. Paul, Minnesota55108

The extracellular lignin peroxidase from the white-

rot basidiomycete Phanerochaetechrysosporium is cy,
(*)$” 1 ($
;oH)(if
thought to play an important role in lignin biodegra-
dation. However, the majority of lignin-derived prep-
arations actually experience overall polymerization at /
OCH, CH@ OCH3
the hands ofthe enzyme in vitro.It has now been found OH OH OH
that, inthe presence of H202at pH 4.0, the monomeric -
1 2
- -
3
lignin precursor coniferyl alcohol is polymerized quan-
titatively by a lignin peroxidase preparation which is a, l = l 3 C
-
uncontaminated with Mn”-dependent peroxidases. 13C
NMR spectrometry of the resulting dehydropolymer- -
b, 2 = 1 3 C
isates from 13C-labeled monolignols confirms that the c , 3=’3C
-
frequencies of different interunit linkages are very Since lignins occupy a pivotal position in the carbon cycle
similar to those engendered through the action of of the biosphere, it is not surprising that thesubject of lignin
horseradish peroxidase with H202.Indeed, lignin per-
oxidase does not ultimately seem to be a prerequisite biodegradation has commanded attention for a considerable
for lignin degradation in vivo,yet its activitycan still period of time (6). In 1983, an apparent breakthrough in
accelerate the conversion of lignin-derived prepara- establishing the mechanism by which the white-rot basidi-
tions by P . chrysosporium to COz. Consequently,lignin omycete Phunerochaete chrysosporium degrades lignin was
peroxidase can provisionally be expected to fulfill two reported a putatively ligninolytic extracellular protein had
important functions. On the one hand, the enzyme may been isolated (7, 8). This HPOz-requiringenzyme was subse-
detoxify lower molecular weight phenolic compounds quently shown to fall within the peroxidase family (9-12),
released from lignins during their fungal decomposi- and up to 15 distinguishable heme-containing lignin peroxi-
tion. On the other hand, through the introduction of dase species have been separated from the extracellular me-
suitable functional groups, lignin peroxidase could in- dium of the microorganism (13). Furthermore, the gene en-
directly enhance the susceptibility of macromolecular coding the predominant isozyme has been cloned and se-
lignin structures toward depolymerization by another quenced (14,15).
enzyme. Early reports (7, 8) about lignin peroxidase seemed to
indicate that the enzyme was capable of degrading two quite
different kinds of lignin-like preparation. First, in the pres-
Lignins are found as cell-wall components in all vascular ence of concentrated extracellular P. chrysosporium culture
plants and woody tissues and, while embodying significant fluid containing 0.05 g of proteinfliter with 0.2 mM HzOa at
structural diversity (1-5), presumably represent the second pH 3.0, -22% of a I4C-methylated aqueous acetone extract
most abundant group of biopolymers. Theyare generally from spruce (Picea engelmanii Parry) wood appeared to
considered to be formed via the random dehydrogenative undergo depolymerization in 1 h at 37 “C (7). The enzymatic
polymerization of monolignols, a reaction catalyzed by per- activity deemed responsible for the effect was assigned to a
oxidases in the presence of HzO2. There are three known single polyacrylamide gel electrophoretic band. This conclu-
monolignol precursors, uiz. p-coumaryl 1, coniferyl 2, and sion was reached on the basis of veratraldehyde liberation
sinapyl 3 alcohols. Interestingly, the ratios of monolignols from the spruce wood extract, albeit at a severely attenuated
incorporated into thepolymers can vary with the plantspecies rate when compared with the original enzymatic activity of
and subcellular compartment (1). the crude extracellular fluid (7). Second, at pH 4.5, the en-
zymes in the extracellular medium of P. chrysosporium cul-
* This work was supported by Grant DE-FG05-88ER13883 from tures together with glucose oxidase (0.02 unit/ml)/glucose (3
the United States Department of Energy, a grant from the Virginia mM)/dioxygen as a means of producing HzOt purportedly
Center for Innovative Technology (both to N. G. L.),Grant 86- engendered 10% degradation during 18 h at 37 “C (8) of a
FSTY-9-0166 from the United States Department of Agriculture, and 0.17-mgring-14C-labeled dehydropolymerisate preparation
a grant from the Legislative Commission on Minnesota Resources
(both to S. S.). This is Paper 18,630 of the Scientific Journal Series from coniferyl alcohol (16). On the other hand, anessentially
of the Minnesota Agricultural Experiment Station funded through complete degradation of such a (17 g/liter) dehydropolymer-
Minnesota Agricultural ExperimentStationProject 43-068, sup- isate sample was achieved with the hydroxyl radical-generat-
ported by Hatch funds. The costs of publication of this article were ing system 1 M HZ02,10 mM FeS04 (i.e. Fenton’s reagent)
defrayed in part by the payment of page charges. This article must (17). It should be borne in mind that ferrous ion in open
therefore be hereby marked “aduertisement” in accordance with 18 solution would inevitably react with hydrogen peroxide dif-
U.S.C. Section 1734 solely to indicate this fact.
ll To whom correspondence should be addressed Inst. of Biological ferently from the ferric ion surrounded by porphyrin and
Chemistry, 467 Clark Hall, Washington State University, Pullman, protein in lignin peroxidase.
WA 99164-6340. Model compound studies (18-20) have since demonstrated

3636
 On the Role of Lignin Peroxidase
that the noteworthiness of lignin peroxidase from P. chryso-
sporium lies in its ability to oxidize phenylpropanoid (and
other aromatic) compounds without free phenolic hydroxyl
groups to the corresponding cation radicals. Normally these
model compounds embody lignan- or neolignan-like struc-
tures, i.e. the substrates used to study lignin biodegradation
are frequently dimeric rather than polymeric. Such cation
radical intermediates, derived from dimers incorporating in-
terunit linkages representative of those found in lignins, may
then undergo C,,-Co cleavage and other transformations cir-
cumscribed by the existent molecular framework. On the other
hand, enzymes confined to phenol oxidase behavior, such as
horseradish peroxidase (21), facilitate single-electron trans-
fers from p-hydroxyphenylpropanoidmoieties to yield phen-
oxy radical intermediates that could either incur C,-arene
cleavage or become coupled to one another, forming new
intermolecular covalent bonds (22). The difference between
the enzymatic activities arises from the redox potential of
lignin peroxidase, which, for the suitably oxidized "compound
I" form (ll),is greater than that of horseradish peroxidase,
but evidently less than that of chloroperoxidase (23).
Only 3 years after the original claims that lignin depolym-
erization i n vivo is under the direct enzymatic control of lignin
peroxidase, a very different picture began to emerge. It was
disclosed that 0.8 unit of lignin peroxidase/ml (as a con-
centrated extracellular solution from a carbon-limited P.
chrysosporium culture medium) engenders the net polymeri-
zation of 1 mg of alkali-isolated straw lignin/ml at pH 4.0 in
the presence of 0.54-NmolHz02portions added at 2-h intervals
(24). Curiously, the introduction of 1.2 pmol of veratryl alco-
hol (3,4-dimethoxybenzyl alcohol)/ml enhanced the effect
considerably. A more rapid rate of polymerization was ob-
served when 1 mg of milled wood spruce lignin/ml was inves-
tigated under these conditions. Additionally, four lignin per-
oxidase fractions, separated from the extracellular enzyme
solution through isoelectric focusing, were found to polymer-
ize the milled wood lignin sample in a like manner.
It must be emphasized that thealkali-isolated straw lignin
and milled wood spruce lignin (24) arestructurally more
closely related to thenative biopolymer than thepreparations
originally investigated (7, 8). Thus, it now seems reasonably
clear that, in uitro, lignin peroxidase causes net polymeriza-
tion rather than degradation of lignin derivatives (24).
The obvious concerns raised by the foregoing observations
have prompted an examination of the effect of partially pu-
rified lignin peroxidases on the monolignol coniferyl alcohol
2: it was deemed instructive to establish whether or not the
products would bear any resemblance to thedehydrogenative
polymerisates formed from the same precursor through the
action of horseradish peroxidase in the presence of H,O,.
These preparations embody many of the structural features
encountered in native lignin biopolymers, and so the effect
upon such dehydropolymerisates from extended exposure to
the lignin peroxidases was also investigated.
EXPERIMENTALPROCEDURES
Enzyme Preparation-Extracellular lignin peroxidases from P.
chrysosporium ME 446 (ATCC 34541) were purified and stored as a
5 mg/ml solution in 0.4 M sodium acetate buffer (pH 6.4 at 4 "C)
containing 10 mM veratryl alcohol as a putative stabilizer.
Assay Procedures for Lignin Peroxidase Actiuity-Lignin peroxi-
dase activity was measured through the rate of oxidation of either
veratryl alcohol (25) or guaiacol (26). With veratryl alcohol, units of
activity were equivalent to micromoles of substrate oxidized at pH
3.0 and 30 "C to veratraldehyde (extinction coefficient at 310 nm of
9300 M" cm") in 1 min followed spectrophotometrically. The assay
mixture consisted of enzyme solution diluted with distilled deionized
water (2 ml, -0.25-0.30 unit), veratryl alcohol solution (1 ml, 3 mM
3637
in 0.33 M sodium tartrate buffer (pH 3.0) at 25 "C), and freshly
prepared 54 mM HzOz(15.4 pl).
Alternatively, the oxidation of 1.32 mM guaiacol (extinction coef-
ficient for product at 436 nm of 6390 M" cm") dissolved in 0.33 M
sodium tartrate buffer (pH 4.0) was likewise monitored (26) spectro-
photometrically. Here, veratryl alcohol (1Fmol), originally contained
in the enzyme solution, was also present in the reaction mixture. In
this second assay, units of activity were taken as micromoles of
guaiacol oxidized at pH4.0 by lignin peroxidase per minute at 30 "C.
Rate measurements were recorded with a Perkin-Elmer Lambda
6/PECSS system consisting of a Lambda 6 UV-visible spectropho-
tometer with a Lambda accessory interface connected to an Epson
Equity 1+personal computer with an Epson EX800 printer. Absorb-
ance at 310 or 436 nm wasrecorded for 1 min with the slope calculated
from 20 to 32 s after initiation of the reaction.
Partial Purification of Lignin Peroxidase-By using a nitrocellulose
membrane and a collodion apparatus (Schleicher & Schuell), 1 ml of
the lignin peroxidase solution was dialyzed overnight (3 X 500-ml
solvent changes) against 20 mM acetic acid (13). The solution was
then concentrated, at 600 mmHg, in the nitrocellulose membrane
(UH100/25 grade, nominal M, cutoff of 25,000, 8-ml capacity) for
almost 8 h to give a final protein concentration of 1 mg/ml.
Isoelectric Focusing (13)"The concentrated lignin peroxidase prep-
aration was separated by isoelectric focusing on a premade 5% poly-
acrylamide gel (Ampholine PAGplate, Pharmacia LKB Biotechnol-
ogy Inc.) containing 2.4%ampholytes for the pH range 3.5-9.5. Using
an Eppendorf micropipettor, the enzyme solution (15 pl) was delivered
onto sample application pieces placed 5 mm apart on the PAGplate
and -3 cm from the anode. Pharmacia LKB Biotechnology PI mark-
ers (PI range 2.4-5.65) were diluted 4-fold in distilled water, and 5 - p l
samples were placed on sample application pieces at selected positions
along the plate. The gel was focused at a constant voltage of 600 V
during the first 30 min and then at 1100 V for a further 1.5 h. With
a surgical blade, the PAGplate wasdivided into five sections for
visualization by protein and activity staining.
Protein and Activity Staining-For protein staining, the gel was
lowered into a fixing solution containing 0.7 M trichloroacetic acid
and 0.14 M sulfosalicylic acid for 0.5-1 h to precipitate the proteins
and toallow the ampholytes to diffuse out; the platewas subsequently
placed in ethanol/glacial acetic acid/H20 (258:67) for 5 min and then
stained for at least 1 h with Coomassie Blue (0.23 g in 200 ml of the
same solvent mixture) at room temperature. Activity staining with
guaiacol was carried out in a solution (50 ml) containing 50 mM
sodium tartrate buffer (pH 4.0 at room temperature), 12 mM guaiacol
(Fluka), and 2.5 mM H20,. Activity staining with 4-chloro-1-naphthol
(13),on the other hand, was performed in a solution (50 ml) contain-
ing 50 mM sodium tartrate buffer (pH 3.0 a t room temperature), 0.45
mM 4-chloro-1-naphthol (Bio-Rad), and50 p M H202;the 4-chloro-l-
naphthol solution was prepared by adding 60 mg of 4-chloro-l-
naphthol dissolved in 20 ml of MeOH to 100 ml of Tris/saline solution
containing 20 mM Trisand 500 mM NaCl at pH 7.5 and room
temperature. Phenolred staining (13) was carried out in the presence
and absence of Mn"; thus, two mixtures (50 ml each) were prepared
consisting of 50 mM sodium succinate buffer (pH 4.5 at room tem-
perature), 0.01% phenol red, and 50 p~ Hz02,with MnS04 (100 p ~ )
added to one of the mixtures.
Detection of Mn"-dependent Enzyme Activity in Lignin Peroxidase
Preparation 127)"The purpose of this assay was to determine
whether the oxidation of phenol red was influenced by any Mn"-
dependent peroxidases contaminating the lignin peroxidase prepara-
tion. Solutions were prepared so as to contain 0.01% phenol red
(Sigma), 25 mM lactic acid (J. T. Baker Inc.), 0.1% chicken egg
albumin (Sigma), and 100 p~ H202; MnS04 (3.34 mM, 30 p l ) was
added to one of these solutions to give a final 100 p~ concentration
of Mn", whereas an equal volume of distilled deionized water was
added to theother.
Sodium succinate buffer (20 mM, 1.0 ml, pH 4.5 a t 30 "C) was
added to respective aliquots (1 ml), and the resulting assay mixtures
(2 ml) were then separately transferred to cuvettes containing lignin
peroxidase (10 pl, with an activity equivalent to 0.08 pmol of guaiacol
consumed per min at pH 4.0 and 30 "C). Toestablish the initial value
of absorbance at 610 nm, 2 M NaOH (40 p1) was added immediately
to a cuvette containing the assay mixture, which was then inverted
three times prior to making a spectrophotometric reading. Otherwise,
the reaction was allowedto proceed for 5 min at 30 "C and terminated
with the addition of 2 M NaOH (40 p l ) , after which the absorbance
was measured as before.
The foregoing assays were also conducted under conditions where
3638 On the Role of Lignin Peroxidase
lactate, H20z,and enzyme were omitted individually. Aseparate
mixture containing only the enzyme and buffer was prepared as well.
In all cases, the absorbance a t 610 nm was determined initially and
after 5 min upon termination of the reaction with 2 M NaOH (40 pl)
as described above. The results are summarized in Table I.
Lignin Peroxidase S~bstrates-[l-'~C]-,[2-13C]-, and [3-13C]coni-
feryl alcohols 2a-c were synthesized as previously described (28) and
either used directly or diluted with natural abundance material as
follows. Unlabeled coniferyl alcohol (40.2 mg) was mixed with[I-"C]
coniferyl alcohol (25.0 mg, 99 atom % "C) to give [l-'3C]coniferyl
alcohol (65.2mg,0.36mmol, 24 atom % MSm/z 181 (M+,40%),
180 (89%),137 (M' + H, -CH13CHz0H, 100%);I3C NMR (CDC13) 6
63.8 ppm.
Spectral data for [2-'3C]coniferylalcohol (96 atom % 13C)afforded
MS m / z 181 (M', 70%), 180 (3%), 137 (M' i H, -J3CHCH20H,
100%);I3C NMR (CDCb) 6 126.5 ppm.
Spectral data for [3-13C]coniferylalcohol (74 atom % I3C)furnished
MS m/z 181 (M+, 70%), 180 (23%), 138 (M' + H, -CHCHzOH,
100%);I3C NMR (CDCl,) 6 131.7 ppm.
Treatment of [1-l3C]-, [2-13C]-, and [3-13C]Coniferyl Alcohols2a-c
with Lignin Peroxidase-A solution of lignin peroxidase (7.1 ml, 54-
60 guaiacol units) containing 10 mM veratryl alcohol dissolved in 0.4
M sodium acetate buffer (pH 6.4 at 4 "C) was introduced under Nz
into a three-necked round-bottomed flask (250 ml) equipped with a
magnetic stirring bar (7.9 X 25 mm) and two polyethylene capillary
inlet tubes. Sodium tartrate buffer (25 mM, 56.9 ml, pH 3.0 at room
temperature) was added until pH 4.0was reached. Two solutions,
designated A and B, were prepared as follows. [l-13C]Coniferylalcohol
2a (30.4mg, 0.17 mmol, 24 atom % 13C)was dissolved in sodium
tartrate buffer (25 RIM, 25 ml, pH 4.0 a t room temperature) to give
solution A; solution B was prepared by adding 30% H,O, (22.7 pl,
0.20 mmol) to sodium tartrate buffer (25 mM, 25 ml, pH 4.0 at room
temperature). These two solutions were simultaneously delivered by
means of Pharmacia LKB Biotechnology Model 2232 or 2132 Micro-
perpex peristaltic pumps (0.52 ml/h flow rate) into the flask contain-
ing lignin peroxidase through the separate polyethylene capillary
tubing connections. The reaction was allowedto proceed at 25 "C for
48 h with the mixture constantly being stirred under N,. The reaction
mixture became slightly turbid within 1 h. Aliquots (-0.08 guaiacol
unit) were removed to determine enzyme activity at 0, 24, and 48 h.
After 48 h, the mixture was centrifuged at 9750 X g for 2 h at 10 "C
using a Sorvall SS-34 rotor, and the supernatantwas returned to the
three-necked round-bottomed flask assembly to determine whether
more polymeric material would separate from solution. The residual
pellet, on the other hand, was resuspended in distilled water (-10-15
ml) and centrifuged (9750 X g) for 2 h. The resuiting supernatant
wash water was discarded, whereas the pellet itself was frozen (liquid
nitrogen) and lyophilized to afford a dry, light-beige product (7.5 mg).
To the original supernatant in the three-necked round-bottomed flask
was next added a solution of 8.0 mM H,Oz in sodium tartrate buffer
(25 mM, 25 ml, pH 4.0 at room temperature) over a 24-h period (1.04
ml/h flow rate). Thesuspension thereupon obtained was centrifuged,
and thepellet was washed as before to give an additional 23.7 mg of
the dehydrogenativelypolymerized product (furnishing a quantitative
yield overall). An aliquot from the final reaction mixture was also
removed to assay for the remaining enzyme activity after 72 h.
A similar procedure was separately carried out with [2-13C]coniferyl
alcohol 2b (96 atom % 13C)to afford a light-beige dehydropolymerized
product in quantitative yield. With [3-'3C]coniferyl alcohol 2c (74
atom % "C), the reaction was continued for 48 h as described above,
and then more H202(25 ml, 8.0 mM in 25 mM sodium tartrate buffer
(pH 4.0) a t room temperature) was delivered by means of a peristaltic
pump during a further 8.3 h. Following centrifugation and lyophili-
zation of the resulting washed pellet, a quantitativeyield of precipitate
was again obtained.
Incubation of ConiferylAlcohol 2 in 25 mM Sodium Tartrate Buffer
(pH 4.0) without Lignin Peroxidase-To a three-necked round-bot-
tomed flask (250 ml) was added 10 mM veratryl alcohol in sodium
acetate buffer (0.4 mM, 7.1 ml,pH 6.4 at 4 "C). Sodium tartrate buffer
(25 mM, 56.9 ml, pH 3.0 at room temperature) was then added until
pH 4.0 was reached. Coniferyl alcohol 2 (30.66 mg, 0.17 mmol) was
dissolved in sodium tartrate buffer (25 mM, 25 ml, pH 4.0 at room
temperature) as in solution A described above. Likewise, a fresh
preparation of 8.0 mM H20, in sodium tartrate buffer (25 mM, 25 ml,
pH 4.0 at room temperature) was designated as solution B. The two
solutions were then delivered over a 48-h period into the round-
bottomed flask with constant stirring under N,.
No precipitate was observed in the reaction mixture. An aliquot (2
ml) was removed after 48 h and filtered through an Acro LC3A filter
assembly (0.45 pm). Next, 10 p1 of the filtrate were applied to a Nova-
Pak CISreversed-phase column, protected by a WatersBondapak CIS
Corasil guard column, through which it was eluted with MeOH/HzO
(15235) at a flow rate of 1.3 ml/min. The area of the absorbance peak
detected at 262 nm arising from the eluted coniferyl alcohol 2 revealed
that no reaction had occurred.
Effect of Further Exposure to Lignin Peroxidase on Dehydropo-
lymerisate from Coniferyl Alcohol-The lignin-like dehydrogenative
polymerisate (10 mg) prepared from [2-13C]coniferylalcohol 2b with
lignin peroxidase in the presence of H,Oz was introduced into a
round-bottomed flask (250 ml) containing lignin peroxidase (-18
guaiacol units of enzyme activity) and sodium tartrate buffer (25 mM,
19.0 ml, pH 3.0 at room temperature). Hydrogen peroxide solution
(15 p1 of 30% aqueous H,Oz in 16.7 ml of sodium tartrate buffer (25
mM, pH 4.0 a t room temperature)) was added directly to the suspen-
sion of the dehydropolymerisate, and thereaction mixture was stirred
for 5 h under Nz. 30% H20, (15 pl) dissolved in distilled dioxane (10
ml) was then added to the flask, and the incubation was continued
for another 15 h. The dioxane was removed under reduced pressure,
and the resulting aqueous suspension was centrifuged (9750 X g) for
2 h at 10 "C. The pellet thus obtained was resuspended in distilled
water (5 ml) and centrifuged as before. After discarding the super-
natant, thepellet was again suspended in water (-500 pl) and freeze-
dried to give a quantitative yield of product.
Molecular Weight Distributions of Dehydropolymerisates Formed
from Coniferyl Alcohol by Lignin Peroxidase-The dehydrogenative
polymerisates obtained by incubating [2-13C]coniferyl alcohol 2b with
lignin peroxidase in the presence of Hz0, were dissolved, at concen-
trations ranging between 3 and 5 g/liter, in carbonate-free aqueous
NaOH (0.1 M, 1 ml) and individually applied to a Sephadex G-100
column (2.5 X 100 cm). Elution with carbonate-free aqueous NaOH
(0.1 M) at a flow rate of30 ml/h was monitored at 280 nm with a
double-beam ISCO Model V' detector at the column outlet. The
column dispersion for the species with M, of -500 was 4 % of the
relative column retention volume. The raw data were digitized and
transformed to elution profiles of absorbance (AZM ",) uersus relative
retention volume (VR).Calibration was achieved by correlation with
paucidisperse fractions from a dissociated kraft lignin preparation,
the molecular weight distribution of which had been exhaustively
characterized through ultracentrifuge sedimentation equilibrium
measurements (29). The effective incubation times represent the
periods elapsed between dissolution of the sample and appearance of
the profile.
Solution-state I3CNMRof Dehydropolymerisates from Coniferyl
Alcohol-Solution-state 13CNMR spectra were recorded with a FOUI-
ier transform Briiker WP 270 SY spectrometer. Each dehydropo-
lymerisate preparation (7.5-16.9 mg) was dissolved in deuterated (99.9
atom % D) dimethyl sulfoxide (1 g, MSD Isotopes, Montreal) to
afford a slightly cloudy solution. With broad-band carbon-proton
decoupling, between 18,000 and 32,000 transients were acquired at 68
MHz employing a 2-ps pulse width, a 0.524-s acquisition time, and a
15.625-kHz spectral width.
RESULTS
Partial Purification of Lignin Peroxidase Isozymes: Isoelec-
tric Focusing and ProteinlActiuity Staining-Following con-
centration of the partially purified lignin peroxidase sample
by dialysis, aliquots were applied to a 5% polyacrylamide gel
containing 2.4% ampholytes to separate the proteins by iso-
electric focusing. The resulting gel was then sectioned for
visualization by protein and activity staining. WithCoomassie
Blue, 10 distinct protein bands were evident. On the other
hand, with 4-chloro-1-naphthol, which can be used to stain
for lignin peroxidase in the presence of Hz02 (13), only seven
red bands were observed. With guaiacol, a substrate widely
used in spectrophotometric assays to determine peroxidase
activity (26), five brown bands appeared that seemed to co-
incide with five of thosestained by 4-chloro-1-naphthol.
Staining with phenol red was also carried out to determine if
any Mn"-dependent peroxidases were contaminating the en-
zyme preparation (27). Two assay mixtures were prepared,
one with and the other without Mn" (as MnSOJ. Two gel
sections, upon being immersed separately in the mixtures,
 On the Role of Lignin Peroxidase 3639
revealed the presenceof six bands that stained faintlyyellow 62.9, and 70.8 ppm (Fig. l a andTable 11, part la). The
in both cases, so that no Mn”-dependent enzymes were pres- corresponding product derived from [2-13C]coniferyl alcohol
ent. Moreover, these faintlyvisible bands appeared to coincide 2b gave two large resonances at -128 and -53 ppm, together
with the locationsof six of the 4-chloro-1-naphthol stains. with a smaller broad signal at 83.8-84.5 ppm (Fig. 2a and
Spectrophotometric Assay for Mn’I-dependent Enzyme Table 11, part 2a). Finally, with the preparation originating
Activity in LigninPeroxidase Preparation-In establishing from [3-’3C]coniferyl alcohol 2c, the solution-state I3C NMR
whether phenol red oxidation was influenced by any Mn”- spectrum displayedsignals at 71.1,71.7,85-87, and 128.9
dependent peroxidases in thedialyzed lignin peroxidaseprep- ppm, respectively (Fig. 3a and Table11, part 3a).
aration, assays were conductedunderdifferentconditions Molecular Weight Distributions of Dehydropolymerisates
where the cosubstrates and cofactor (Mn”’ chelator) had been Formed from Coniferyl Alcohol 2 by Action of Lignin Peroxi-
individually omitted (Table I). It can be seen that theoxida- dase-The molecularweight distributions of the products
tion of phenol red was unaffected by the presenceof MnSO, resulting from incubation of coniferyl alcohol 2 with lignin
(Table I, lines 1 and 4). No significant change in the degree peroxidase/H202 were determined size-exclusion chromato-
of oxidation occurred when lactate was absent from the assay graphically. Thesamples were eluted withaqueous 0.1 M
mixture (Table I, line 3). The phenol redwas not oxidized at NaOH through a Sephadex G-100 column calibrated with a
all when either the enzyme preparation or H202 was omitted kraft lignin preparation that had been exhaustively charac-
(Table I, lines 2,5, and6). terized by ultracentrifuge sedimentationequilibrium measure-
Treatment of[1-l3C]-, [2-l3C/-, and [3-’3CJConiferyl Alco- ments.
hols 2a-c withLignin Per~xidase-[l-’~C]-, [2-I3C]-, and Elution profile I in Fig. 4 represents the dehydropolymer-
[3-13C]coniferylalcohols 2a-c were synthesized aspreviously isate formed by the action of ligninperoxidase on [2-13C]
described(28) and diluted with unlabeled material to the coniferyl alcohol 2b in the presence of H202during a 72-h
desired level (see “Experimental Procedures”). Each sample period. It is clearly polymeric. Profile 2 delineates the effect
of coniferyl alcohol (0.17 mmol) was individually dissolved in of further treating this dehydropolymerisate, in sodium tar-
sodium tartrate buffer (25 mM, 25 ml, pH 4.0 at room tem- trate buffer containing dioxane as cosolvent, with lignin per-
perature) and delivered (0.52 ml/h for 48 h) into a three- oxidase/H202 for 20 h(see “ExperimentalProcedures”).
necked (250 ml) round-bottomed flask containing lignin per- Clearly, the degree of polymerization has increased substan-
oxidase (54-60 guaiacol units). Simultaneously, a 8 mM H z 0 2 tially with no sign of concomitant degradative transforma-
solution in sodium tartrate buffer (25 mM, p H 4.0 at room tions takingplace.
temperature) was delivered at the same rate(0.52 ml/h for 48
h) over the same timeperiod. DISCUSSION
Aliquots of the reaction mixture were removed 0, 24, and The results obtained convincingly demonstrate that lignin
48 h after initiation of the reaction to determine enzymatic peroxidase isozymes, uncontaminated with Mn”-dependent
activity towardguaiacol. Almost one-third of the activitywas peroxidases, in the presence of H202engender net polymeri-
lost after24 h, but theresidual activity beyond this pointwas zation of the monolignol coniferyl alcohol 2 to afford at pH
relatively unchanged over the next 24-h period. After 48 h, 4.0 awater-insolublepolymeric preparation. This reaction
the precipitate in the flask was recovered by centrifugation, does not occur in the absence of lignin peroxidase. That the
and H202(8.0 mM) was reintroduced into the resulting super-material is clearly polymeric has been established from the
natant for a n additional 24 h, following which the precipitate profiles generated by eluting the product through a Sephadex
was againremoved. At this point,-50% of the original enzyme G-100 columncalibrated with a kraft lignin preparation char-
activity hadbeen lost. Importantly, theyields of the dehydro-
polymerisates obtained from each of the three incubations
with lignin peroxidase in the presence of Hz02were quanti- D.E /
tative relative to the weights of coniferyl alcohol 2 starting
material.
Solution-state 13CNMR of Dehydropolymerisates from 13C-
Labeled Coniferyl Alcohols-Figs. 1-3 depict the solution-state
I3C NMR spectra of the reaction products obtained following
incubation of [l-”C]-, [2-13C]-,and [3-13C]coniferyl alcohols
2a-c with lignin peroxidase in the presence of H202. The
preparation obtained by treating [ l-13C]coniferyl alcohol 2a
with lignin peroxidase/H202showed resonances at 60.1, 61.5, I-*
TABLE I
Spectrophotometric assay for Mn“-dependent peroxidase
activity in the enzyme preparation
AGlOnm
Assay condition aAe,,,,
t = 0 rnin t =5 min
1. Complete” 0.151 1.493 1.342
2. -Enzyme 0.141 0.148 0.007
3. -Lactic acid 0.146 0.283 1.137
4. -MnS04 0.138 1.528 1.390
5. -HZ01 0.158 0.158 0.000 1 bo 160 5’0 b PPM
6. Enzyme + buffer 0.004
0.004 0.000
FIG.1. 13C NMR solutionspectraofdehydrogenative
Complete assay mixture consisted of 20 mM sodium succinate polymerisates formed from [ l-’3C]coniferyl alcohol with HzOz
buffer (1.0ml)addedto a solution (1 ml)containingphenol red in presence of lignin peroxidase at pH 4.0 (a)and horseradish
(0.01%),lactic acid (25mM), MnSOl (100mM), egg albumin (O.l%), peroxidase at pH 6.5 ( 6 ) . S denotes solvent; substructural assign-
H20z (100mM), and theenzyme. ments are listed in Table11, parts l a and Ib.
3640 On the Role of Lignin Peroxidase
TABLE I1
I3C N M R assignments for dehydrogenative polymerisates fromconiferyl alcohols 2a-c formed
through the action of lignin peroxidase/H202and horseradish peroxidase/H202
Lignin peroxidase/H202 Horseradish peroxidase/Hn02
Chemical shift Lignin substructure" Chemical shift Lignin substructure"
PPm ppm
la. [l-'3C]Coniferyl alcohol 2a lb. [l-13C]Coniferylalcohol 2a
70.8 C 70.9 C
62.9 D and E 62.9 D or E
62.6 D or E
61.5 A 61.6 A
60.1 B 60.0 B

2a. [2-'3C]Coniferylalcohol 2b 2b. [2-13C]Coniferylalcohol 2b

128.56 A 128.33 A
128.00 A 127.75 A
83.8-84.5 B 83-84 B
53-53.6

3a. [3-13C]Coniferylalcohol 2c 3b. alcohol 2c

[3-L3C]Coniferyl
128.85 A 128.90 A
128.43 A 128.47 A
86.8-87 D 86.91 D
84.8-85 C 84.91 C
71.7 E 71.47 E
71.1 B 70.83 B
Lignin substructures A-E are shown in Scheme 1.

C.D, E
1

150 100 50 OPPM

FIG. 2. I3C NMR solutionspectraofdehydrogenative

polymerisates formed from [2-"C]coniferylalcohol with Hz02
in presence of lignin peroxidase at pH 4.0 (a) and horseradish
peroxidase at pH 6.5 ( b ) .S denotes solvent; substructural assign-
ments are listed in Table 11, parts 2a and 2b.
acterized through ultracentrifuge sedimentation equilibrium
measurements (Fig. 4, profile 1).
Further treatment of this lignin-like dehydropolymerisate
with lignin peroxidase(s)/HzO2was also examined. As can be
seen from profile 2 in Fig. 4, the only outcome was an increase
in the degree of polymerization of the preparation. This is
indeed in keeping with previously reported effects of lignin
peroxidase on lignin-derived preparations (24).
It was next of interest to establish whether the bonding
environments within these polymers resembled those in syn-
thetic dehydrogenative polymerisates obtained through horse-
radish peroxidase/H202action on monolignols such as coni-
feryl alcohol 2. In this regard, Scheme 1 shows the main
substructural assignments previously made for dehydropo-
lymerisates from coniferyl alcohol 2, the formation of which
was catalyzed by horseradish peroxidase/HZO2(30).Thus, the
solution-state I3C NMR spectra of the dehydropolymerisates,
L
50 6 PPM
FIG. 3. I3C NMR solutionspectra of dehydrogenative
polymerisates formed from [3-I3C]coniferylalcohol with Hz02
in presence of lignin peroxidase at pH 4.0 ( a )and horseradish
peroxidase at pH 6.5 ( b ) .S denotes solvent; substructural assign-
ments are listed in Table 11, parts 3a and 3b.
obtained upon incubation of [1-13C]-, [2-13C]-, and [3-13C]
coniferyl alcohols 2a-c individually with lignin peroxidase/
HzO2,are depicted in Figs. l a , 2a, and 3a. Although there are
some minor differences in the intensities of individual reso-
nances, it can immediately be seen that there are striking
similarities between these spectra and those of the corre-
sponding polymers (Figs. lb, 2b, and 3b) formed by the action
of horseradish peroxidase/H2O2(30). This confirms that the
polymerization of the monolignol precursor coniferyl alcohol
at the hands of lignin peroxidase/H2O2 affords synthetic
lignin-like preparations with bonding environments more or
less identical to those obtained by treatment with horseradish
peroxidase/H2O2.
In no case wereany new bonding environments detected as
 On the Role of L,igninPeroxidase 3641
In a similar manner, when [2-'3C]coniferyl alcohol 2b was
used asasubstrate for lignin peroxidase(s)/HzOz, the 13C
NMR spectrum of the product gave two large resonances at
-128 and -53 ppm and smaller broad signals at 83.8-84.5
ppm (Fig. 2a and Table 11, part 2a). Once again, these match
closely with those of the dehydropolymerisate obtained from
[2-13C]coniferylalcohol 2b upon incubation with horseradish
peroxidase/H202 (Fig. 26 and Table 11, part 2b) (30). The
resonance at 128 ppm corresponds to substructure A, those
at 83-84 ppm to substructure B, and signals at 53-54 ppm to
1 substructures C-E.
20 10 5 2 1 0.5 In a comparable way, the 13CNMR spectrum of the prepa-
Uwx 1 0 - 3 ration derived from incubating [3-13C]coniferylalcohol 2c
with lignin peroxidase(s)/H202(Fig. 3a and Table 11, part 3a)
FIG. 4. Molecular weight distribution of dehydrogenative bears a great similarity to that obtained in the presence of
polymerisate (profileI ) formed from [2-13C]coniferylalcohol
with H2Oz in presence of lignin peroxidase during 72-h period horseradish peroxidase/H2O2(Fig. 36 and Table 11, part 3b).
at pH 4.0 and effect of further enzyme action (profile2 ) upon This is to say that the signals at 71.1, 71.7, 85-87, and -128
dehydropolymerisate for another 20 h in presence of HzOz. ppm can be assigned to substructures B, E, C/D, and A,
Shown are Sephadex G-lOO/aqueous 0.1 M NaOH elution profiles respectively (30).
monitored at 280 nm after 6.5-h incubation times (dotted line, blue
dextran). This figure was reprinted with permission from Ref. 46.
It can therefore be concluded that the effect of lignin
peroxidase(s) and hydrogen peroxide on the phenolic mono-
lignol coniferyl alcohol 2 is to engender the formation of a
polymeric preparation arguably identical to that of the cor-
responding lignin-like dehydropolymerisate, the formation of
which is catalyzed by horseradish peroxidase/HzOz (30). This
result is consistent with previous observations that, in vitro,
lignin peroxidase/Hz02 converts lignin-derived preparations
from natural sources into higher molecular weight polymers
"L "L (24).
It therefore seems remarkable that such an enzyme can be
A
- -B considered to be lignin-degrading. Yet, lignin peroxidase does
also cleave C,-CB bonds (31,32) and aromatic rings (33) in a
variety of nonphenolic dimeric phenylpropanoid compounds.
This occurs through the initial formation of corresponding
cation radical intermediates (12, 20, 34), which then undergo
a number of degradative transformations involving reactive
oxygen species (35, 36). Obviously, such effects wouldbe
expected to reduce rather than increase the degrees of polym-
erization of lignin-derived preparations. However, depolym-
erization was not observed during this work when either the
phenolic monolignol, coniferyl alcohol, or its dehydropo-
lymerisate were incubated with lignin peroxidase/H20z. This
apparent conflict can be resolved by the realization that the
single-electron oxidation of phenols into corresponding phen-
oxy radicals is more facile as far aslignin peroxidase/HzOzis
concerned.
Indeed, it was recognized at a relatively early stage that
phenolic moieties liberated as aconsequence of homolytic C,-
CBcleavage in arylglycerol @-arylether model compounds
would undergo polymerization at the hands of lignin peroxi-
-D E
- dase and Hz02 (22,25,37). Some evidence has indeed surfaced
suggesting that polymerization may also occur in intact P.
SCHEME I chzysosporium cultures. Upon introduction of a pine kraft
lignin fraction, the highest region in the molecular weight
a result of incubating coniferyl alcohol 2 with lignin peroxi- distribution of a small component subset, which develops a
dase in the presence of H202when compared to the corre- close and sustained association with the mycelium, first in-
sponding dehydropolymerisates formed by horseradish per-
creases appreciably beforebecoming somewhat attenuated
oxidase/H2O2.For example, with the product resulting from
[l-13C]coniferylalcohol 2a in the presence of lignin peroxi- and approaching a stable value (38). Presumably, this is the
dase(s)/H202, a series of resonances were observed at 60.1, first effect caused by the lignin peroxidases.
61.5,62.9, and 70.8 ppm (Fig. la and Table 11, part la).These The ability of lignin peroxidase to polymerize phenolic
signals, originating from lignin substructural environments moieties suggests that one of the major functions of the
B, A, D/E, and C, respectively (Scheme l),closely correspond enzyme lies in detoxifying lower molecular weight phenolic
to those exhibited by the preparation obtained with [1-13C] compounds released from lignins during their fungal decom-
coniferyl alcohol 2a and horseradish peroxidase/H202 (Fig. position (39, 40). One way of accomplishing this task would
16 and Table 11, part lb) (30). be by polymerizing these species into molecular entities suf-
3642 On the Role of Lignin Peroxidase
ficiently large that they can no longer penetrate through the preparations encountered in the presence of the enzyme in
hyphal cell wall. uitro.
Yet P. chrysosporium is quite capable of depolymerizing A reasonable workinghypothesis is suggested by the effects
lignin preparations when no lignin peroxidase activity canbe of a-carbonyl groups that can be formed as one consequence
detected in the culture medium (41). Clearly, there must be of lignin peroxidase-catalyzed oxidation of nonphenolic aro-
other enzymes and/or cofactors controlling the ligninolytic matic moieties to intermediate cationradicals (32). Thus, the
system of P. chrysosporium. However, lignin biodegradation selective introduction of a-carbonyl groups enhances the rate
by the microorganism is definitely influenced by lignin per- of depolymerization of a spruce lignin preparation by P.
oxidase: addition of 2 units (25) of the enzyme to washed P. chrysosporium cultures (44). On the other hand, it has been
chrysosporium pellet cultures was found to elicit a 4-fold convincingly documented that oxidation of the benzylic hy-
enhancement in theoverall rate of the process (41). droxyl in 1-(4’-ethoxy-3’-methoxypheny1)-2-(2”-methoxy-
These considerations prompted the formulation of a pro- phenoxy)propane-1,3-diolto a carbonyl group markedly re-
visional rationale for the ligninolytic pathway contrivedby P. tards metabolism of this nonphenolic @-0-4’-ether lignin
chrysosporium in termsof alternating oxidative and reductive model compound (44). As far as the lignin macromolecule is
events involving lignin peroxidase andanintracellular concerned, however, the presence of a carbonyl group at the
NADP-dependent aryl-alcoholoxidoreductase (41), respec- a’-position in a nonphenolic P-O-4”ether moiety could in-
tively. The viability of such a scheme should be demonstrable crease the rate of nucleophilic displacement of the aryloxy
through investigationsof the twoenzymes acting concertedly group from the@-positionby more than an order of magnitude
in uitro. No report about the matter has hitherto appeared for (45) (Scheme 2).
the aryl-alcoholoxidoreductase, butthe effect of cello- Herein may reside the central insight into how lignin de-
biose:quinone oxidoreductase(42) has beenexplored(37) polymerization can be accelerated indirectly by lignin perox-
since it was suggested that theenzyme may be able toreduce idase in uiuo. As well as detoxifying the extracellular fungal
environment (seeabove),ligninperoxidase may be quite
phenoxy radicals as well as quinones (42).
capable of introducing into the lignin fabric functionalgroups
The influence of cel1obiose:quinone oxidoreductase appears
which render thepolymeric structure more susceptibletoward
t o vary with the lignin peroxidase substrate examined and, degradation by another enzyme (46).
perhaps, the conditions employed. The enzyme was not ob- This possibility warrants decisive experimental investiga-
served to interfere with lignin peroxidase-catalyzed phenoxy tion. No 13CNMR spectral changeshave so far been detected
radical formation from acetosyringone (37); moreover, it did by other workers as a consequence of treating dehydropo-
not affect the apparent product size distribution engendered lymerisates from [1-13C]-,[2-’3C]-, and [3-13C]coniferylalco-
by the polymerization of either guaiacol or a dehydropo- hols or a dehydrogenative copolymer of p-[ring-4’-13C]cou-
lymerisate from coniferyl alcohol at the hands of lignin per- maryl alcohol and coniferyl alcohol at pH3.0 separately with
oxidase (37). extracellular P. chrysosporium culture fluid and purified lignin
Yet cel1obiose:quinone oxidoreductase, alongwith cello- peroxidase in the presence of HZ02(47); moreover, the out-
biose, did seemto mitigate the polymerization of a kraft lignin come, in the case of the dehydropolymerisate from [3-I3C]
preparation catalyzed by the crude extractfrom a P. chryso- coniferylalcohol,was not affected by prior acetylation or
sporium culture (43). Curiously, the total areaof the resulting methylation of the substrate (47).
gel filtration profile (monitored at 280 nm) was attenuated by Nevertheless, the present work has substantiated recent
32%; however, the effectcould hardly have been due, as claims (47, 48) that such dehydropolymerisates are polymer-
claimed, to extensive aromatic ring cleavage since no low ized by lignin peroxidase/HzO2. It now remains t o be con-
molecular weight components were detected (43). It is anyway firmed whether the oxidative transformations throughwhich
not clear with what intermediate species cel1obiose:quinone carbonyl and carboxylgroups areintroducedinto lignins
oxidoreductase directly interacts under these circumstances: during wood decay by P. chrysosporium (49) are also initiated
the enzyme, in the presence of cellobiose, can also diminish by the sameenzyme.
the lignin peroxidase-catalyzed oxidation of veratryl alcohol Yet noobligatory relationship has been established between
t o veratraldehyde (43). lignin peroxidase activity and the overall process of lignin
Thus, cel1obiose:quinone oxidoreductase does appear to be biodegradation. The white-rot fungus Lentinula edodes, for
capable of inhibiting the oxidation of phenolic and nonphe- example, achieves an almost imperceptible rate of lignin bio-
nolic moieties by lignin peroxidase. If the two alternatives are degradation when manifesting a greater specific lignin per-
supposed to bring about a net increase anddecrease in molec- oxidase activity than optimally expressed by P. chrysosporiurn
ular weight of a lignin substrate, respectively, then both even- (46,50,51). Conversely, no extracellularperoxidases whatever
tualities should be hindered by cel1obiose:quinone oxidoreduc- have been detected from Fomes lignosus or Trametes cingu-
tase. Consequently, the accelerative effectof lignin peroxidase lata, both of which engender (in agitated and nonagitated
upon the rate of lignin biodegradation in uiuo still has some- cultures, respectively) quite irreproachable rates of 14C02 ev-
how to be reconciled with the polymerization of lignin-derived olution from HCl/dioxane-isolated I4C-labeled wheat straw
lignin (52). Indeed, T. cingulata was incapable of oxidizing
veratryl alcohol, the substrate most commonly adopted for
assaying lignin peroxidase activity (25), even though it SUS-
tained a rate of lignin biodegradation which was no less than
half of that encountered withP. chrysosporium (52).
In view of these findings, it should come as no surprise that
there is no systematic relationshipbetween detectable lignin
peroxidase activityandtherate of ligninbiodegradation
among different strains of P. chrysosporium.’ It might be
mentioned here that lignin peroxidase is susceptible to inac-

SCHEME
2 0. M. Birch and P. Broda, unpublished data cited in Ref. 53.
 On the Role of Lignin Peroxidase
tivation, the extent of which depends upon the relative con-
centrations present of the electron-donor substrate and Hz02
(54, 55). Be that as it may, the complete absence of a corre-
lation between extracellular lignin peroxidase activity and
lignin biodegradation cannot automatically be ascribed to
differing sensitivities on the part of the corresponding iso-
zymes to inhibition.
There is little doubt that respectable rates of lignin depo-
lymerization can be maintained in vivo without the partici-
pation of extracellular lignin peroxidase, whether by L. edodes
(46, 50, 51), F. ligmsus, T. cingulata (52), or washed P.
chrysosporium pellet cultures (41).It could be argued that,
under these conditions, the enzyme is still operative while
remaining intracellularly confined in vesicles (56, 57) or
bound to the plasmalemma (56), cell wall (57), and mucilagi-
nous exopolysaccharides (57). However, it would be difficult
to envisage how lignin biodegradation could beso successfully
upheld by diffusion of the sparingly soluble substrate com-
ponents to theplaces where the enzyme is located.
Alternatively, it has been proposed that lignin peroxidase
may oxidizelignins indirectly through the mediation of small
molecules like veratryl alcohol (58, 59): single-electron trans-
fer to theimmobilized enzyme would formthe derived cation
radical, which could then diffuse freely to sites in the nearby
macromolecular substrate. Subsequent oxidation of nonphe-
nolic moieties in the lignin structure could be followed by
transfer of the unpaired electron through the arrays of parallel
aromatic rings (55, 59), whichseem to be disposed in an
organized manner (60). The resulting spatial separation be-
tween incipient phenoxy radicals at distant locations within
the macromolecular framework could reduce the incidence of
new interunit covalent bond formation that would tend to
polymerize the lignin further (55,59).
Certainly, charge transfer through macromolecular lignin
structures is a probable consequence of single-electron oxi-
dation, whether directly or indirectly, by lignin peroxidase.
However, such effects are of no avail to T. cingulata: despite
its ability to sustain a rate of lignin biodegradation that is
considerable, the microorganism fails to oxidize veratryl al-
cohol (52). Evidently, the enzyme ultimately responsible for
lignin depolymerization in uiuo remains as elusive at thetime
of writing as it was before the discovery of lignin peroxidase
in 1983.
Acknowledgments-We are indebted to Matthew L. Iwen (Univer-
sity of Minnesota) for determining the molecular weight distributions
of the dehydropolymerisates formedfrom[2-13C] coniferyl alcohol
and to Dr. Matti S. A. Leisola (Cultor Ltd., Kantvik, Finland) for
supplying a sample of lignin peroxidase, providing technical advice,
and offering helpful suggestions.
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