Action of diverse peroxidases and laccases on six cell wall-related phenolic compounds

Article abstract of DOI:10.1016/S0031-9422(99)00342-8

Four peroxidases and four laccases were compared as to reaction rates catalysed with six phenolic substrates of relevance to the plant cell wall. When each phenolic substrate was tested at 670 μM and pH 6.0, in the presence of 670 μM H₂O₂ or ~270 μM O₂ as the electron acceptor, all the peroxidases and laccases had similar substrate preferences: reaction rates were in the order sinapyl > coniferyl > p-coumaryl alcohols, and feruloyl > p-coumaroyl esters. Specific activities were in the order basic peroxidase > acidic peroxidase>>laccase. The data are consistent with the view that peroxidases rather than laccases play a major role in phenolic cross-linking in the cell wall.

Full text of DOI:10.1016/S0031-9422(99)00342-8

Phytochemistry 52 (1999) 769±773
Action of diverse peroxidases and laccases on six cell wall-related
phenolic compounds
Graham Wallace, Stephen C. Fry*
The Edinburgh Cell Wall Group, Institute of Cell and Molecular Biology, The University of Edinburgh, Daniel Rutherford Building, The King's
Buildings, Edinburgh EH9 3JH, UK
Received 15 March 1999; received in revised form 2 June 1999; accepted 2 June 1999
Abstract
Four peroxidases and four laccases were compared as to reaction rates catalysed with six phenolic substrates of relevance to
the plant cell wall. When each phenolic substrate was tested at 670 mM and pH 6.0, in the presence of 670 mM H2O2 or 0270
mM O2 as the electron acceptor, all the peroxidases and laccases had similar substrate preferences: reaction rates were in the
order sinapyl > coniferyl > p-coumaryl alcohols, and feruloyl > p-coumaroyl esters. Speci®c activities were in the order basic
peroxidase > acidic peroxidase>>laccase. The data are consistent with the view that peroxidases rather than laccases play a
major role in phenolic cross-linking in the cell wall. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Peroxidase; Laccase; Phenol oxidase; Cell wall; Coniferyl alcohol; Ferulic acid; p-Coumaric acid; HPLC
1. Introduction
tase, E.C. 1.11.1.7) (Harkin & Obst, 1973). More
recently, however, an oxidase (`laccase', i.e. benzene-
diol : O2 oxidoreductase, E.C. 1.10.3.2), which can cat-
alyse similar coupling reactions of monolignols using
O2 instead of H2O2 as the electron acceptor (Dean &
Eriksson, 1994; McDougall, Stewart & Morrison,
The oxidative coupling of phenolic compounds is
important in the architecture of the plant cell wall
(Fry, 1986; Wallace & Fry, 1994). From the cross-link-
ing
of
hemicellulose-bound
feruloyl
groups
1994; Savidge
&
Udagama-Randeniya, 1992;
(Markwalder & Neukom, 1976; Wallace & Fry, 1995)
and of extensin-bound tyrosine groups in primary cell
walls (Brady, Sadler & Fry, 1998) to the synthesis of
lignin in wood (Sarkanen, 1971), such reactions have a
profound e€ect on the cell wall.
Sterjiades, Dean, Gamble, Himmelsbach & Eriksson,
1993), has been suggested to be responsible for ligni®-
cation, a possibility ®rst raised by Freudenberg and
Neish (1968). There is now confusion as to the relative
contributions of peroxidases and laccases in the cell
wall.
Several substrates have been used routinely for
assaying peroxidase and laccase activities, e.g. guaia-
col, o-dianisidine, pyrogallol, syringaldazine and 2,2'-
azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS)
(McDougall et al., 1994; Sterjiades et al., 1993).
Although such arti®cial, chromogenic substrates are
useful for simple enzyme assays, they give little infor-
mation about the speci®city of the enzymes for par-
ticular substrates of relevance to the plant cell wall.
Natural substrates yield mainly colourless reaction
products and therefore tend not to be favoured for use
However, despite the biological, dietary and indus-
trial implications of the oxidative coupling of plant cell
wall phenolics, many questions remain to be answered
about the enzymology of coupling and about the sub-
sequent nonenzymic rearrangements of the immediate
reaction products (Ralph & Helm, 1993). For many
years the enzyme thought to be principally responsible
for the oxidative polymerisation of monolignols to
form lignin was peroxidase (donor : H2O2 oxidoreduc-
* Corresponding author. Fax: +44-131-650-5392.
E-mail address: s.fry@ed.ac.uk (S.C. Fry)
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00342-8

770
G. Wallace, S.C. Fry / Phytochemistry 52 (1999) 769±773
in routine spectrophotometric assays. Although several
studies have used individually optimised assays, with
`natural' phenolics as peroxidase substrates, e.g. coni-
feryl alcohol (Calderon, Munoz, Morales & Ros
Barcelo, 1992; Ferrer & Ros Barcelo, 1994a, 1994b;
Rasmussen, Dunford & Welinder, 1995; Takahama,
1993), ferulate (Gelinas, 1973; Pickerling, Powell,
Wender & Smith, 1973; Rasmussen et al., 1995;
Whitmore, 1976; Zimmerlin, Wojtaszek & Bolwell,
1994) and ca€eate (Rasmussen et al., 1995), such stu-
dies are in the minority. In addition, peroxidases tend
to have lower anities for many arti®cial substrates
than for wall-related phenolics; for example, KM values
for pyrogallol or guaiacol of 2±7 mM (Kwak et al.,
1995; Zimmerlin et al., 1994) compare with values of
20±460 mM for coniferyl alcohol, p-coumaroyl esters
and ferulic acid (Otter & Polle, 1997; Takahama &
Oniki, 1997; Zimmerlin et al., 1994).
We present here a direct quantitative comparison of
the speci®city of several peroxidases and laccases on a
range of cell wall-related phenolic substrates.
2. Results and discussion
2.1. Internal standardisation
For the analysis of phenylpropanoid reaction pro-
ducts, methyl benzoate ful®lled all the requirements of
an internal standard: it is chemically similar to the
analytes, it was stable and unreactive to peroxidase
and laccase under the conditions used, and it could be
completely resolved from all substrates and products
(Snyder & Kirkland, 1979). Addition of up to 6 mM
methyl benzoate did not a€ect the oxidation of guaia-
col by any of the peroxidases or laccases used in this
study when assayed spectrophotometrically (results not
shown).
2.2. Action of peroxidases and laccases on cell wall-
related substrates
Each enzyme was assayed (Table 1) on a range of
cell wall-related phenolic substrates at a standardised
concentration (670 mM) well above reported KM values
of peroxidases for natural substrates (Otter & Polle,
1997; Takahama & Oniki, 1997). None of the peroxi-
dases exhibited detectable oxidase activity on any of
the six phenolic substrates tested. In the presence of
H2O2, the basic iso-peroxidase was more active than
the acidic isoenzymes, which are often proposed to be
associated with ligni®cation (Ferrer & Pedreno, 1992;
Mader, Nessel & Bopp, 1977). Laccases, when assayed
at 100Â higher enzyme concentrations (mg protein
basis), had activities similar to or lower than those of
the peroxidases. H2O2 was used at 670 mM whereas O2

G. Wallace, S.C. Fry / Phytochemistry 52 (1999) 769±773
771
strong discrimination between ester, aldehyde or alco-
hol substrates (e.g. methyl ferulate 1 coniferalde-
hyde 1 coniferyl alcohol; Table 1).
2.3. Product pro®ling
Peroxidase A2 oxidised each substrate to yield sev-
eral products (Fig. 1). It has been reported that di€er-
ent peroxidases may yield di€erent products
(Zimmerlin et al., 1994); such a phenomenon could
have profound e€ects on lignin synthesis. However, all
the enzymes studied here yielded product pro®les very
similar to those shown in Fig. 1.
2.4. Conclusions
We have established a simple and e€ective method
for assaying oxidase and peroxidase activities on
diverse cell wall-related substrates. Most previously
described routine assays for laccase and peroxidase
have involved the measurement of either one arbitra-
rily selected product or an ill-de®ned mixture of pro-
ducts. The present HPLC-based assay monitors the
disappearance of substrate and thus records total
enzyme activity on the substrate of interest whether or
not all the diverse reaction-products can be eciently
detected. Our assay therefore enables a reliable esti-
mate of the ability of a given enzyme to oxidise a
given phenolic component of the cell wall under the
conditions tested.
Numerous iso-peroxidases and several phenol oxi-
dases are present in plants. Although it remains poss-
ible that other isoforms, not yet tested, will turn out to
exhibit di€erent catalytic preferences or to yield di€er-
ent reaction products, all the peroxidases and laccases
tested by us followed the same pattern, preferring
dimethoxy > monomethoxy > nonmethoxy phenols.
There was relatively little di€erence between alcohols,
aldehydes and esters.
Fig. 1. HPLC pro®les of oxidation products formed by the action of
horseradish peroxidase A2 on (A) methyl p-coumarate (MC), (B)
methyl ferulate (MF), (C) coniferaldehyde (Cald), (D) p-coumaryl
alcohol (CouA), (E) coniferyl alcohol (CA) and (F) sinapyl alcohol
(SA). IS=internal standard (methyl benzoate), P=oxidation pro-
duct, not present in substrate or enzyme preparations alone. A280 is
shown in arbitrary units.
was supplied as air-saturated bu€er (which provides
0270 mM O2 at 258C); however, this di€erence in elec-
tron acceptor concentration between the peroxidases
and the laccases is small compared with the di€erence
in enzyme concentrations. Thus, under the conditions
employed, the peroxidases had a far higher speci®c ac-
tivity than the laccases.
All enzymes tested followed the same pattern of sub-
strate preference. The reaction rates were in the order:
sinapyl > coniferyl > p-coumaryl alcohols, and feru-
loyl > p-coumaroyl esters. These results are in accord-
ance with the ®ndings of Russell, Forrester and
Chesson (1996) but disagree with those of Takahama
(1995) and Ros Barcelo, Munoz and Sabater (1987),
who reported that peroxidases oxidise coniferyl alcohol
much faster than sinapyl alcohol, and p-coumarate fas-
ter than ferulate. None of the enzymes tested showed a
The basic peroxidase was considerably more active
on all the substrates used in this study than the acidic
peroxidases. Acidic peroxidases are often held to be
those most directly involved in ligni®cation (Ferrer &
Pedreno, 1992; Mader et al., 1977), although there is
relatively little lignin in horseradish roots and thus the
predominant physiological role of the acidic isoen-
zymes studied in the present work may be other than
in ligni®cation. At comparable enzyme concentrations
(expressed either per mg of protein or per nmol of pro-
tein), and at the mildly acidic pH and submillimolar
phenolic substrate concentrations likely to be encoun-
tered in vivo, laccases were much less e€ective than
peroxidases. Several independent studies, using diverse
methods, have indicated that H2O2 is produced in the
plant apoplast in vivo (Frahry & Schopfer, 1998; Liu,
Eriksson & Dean, 1995; Olson & Varner, 1993;

772
G. Wallace, S.C. Fry / Phytochemistry 52 (1999) 769±773
Patterson, Macrae & Ferguson, 1984; Schopfer, 1994;
Warm & Laties, 1982) and in solutions mimicking apo-
plastic conditions in vitro (Elstner & Heupel, 1976;
Frahry & Schopfer, 1998; Federico & Angelini, 1986;
Halliwell, 1978; Ishida, Ookubo & Ono, 1987). If these
studies are correct in implying that H2O2 is available
in the cell wall in vivo, and/or if some peroxidases can
under certain circumstances e€ectively act as `oxidases'
(Grambow & Langenbeck-Schwich, 1983), the results
presented here support the view of Ros Barcelo (1995)
that peroxidase and not laccase has the major role in
ligni®cation.
3.3. E€ect of methyl benzoate on peroxidase activity
To 0.5 ml of 30 mM guaiacol, with and without 18
mM methyl benzoate, in 50 mM Pi bu€er (pH 6.8),
was added 0.5 ml 5 mM H2O2 and 0.5 ml of an appro-
priate concentration of enzyme soln. The reaction was
monitored by A436 for 5 min.
3.4. HPLC assays
3.4.1. Peroxidase
To 20 ml of 2 mM phenolic substrate (in 50 mM suc-
cinate (Na+) bu€er, pH 6.0, containing 6 mM methyl
benzoate), was added 20 ml of 2 mM H2O2 and 20 ml
of peroxidase solution (typically 50 ng/ml). Controls
containing H2O in place of the enzyme or H2O2 were
used for time zero. The mixture was incubated at 258C
for 30 min and the reaction stopped by the addition of
60 ml MeOH. All assays were performed in quadrupli-
cate. The choice of pH for the assays was necessarily
arbitrary because the precise natural pH of mature cell
walls during ligni®cation or other phenolic cross-link-
ing is unknown. A value of 6.0 was selected bearing in
mind that the mature cell wall is not likely to be as
acidic as the rapidly growing wall (Schopfer, 1993).
3. Experimental
3.1. Materials
Methyl benzoate, ferulic and p-coumaric acids were
from Aldrich, Poole, Dorset. Sinapyl alcohol was from
Dr W.R. Russell (Rowett Research Institute,
Aberdeen). Coniferaldehyde, horseradish peroxidases
(acidic types VII [RZ 3.0] and type VIII [RZ 3.0];
basic type IX [RZ 3.2]), bovine liver catalase, and
crude laccase from the fungus Pyricularia oryzae were
purchased from Sigma, Poole, Dorset. A highly puri-
®ed prepn of horseradish peroxidase A2 (acidic; RZ
3.67) (éstergaard, Abelskov, Mattsson & Welinder,
1996) was from Professor K. Welinder (University of
Aalborg). Highly puri®ed laccases from the white rot
fungus Rigidoporus lignosus (Bonomo et al., 1998) and
the trees Acer pseudoplatanus and Populus eurameri-
cana (Ranocha et al., 1999) were from Dr D. Go€ner,
Dr R. Sterjiades and Professor A.-M. Boudet
(University of Toulouse). All laccase preparations were
stored in Eppendorf tubes previously treated with
0.1% polylysine soln (Sigma) to prevent adsorption of
the enzyme.
3.4.2. Laccase
The assay was as above but the enzyme solns added
typically contained 10 mg/ml, and 20 ml of a 5 mg/ml
soln of catalase was added instead of the H2O2.
3.5. Product pro®ling
HPLC was performed on a 25 Â 0.46 cm column of
Spherisorb 5ODS2 using a linear 30-min gradient (0±
100%) of H2O/BuOH/HOAc (98.3:1.2:0.5) to MeCN/
BuOH/HOAc (98.3:1.2:0.5) with detection by A280
.
3.2. Synthesis of methyl ferulate, methyl p-coumarate
and coniferyl and p-coumaryl alcohols
Acknowledgements
We thank Professor K. Welinder (University of
Aalborg), Dr D. Go€ner, Dr R. Sterjiades and
Professor A.M. Boudet (University of Toulouse) and
Dr W.R. Russell (Rowett Research Institute,
Aberdeen) for kindly providing enzymes and sub-
strates. We thank the Commission of the European
Communities for funding this work (AIR2 Contract
CT93 1661).
Methyl ferulate and methyl p-coumarate were prepd
as described (Wallace & Fry, 1995). Ethyl ferulate and
ethyl p-coumarate were prepd by the same method
using EtOH instead of MeOH. Ethyl ferulate and
ethyl p-coumarate (5 g) were dissolved in 150 ml dry
Et2O and stirred under a stream of N2 at 108C.
LiAlH4 (1.5 g) in 100 ml dry Et2O was then slowly
dripped into the soln, over 1 h, to reduce the ester to
the corresponding alcohol. The mixture was stirred for
18 h at 08C. H2O was slowly added to the mixture
until all the LiAlH4 was destroyed. The Et2O phase
was then extracted 3Â with satd aq. NaCl, dried with
Na2SO4 and evapd to dryness. The alcohols were
recrystallised from CHCl3.
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