Oxidation of lignin model compounds by organic and transition metal-based electron transfer mediators

Article abstract of DOI:10.1039/b202621j

We have studied the oxidation of lignin model compounds by organic and transition metal-based mediators using either an enzyme or an electrolysis cell as the mediator oxidizing agent. Electrolysis of inorganic mediator seems a promising technology for pulp delignification.

Full text of DOI:10.1039/b202621j

Oxidation of lignin model compounds by organic and transition
metal-based electron transfer mediators
ab
b
c
b
Dominic Rochefort, Robert Bourbonnais, Dónal Leech* and Michael G. Paice
a
Département de chimie, Université de Montréal, Montréal, Canada.
E-mail: dominic.rochefort@umontreal.ca
Pulp & Paper Research Institute of Canada, Pointe-Claire, Canada. E-mail: rbourbonnais@paprican.ca;
mpaice@paprican.ca
Department of Chemistry, National University of Ireland, Galway, Ireland.
E-mail: donal.leech@nuigalway.ie
b
c
Received (in Cambridge, UK) 20th March 2002, Accepted 17th April 2002
First published as an Advance Article on the web 26th April 2002
4
We have studied the oxidation of lignin model compounds
by organic and transition metal-based mediators using either
an enzyme or an electrolysis cell as the mediator oxidizing
agent. Electrolysis of inorganic mediator seems a promising
technology for pulp delignification.
by laccase. This permits evaluation of the effect of dioxygen on
oxidation of LMCs.
The reaction mixtures are composed of 2 mM of the LMC and
0.5 mM of mediator dissolved in citrate buffer (100 mM) at pH
4.5. For enzymatic treatments, 0.5 units mL of laccase is
added to the solution while an electrolysis cell (Bioanalytical
Systems Inc.) is used for electrochemical treatments. The
laccase activity is measured spectrophotometrically using
2
1
The use of a laccase† enzyme with a mediator as a chlorine-free
alternative method for pulp delignification (paper bleaching)
was proposed by us over a decade ago.1 Although research on
such systems has been underway since then, the mechanisms
leading to lignin oxidation by the laccase-mediator system are
still unclear. A proposed simplified mechanism is presented in
Scheme 1.
,2
2
ABTS as described earlier. All reactions are for 20 hours and
the reaction products are analysed by reversed-phase HPLC.
5
Initially, we compare the ability of four mediators to oxidize
the b-1 dimer LMC (Fig. 2). As expected, electrolytic oxidation
(E = 615 mV vs Ag/AgCl) of mediators possessing oxidation
potentials more positive than the applied potential gives similar
yields of oxidized LMC to the control, as they are not
catalytically oxidized. The control is the % of LMC oxidized
directly at the electrode. Potassium octacyanomolybdate(IV
)
(
MoCN) can mediate the LMC oxidation, when electrolytically
and enzymatically oxidized. ABTS gives particularly high
yields of oxidation, even though the radical cation formed at this
applied potential or in the presence of laccase is unreactive
towards the LMC. The ABTS· can undergo a disproportiona-
tion reaction (eqn. (1) producing the dication, which is more
reactive and oxidizes the LMC efficiently.6
Scheme 1 Proposed reaction pathways for mediated lignin and model
compound oxidation.
1
+
Several hypotheses of lignin oxidation mechanisms have
been proposed based on results from the oxidation of lignin
model compounds (LMC)‡ by radical-forming organic media-
tors such as 2,2A-azinobis(3-ethylbenzthiazoline-6-sulfonate)
+
2+
2 ABTS· Û ABTS + ABTS
(1)
HBT gives high LMC oxidation yields with laccase, but not
when electrolytically oxidized. HBT oxidation at carbon
electrodes is irreversible and occurs at more positive potential
than the applied potential. Laccase, however, can rapidly
oxidize HBT to the radical cation, which then oxidizes the
LMC. Increased LMC oxidation is obtained upon increasing the
electrolysis potential. In all cases, the mechanism involved is
(
ABTS), 1-hydroxybenzotriazole (HBT) and N-hydroxyaceta-
nilide (NHA). We recently reported on the use of transition
metal-based mediators for pulp delignification.3 These new
mediators are coordination complexes of a transition metal (Fe,
Mo or Ru) and ligands such as cyanide or 2,2A-bipyridine. These
compounds do not form radicals when oxidized by laccase or by
an electrode. The products resulting from the oxidation of lignin
and its model compounds with these mediators may then differ
from that of the radical-forming organic mediators. We have
studied the oxidation of three LMCs, 3,4-dimethoxybenzyl
alcohol (veratryl alcohol), 1-(3,4-dimethoxyphenyl)-2-(2-me-
thoxyphenoxy)propane-1,3-diol and 1-(3,4-dimethoxyphenyl)-
2
-phenylethanediol, the last two being compounds representing
b-O-4 and b-1 linkages in the lignin, respectively (see Fig. 1).
Bulk electrolysis is used to simulate oxidation of the mediator
Fig. 2 Percentage of b-1 dimer oxidized after a 20 hour treatment with
21
different mediators, using either laccase (0.5 U mL ) or electrolysis (E =
6
15 mV vs. Ag/AgCl) as the oxidizing agent. The error bars represent the
Fig. 1 Structure of the lignin model compounds used in this study.
standard deviation of triplicates.
1
182
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Ca–Cb cleavage (see Fig. 1) and the products identified as
Table 2 Amount in millimolar (percentage) of VA oxidized (initial
concentration of 2 mM) after a 20 hour treatment using mediators (0.5 mM)
veratraldehyde and benzaldehyde in stoichiometric ratio to the
0
with a high E A
amount of LMC oxidized. Removing O
7
1
2
during electrolysis at
50 mV has little effect on MoCN-mediated oxidation of the b-
dimer (86% anaerobic vs 77% aerobic), but a decrease in the
Enzymatic treatment
Laccase 0.5 U mL
Electrolysis
(E = 900 mV)
Mediator (E0A)a
amount of benzaldehyde formed is observed. This effect has
already been reported.7
No mediator
HBT (850)
FeDMBPY (689)
RuCN (684)
0.00 (0 %)
1.10 (55 %)
0.01 ( ~ 0%)
0.01( ~ 0%)
0.00 (0%)
0.99 (50%)
0.31 (16%)
1.98 (99%)
The b-O-4 dimer is often used as a lignin model compound
since this linkage is the most abundant (near 50%) in kraft lignin
chemical structure. The b-O-4 dimer is not easily oxidized,
b
(oxidation potential 1200 mV vs. Ag/AgCl), and only ABTS
a
Formal potential measured vs Ag/AgCl.
(oxidation potential).
b
HBT oxidation is irreversible
and HBT mediators are able to do so (Table 1). There is a
difference in b-O-4 dimer oxidation mechanism when laccase
and electrolytic oxidation of the mediator is used. With laccase
only one oxidation product (Ca-ketone) is detected, whilst
veratraldehyde, a cleavage product, is detected with electro-
chemical treatments. It is known that these oxidations occur via
a two-electron reaction.6 The reaction probably consists of two
different electron transfer events: a first structure-specific
to study delignification by transition metal-based mediators
compared to HBT (Table 2).
Neither of the two mediators, hexacyanoruthenate(II) (RuCN)
,8
and tris-(4,4A-dimethyl-2,2A-bipyridine)iron(II) (FeDMBPY)
can oxidize the VA when oxidized by laccase, most likely
because the overall reaction (Scheme 1) is limited by slow
1
-electron oxidation by a mediator that could be followed by a
second oxidation. The oxidation at the electrode is probably
non-specific toward an element of structure of the dimer since
several products are detected. Two of these products, the most
abundant, have been identified (Table 1) but several small
unidentified HPLC peaks were also observed. These products
could explain the difference existing between the concentration
of the dimer oxidized and of the identified products. The
mediated laccase oxidation is specific since only one oxidation
product is found, and in stoichiometric ratio to the model
compound. These issues are presently under further investiga-
tion in our laboratories.
13
oxidation of mediator by the enzyme. Nevertheless, when
electrolysis at 900 mV is used, RuCN oxidizes VA completely.
HBT can oxidize only 50% of VA, probably because of the
instability of the oxidized HBT, as it is known that the HBT
radical decays eventually to benzotriazole, which is unreactive
6
towards the model compounds. Indeed, HPLC analysis shows
a benzotriazole concentration corresponding to a 50% decrease
in HBT concentration. The results in Table 2 show therefore that
electrocatalytic oxidation of VA by the mediators occurs.
Oxidation of paper pulps using electrolysis and these inorganic
mediators is thus a promising route for chlorine-free delignifi-
cation.
Table 1 Comparison of mediated enzymatic (laccase 0.5 U mL21) and
electrolytic (E = 900 mV vs. Ag/AgCl) oxidation of the b-O-4 dimer (2
mM) showing amount (percentage) oxidized after 20 h. Amounts of major
oxidation products detected are also shown
Notes and references
†
Laccase is a blue copper oxidase that can oxidise substrates via the four
Oxidizing agent/
mediator (0.5 mM)
Dimer oxidized Ca-ketone
Veratraldehyde
(mM)
electron-reduction of dioxygen to water. Laccases are commonly found in
(mM)
(mM)
wood-rotting fungi.
Lignin is an irregular, non-soluble biopolymer that surrounds the cellulose
14,15
‡
Laccase/controla
Laccase/HBT
Laccase/ABTS
Electrolysis/control
Electrolysis/HBT
Electrolysis/ABTS
0.00 (0 %)
0.95 (48 %)
0.39 (19 %)
0.28 (14 %)
0.85 (42 %)
1.68 (84 %)
n.d.b
0.89
0.42
0.12
0.30
0.20
n.d.
n.d.
n.d.
0.07
0.16
0.65
fibres in wood. The insolubility of lignin hampers the study of oxidation
mechanisms using the intact biopolymer. Model compounds of lignin that
are small, soluble and represent some key chemical linkages in pulp lignin,
can thus be used to study lignin oxidation mechanisms.
1 R. Bourbonnais and M. G. Paice, FEBS Lett., 1990, 267, 99.
2 R. Bourbonnais, M. G. Paice, B. Freiermuth, E. Bodie and S. Borneman,
Appl. Environ. Microbiol., 1997, 63, 4627.
a
The control represents an assay without mediator. b Not detected.
3
R. Bourbonnais, D. Rochefort, M. Paice, S. Renaud and D. Leech,
TAPPI J., 2000, 83, 68.
According to the synthesis procedure,9 the b-O-4 dimer
exists in two diastereomeric forms, erythro and threo, which
4 D. Rochefort, R. Bourbonnais, D. Leech, S. Renaud and M. Paice, J.
Electrochem. Soc., 2002, 149, D15.
5 R. Bourbonnais and M. G. Paice, Appl. Microbiol. Biotechnol., 1987,
appear as two resolved peaks by HPLC with the erythro being
eluted first.10 Under normal conditions and for all mediators,
2
6, 164.
R. Bourbonnais, D. Leech and M. G. Paice, Biochim. Biophys. Acta,
998, 1379, 381.
both isomers are equally oxidized, as both peak areas decrease
evenly after the treatment. Under anaerobic conditions, the
oxidation of the dimer with HBT becomes stereospecific to the
threo form. This effect has not been observed with other
mediators. When HBT is oxidized in the presence of oxygen, it
rapidly forms a dioxide.11 It is therefore possible that the
monoxide, which is the only oxidized form of HBT found under
anaerobic conditions, is only reactive toward the threo form of
6
1
7
8
H. E. Schoemaker, Recl. Trav. Chim. Pays-Bas, 1990, 109, 255.
A. Majcherczyk, C. Johannes and A. Hüttermann, Appl. Microbiol.
Biotechnol., 1999, 51, 267.
9 F. Nakatsubo, in Methods in Enzymology, 1988, 161, 57.
10 S. Omori, M. Aoyama and A. Sakakibara, Holzforschung, 1998, 52,
391.
1
1 H. G. Aurich and W. Weiss, Chem. Ber., 1973, 106, 2408.
12 S. Descombes, K. Mazeau, D. Robert, F. R. Taravel and J. P. Utille, in
the b-O-4 dimer. Since the interphenyl chain in the threo form
is more exposed than in the erythro form,12 this suggests that the
Proc. 11th Int. Symp. Wood Pulp. Chem., France, 2001, III, 581.
reaction is initiated on this chain and not on the phenyl units,
which in both isomers are equally exposed to a reaction with the
mediator.
Veratryl alcohol is a non-phenolic LMC that can be
irreversibly oxidized at carbon electrodes to veratraldehyde at
approximately 1200 mV (vs Ag/AgCl). We have used this LMC
1
1
3 D. Rochefort, , R. Bourbonnais, , M. Paice and D. Leech, manuscript in
preparation.
4 E. I. Solomon, U. M. Sundaram and T. E. Machonkin, Chem. Rev., 1996,
9
6, 2577.
15 B. Reinhammar, in Copper Proteins and Copper Enzymes, ed. R.
Lontie, CRC Press, Boca Raton, 1984, vol. 3, p. 2.
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