Oxidation of phenols by laccase and laccase-mediator systems
Solubility and steric issues
Francesca d’Acunzo, Carlo Galli and Bernardo Masci
Dipartimento di Chimica and Centro CNR Meccanismi di Reazione, Universita
`
ÔLa SapienzaÕ, 00185 Roma, Italy
To investigate how solubility and steric issues affect the
laccase-catalysed oxidation of phenols, a series of oligomeric
polyphenol compounds, having increasing size and
decreasing solubility in water, was incubated with laccase.
The extent of substrate conversion, and the nature of the
products formed in buffered aqueous solutions, were com-
pared to those obtained in the presence of an organic
cosolvent, and also in the presence of two mediating species,
i.e. N-hydroxyphthalimide (HPI) and 2,2,6,6-tetramethyl-
piperidin-1-yloxy (TEMPO). This approach showed not
only an obvious role of solubility, but also a significant role
of the dimension of the substrate upon the enzymatic reac-
tivity. In fact, reactivity decreases as substrate size increases
even when solubility is enhanced by a cosolvent. This effect
may be ascribed to limited accessibility of encumbered sub-
strates to the enzyme active site, and can be compensated
through the use of the appropriate mediator. While TEMPO
was highly efficient at enhancing the reactivity of large, less
soluble substrates, HPI proved less effective. In addition,
whereas the laccase/HPI system afforded the same products
as laccase alone, the use of TEMPO provided a different
product with high specificity. These results offer the first
evidence of the role of Ôoxidation shuttlesÕ that the media-
tors of laccase may have, but also suggest two promising
routes towards an environmentally friendly process for

or also in selective organic transformations [16–19]. The
study presented here is part of our efforts to elucidate the
mechanisms of action of the laccase/mediator systems [20] in
the oxidation of lignin model compounds, as well as non-
lignin-related structures (Fig. 1).
A conceivable role of the mediator could be that of a sort
of Ôelectron shuttleÕ between the enzyme and the substrate
[21]. Once the mediator is oxidized by the enzyme, it diffuses
away from the enzymatic pocket and in turn oxidizes
substrates that, due to their size, could not directly enter the
enzymatic pocket. Within this framework, we wished to
investigate the influence of substrate size and solubility on
the effectiveness of laccase oxidation, and also the effect of
mediators endowed with possibly different mechanisms of
action. To this aim, we needed to start from a simple
phenolic structure, which laccase could recognize as a
ÔnaturalÕ substrate, and modify it into bigger and more
insoluble derivatives. The oligomeric series shown in Fig. 2
served our purposes for the following reasons: (a) each
repeat unit is a phenol, and therefore subject to oxidation
by laccase, at least in terms of redox potential; (b) the
number of repeat units in each oligomer, and therefore its
size, is exactly determined, because directed synthesis and
Fig. 1. Catalytic cycle of a laccase-mediator oxidation system.
Correspondence to C. Galli, Dipartimento di Chimica and Centro
CNR Meccanismi di Reazione, Universita
`
ÔLa SapienzaÕ,
00185 Roma, Italy. Fax: + 39 06 490421,
E-mail:

may prove useful for synthetic purposes, as well as in lignin
degradation. The mechanism reported in Fig. 3B matches
the well-established one reported for the oxidation of
alcohols by catalytic amounts of TEMPO with stoichio-
metric co-oxidants [25–27]. In all these cases the oxoam-
monium form of TEMPO is involved. In our particular
case, laccase would be the catalytic oxidant of TEMPO [17].
Following a nucleophilic attack of the lone-pair of the
alcohol onto the TEMPO-oxoammonium ion, the interme-
diate adduct is deprotonated at the a-C-H benzylic bond by
the base-form of the buffer B [17,25–27].
In order to decouple the effect of increasing substrate-size
and decreasing solubility, the oxidations were carried out in
1 : 1 aqueous buffer/dioxane, and the results compared with
those obtained in 100% aqueous buffer.
MATERIALS AND METHODS
Laccase
Laccase from Poliporus pinsitus was kindly donated by
Novo Nordisk Biotech and purified by ion-exchange
chromatography on Q-Sepharose by elution with phosphate
buffer [5,20], and an activity of 10 000 UÆmL
)1
determined
spectrophotometrically by the standard reaction with ABTS
[28]. Laccase having an absorption ratio A
280
/A
610
of 20–30
was considered sufficiently pure [5].

Fig. 3. Mechanisms of the O
2
-laccase-HPI (A) and O
2-
laccase-
TEMPO (B) oxidation of substrates 1–5 (Fig. 2).
Fig. 2. Oligomeric compounds 1–5.
Ó FEBS 2002 Oxidation of phenols (Eur. J. Biochem. 269) 5331
from ethanol prior to use. A stock solution was prepared by
dissolving 3 mg of ABTS in 10 mL 0.1
M
citrate buffer at
pH 5.0; 200 lL of the stock solution were added to 3 mL
citrate buffer in a quartz cuvette (10 mm pathlength); 1 lL
of laccase solution, approximately 10–20 UÆmL
)1
,was
then added, and the initial rate of ABTS
•+
formation
was determined. The 10–20 UÆmL
)1
laccase activity was
achieved by diluting the purified laccase solution in citrate
buffer.
Evaluation of substrates solubility
The solubility of substrates 2, 3, 5 was evaluated by UV-Vis
spectrometry. A Hewlett Packard 8453 diode-array single
beam spectrophotometer was used. A 2.0-m
M

gradients of water/methanol/isopropanol mixtures, contain-
ing 0.03% trifluoroacetic acid, at 0.5–1 mLÆmin
)1
flow rate.
Quantitation of unreacted substrate was achieved by using
2-bromonaphtalene (Aldrich) as the internal standard. The
standard was added to the reaction crude, which was then
diluted in the mobile phase and filtered through 0.2 lm
Teflon syringe filters (Superchrom Varisep) prior to
analysis.
Product analysis: liquid mass spectrometry (LC-MS)
The analysis was carried out using a triple quadrupole
Perkin Elmer Sciex API 365 spectrometer with a turbo–ion
spray interface. Samples were diluted in HPLC-grade
methanol and filtered through 0.2 lm Teflon syringe filters
prior to injecting. The samples were directly injected into the
ion spray chamber without chromatographic separation.
Product analysis:
1
H-NMR
Samples were dissolved in dimethylsulfoxide-d
6
(Merck)
and spectra were acquired using a Varian 300 MHz
spectrometer with a Mercury console.
RESULTS AND DISCUSSION
Solubility of substrates
This was assessed by a UV-Vis spectrophotometric experi-
ment, aimed at verifying that the substrates were soluble in
the buffer/dioxane mixed solvent up to the concentration

4 00 0 0 687
5 00 0 0 040
5332 F. d’Acunzo et al.(Eur. J. Biochem. 269) Ó FEBS 2002
1, which is the smallest and the most soluble, is metabolized
quantitatively in buffered water. This is reasonable, in view
of its phenolic nature. With phenols 2 and 3, however, the
extent of substrate oxidation is much lower; the use of the
cosolvent makes the consumption of 2 quantitative, whereas
that of 3 remains around 55%, and no conversion is
observed for substrates 4 and 5. Moving to the experiments
with mediators, we observe that HPI does not provide any
significant improvement in the extent of substrate oxidation
with respect to laccase alone, nor promote the oxidation of
the bulkiest substrates (4 and 5). On the other hand,
literature evidence supporting the ability of HPI to act as a
mediator in laccase-catalysed oxidations of different sub-
strates is available [18,22,23].
(b) Laccase/TEMPO. The effect of TEMPO is much
more remarkable than that of HPI (Table 1). In fact, when
TEMPO is used in the mixed solvent, substrate 3 is
quantitatively consumed, and substrates 4 and 5 are also
significantly oxidized. A small amount of 4 is oxidized by
the laccase/TEMPO system even in the simple buffered
solution, namely, in the absence of cosolvent. This proves
that the appropriate choice of a mediator is of utmost
importance with substrates of limited solubility. In partic-
ular, the formation of the TEMPO-substrate adduct
(Fig. 3B) may account for the enhanced reactivity of poorly
soluble substrates. Conversely, HPI, which does not form
adducts, can only react with the amount of substrate

resented in the Product column.
Fig. 5. Oxidation of substrates 1–5 by O
2
-laccase and O
2
-laccase-HPI.
Phenol coupling with loss of formaldehyde (A) and ring-opening (B).
Ó FEBS 2002 Oxidation of phenols (Eur. J. Biochem. 269) 5333
solvent, namely, in conditions in which substrate con-
sumption is nearly quantitative and dimerization products
are likely to undergo further reaction, both of type (A) and
(B) (in Fig. 5). Complex reaction mixtures are expected
and the spectra can be further complicated by the presence
of slowly interconverting conformers, so that we do not
attempt to assign the observed peaks to specific structures.
In the product mixtures from both substrates 1 and 2,
peaks are found in the aromatic (6.6–7.8 p.p.m.) and in the
vinylic (6–6.6 p.p.m.) regions. A signal from an acidic
proton at 11.5 p.p.m. (not shown), which is suppressed by
the addition of D
2
O, is also detected. The peaks in the
aromatic region are attributed to products from the
dimerization (and the like) reaction shown in Fig. 5A,
while the vinylic signals and the peak at 11.5 p.p.m. are
assigned to the unsaturated carboxylic acids resulting from
the ring-opening reactions, as reported in Fig. 5B. No
specific ring-opening product from substrate 2 could be
detected by LC-MS, even though the formation of small
amounts of olefinic products is indicated by

products are observed, the aldehydic signals are prominent,
and several signals are present in the aromatic region. It is
quite likely that TEMPO not only acts as a mediator (which
accounts for aldehyde formation) but, in view of its known
role as an inhibitor of free-radical chains, it also traps the
phenoxy radicals formed by the direct interaction of
substrate 1 with laccase [26,30].
CONCLUSIONS
The oxidation of an oligomeric series of phenols with
laccase alone, or in combination with two mediators (HPI
and TEMPO) was investigated in buffered water solution or
in a 1 : 1 buffer/dioxane mixed solvent. HPI, a mediator
that promotes the formation of the same phenoxy radical
intermediate as laccase, yields the same products as the
enzyme alone, namely, ring-opening and phenol coupling
products, with a comparable extent of substrate consump-
tion. On the other hand, the laccase/TEMPO system
performs the selective oxidation of the hydroxymethyl
groups of the substrate to aldehydes. Both with laccase
alone and with the laccase/HPI system, a limited enhance-
ment of the extent of oxidation was obtained by the use of
the cosolvent with the smaller substrates 1–3, but no
oxidation was obtained with the bulkiest substrates 4 and 5.
We therefore conclude that no benefit derives from the use
ofamediatorsuchasHPI,whichformsthesame
intermediate as the enzyme, whenever size and solubility
issues need be addressed. On the other hand, the laccase/
TEMPO system not only affords the oxidation of the
bulkiest and least soluble substrates, but it also benefits from
the enhancement of substrate solubility achieved with the

hymenomycete Phanerochaete chrysosporium burds. Science 221,
661–663.
3. Kuwahara,M.,Glenn,J.K.,Morgan,A.&Gold,M.H.(1984)
Separation and characterization of two extracellular H
2
O
2
-
dependent oxidases from ligninolytic cultures of Phanerochaete
chrysosporium. FEBS Lett. 169, 247–250.
4. Messerschmidt, A. (1997) Multi-Copper Oxidases, World Scienti-
fic, Singapore.
5. Xu, F. (1996) Oxidation of phenols, anilines, and benzenethiols by
fungal laccases: correlation between activity and redox potentials
as well as halide inhibition. Biochemistry 35, 7608–7614.
6. Itoh, K., Fujita, M., Kumano, K., Suyama, K. & Yamamoto, H.
(2000) Phenolic acids affect transformations of chlorophenols by a
Coriolus Versicolor laccase. Soil Biol. Biochem. 32, 85–91.
7. Guille
´
n, F., Mun
˜
oz, C., Go
´
mez-Toribio, V., Martı
´
nez, A.T. &
Martı
´
nez, M.J. (2000) Oxygen activation during oxidation of

tional Patent WO 0027204, A1. European Patent Office, Munich,
Germany.
15. Call, H P. & Mu
¨
cke, I. (1997) History, overview and applications
of mediated lignolytic systems, especially laccase-mediator-sys-
tems (Lignozym-process). J. Biotechnol. 53, 163–202.
16. Potthast, A., Rosenau, T., Chen, C.L. & Gratzl, J.S. (1995)
Selective enzymatic oxidation of aromatic methyl groups to
aldehydes. J. Org. Chem. 60, 4320–4321.
17. Fabbrini, M., Galli, C., Gentili, P. & Macchitella, D. (2001)
An oxidation of alcohols by oxygen with the enzyme
laccase, and mediation by TEMPO. Tetrahedron Lett. 42, 7551–
7553.
18. Fritz-Langhals, E. & Kunath, B. (1998) Synthesis of aromatic
aldehydes by laccase-mediator assisted oxidation. Tetrahedron
Lett. 39, 5955–5956.
19. Johannes, C. & Majcherczyk, A. (2000) Natural mediators in the
oxidation of polycyclic aromatic hydrocarbons by laccase med-
iator systems. Appl. Environ. Microb. 66, 524–528.
20. Fabbrini, M., Galli, C. & Gentili, P. (2002) Comparing the effi-
ciency of some mediators of laccase. J. Mol. Catal. B: Enzym. 16,
231–240.
21. Banci, L., Ciofi-Baffoni, S. & Tien, M. (1999) Lignin and Mn
peroxidase-catalysed oxidation of phenolic lignin oligomers. Bio-
chemistry 38, 3205–3210.
22. Xu,F.,Kulys,J.J.,Duke,K.,Li,K.,Krikstopaitis,K.,Deussen,
H J.W., Abbate, E., Galinyte, V. & Schneider, P. (2000) Redox
chemistry in laccase-catalysed oxidation of N-hydroxy com-
pounds. Appl. Environ. Microb. 66, 2052–2056.