Oxidation of phenols by laccase and laccase-mediator systemsSolubility and steric issues Francesca d’Acunzo, Carlo Galli and Bernardo Masci Dipartimento di Chimica and Centro CNR Meccani
Trang 1Oxidation 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` La Sapienza, 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 oxidation shuttles that the media-tors of laccase may have, but also suggest two promising routes towards an environmentally friendly process for kraft pulp bleaching: (a) the identification of mediators which, once oxidized by laccase, are able to target strategic functional groups present in lignin, and (b) the introduction
of those strategic functional groups in an appropriate pretreatment
Keywords: laccase; phenols; lignin degradation; HPI; TEMPO
Lignin is a three-dimensional, insoluble aromatic polymer
that constitutes 15–33% of biomass Its structure
encom-passes a number of different types of links between its
constituents, namely ether and C-C diaryl linkages [1]
White-rot fungi achieve the oxidative depolymerization of
lignin by secreting several enzymes, such as lignin peroxidase
[2], manganese peroxidase [3], and laccase (EC 1.10.3.2) [4]
In contrast with lignin peroxidase and manganese
peroxi-dase, laccase can only oxidize the phenolic constituents of
lignin, due to its lower oxidation potential On the other
hand, it is more readily available and easier to manipulate
than the other two enzymes, and its substrate specificity is
low, as long as a good match of oxidation potentials is
provided [5–8] In addition, the use of appropriate low
molecular-mass compounds (viz., mediators), in
combina-tion with laccase, makes this enzyme competent for the
oxidation of non-natural nonphenolic substrates [9–12] In
fact, the oxidized mediator (Fig 1) can rely on an oxidation
mechanism that is not available to the enzyme [13]
Laccase can therefore be turned into a much more
versatile enzyme, and this opens up various possible
applications, as in the textile dye bleaching [14], or for environmentally respectful kraft pulp delignification [10,15],
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 electron shuttle 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
natural 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` La Sapienza,
00185 Roma, Italy Fax: + 39 06 490421,
E-mail: carlo.galli@uniroma1.it
Abbreviations: HP I, N-hydroxyphthalimide; TEMPO,
2,2,6,6-tetramethylpiperidin-1-yloxy; ABTS,
2,2¢-azinobis-(3-ethylbenzothi-azoline-6-sulfonate).
(Received 18 June 2002, revised 9 September 2002,
accepted 12 September 2002)
Trang 2suitable purification allowed pure monodisperse oligomers
to be available; (c) solubility in water decreases as size
increases; and (d) o-o-p-substitution should inhibit C-C
diaryl bond formation, a well-known reaction pathway of
phenoxy radicals [1,12]
We chose N-hydroxyphtalimide (HPI) and
2,2,6,6-tetramethylpiperidin-1-yloxy free radical (TEMPO) as
mediators, since they greatly differ both in their
mechan-ism of action and in their specificity In fact, HPI is
representative of a mediator that, after having been oxidized
by laccase, should only induce the generation of phenoxy
radical(s) from the substrate (Fig 3A) [18,20,22–24] This
radical role of the oxidized HPI would be comparable to the
natural oxidation role of the enzyme, with the only difference of presenting fewer stringent steric and solubility requirements The phenoxy radical of the product, in turn, should evolve towards end-products with little or no further intervention from HPI
On the other hand, TEMPO is representative of a mediator that, by selectively interacting with specific func-tional groups [25] (i.e alcohols) (Fig 3B), not only acts as a shuttle for the oxidizing power of laccase towards insoluble
or bulky substrates, but also induces the formation of different products than the physiological ones This aspect 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
M A T E R I A L S A N D M E T H O D S
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)1determined spectrophotometrically by the standard reaction with ABTS [28] Laccase having an absorption ratio A280/A610of 20–30 was considered sufficiently pure [5]
Materials HPI and TEMPO were used as received from Aldrich The solvents were from Aldrich, Merck or Carlo Erba Synthesis of substrates
Compounds 2–4 were prepared according to a literature procedure [29] The latter procedure was also extended to the stepwise synthesis of compound 5, the pentaphenol condensation product obtained from compound 3 and 4-tert-butylphenol being bis-hydroxymethylated The purity
of these compounds (> 96%) was checked by1H NMR and HPLC
Spectrophotometric determination of laccase activity The activity of laccase was determined by following the rate
of oxidation of 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) to ABTS radical cation (ABTS•+), by plotting the absorbance at 414 nm against time [28] The extinction coefficient of ABTS•+ at 414 nm is 3.15· 10)4M )1Æcm)1 ABTS (Aldrich) was recrystallized
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.
Trang 3from ethanol prior to use A stock solution was prepared by
dissolving 3 mg of ABTS in 10 mL 0.1Mcitrate 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-mMstock solution
of each substrate was prepared in dioxane; aliquots (5.0 lL)
of the stock solution were added to 2.5 mL of 1 : 1 (v/v) 0.1
Mcitrate buffer pH 5.0/dioxane mixed solvent in a 10-mm
quartz cuvette Spectra were recorded in the 220–320 nm
range, and the absorbance at 280 nm was plotted against
concentration
Oxidations catalysed by laccase and laccase/mediator
systems
In a typical experiment, 60 lmol of substrates 1–5 were
weighed in a 2-mL screw-cap vial equipped with a stirring
bar In the experiments with mediators, 20 lmol HPI or
TEMPO were also added In the experiments with aqueous
solvent only, 0.3 mL of 0.1Mcitrate buffer, pH 5.0, were
added at this point, followed by 9–10 U of purified laccase
In the experiments with the mixed solvent, 0.15 mL of
dioxane were added first, followed by an equal amount of
citrate buffer and 9–10 U laccase The reaction mixture was
allowed to react at room temperature for 24 h The vials
were left uncapped and vigorous stirring was maintained in
order to ensure oxygen saturation
HPLC determination of substrate conversion
Substrate consumption after a 24-h reaction time was
determined using a Hewlett-Packard 1050 HPLC system
(pump, detector, and solvent delivery system) equipped with
a Supelcosil LC-18-DB 25 cm· 4.6 mm column and a HP
3395B integrator The analyses were carried out with
gradients of water/methanol/isopropanol mixtures,
contain-ing 0.03% trifluoroacetic acid, at 0.5–1 mLÆmin)1flow 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:1H-NMR
Samples were dissolved in dimethylsulfoxide-d6 (Merck) and spectra were acquired using a Varian 300 MHz spectrometer with a Mercury console
R E S U L T S A N D D I S C U S S I O N
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 used in the laccase-catalysed reaction We checked that the absorbance at 280 nm, corresponding to the maximum absorbance of the substrates, increases linearly with sub-strate concentration without scatter from precipitation Furthermore, we checked that the absorbance falls to zero outside the peak, i.e that no wavelength-independent turbidity arises from precipitation We thus verified that solutions of substrates 2–5 as concentrated as 85 lMcan be prepared However, the solution containing substrate 5 became visibly turbid after 24 h We therefore concluded that the heaviest of our substrates can yield over-saturated solutions in the buffer/dioxane mixed solvent, at the concentrations used for the oxidation reaction
Substrate consumption Table 1 summarizes the results of our laccase oxidations of substrates 1–5, and the effect of the cosolvent and of the mediators on the amount of substrate metabolized by the enzyme
(a) Laccase and laccase/HPI In general, the use of the buffer/dioxane mixed solvent enhances substrate conversion both with and without mediator The monomeric substrate
Table 1 Percent of substrate metabolized by laccase or laccase/mediator systems with and without an organic cosolvent Substrate recovery was determined by HPLC.
Trang 41, 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
dissolved in solution, thus showing no advantage over
laccase alone
Product identification
(a) Laccase and laccase/HPI 1H-NMR (Fig 4) and
LC-MS (Table 2) spectra indicate that with substrates 1 and
2 the same products are formed both in the absence and in
the presence of HPI
The general reaction schemes that rationalize the prod-ucts observed are sketched in Fig 5 Figure 5A shows a dimerization process with loss of formaldehyde, while the ring-opening products indicated in Fig 5B derive from the attack of dioxygen on the phenoxy radical [12] The spectra
in Fig 4 refer to oxidations carried out in the mixed
Fig 4 Expansion of1H-NMR spectra run in dimethylsulfoxide-d 6 of
the product mixtures from the laccase-catalysed oxidation of substrates 1
and 2 Peaks in the aromatic region (6.6–7.8 p.p.m.) are assigned to
arylether products (Fig 5A) Peaks in the 6.0–6.6 p.p.m region are
attributed to vinylic protons of unsaturated carboxylic acids (Fig 5B).
A signal from a mobile proton is also present at 11.5 p.p.m., which is
assigned to the carboxylic moieties.
Table 2 LC-MS data for the main product detected in the reaction mixture from the laccase-catalysed oxidation of substrates 1 and 2.
a M stands for molecular ion; A, B and C are the fragments rep-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).
Trang 5solvent, 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 D2O, 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 1H-NMR
spectra also in this case (Fig 4) There is no evidence of
oxidation of the internal methylenes In conclusion, the1
H-NMR spectra are compatible with product formation
according to the reactions sketched in Fig 5, which we
expected on the basis of literature data [12], and for which
LC-MS (Table 2) provides evidence
(b) Laccase/TEMPO TEMPO is known to selectively
oxidize alcohols to aldehydes [17,25–27] Consistent with
this evidence, and in contrast with the outcome of the
laccase and laccase/HPI reactions, the only product
observed (by1H-NMR), when substrate 4 is reacted with
the laccase/TEMPO system in the mixed solvent, results
from the oxidation of the hydroxymethyl groups to
aldehydes It is worth mentioning that substrate 4 does
not react with laccase alone in the mixed solvent, whereas
TEMPO does not react with the substrate in the absence of
laccase Therefore, this is a simple setting in which the
products observed can unambiguously be ascribed to the
mediating action of TEMPO on the enzyme On the other
hand, when the monomeric substrate 1 is reacted with the
laccase/TEMPO system,1H-NMR product analysis shows
a more complex situation Specifically, no phenolic coupling
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]
C O N C L U S I O N S
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
of a mediator such as HP I, which forms the same 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 cosolvent Aldehydes are exclusively obtained with those substrates that are only oxidized in the presence of TEMPO (4 and 5), while a more complex product mixture results with substrate 1, which can be oxidized not only by TEMPO but also by laccase directly We conclude that the laccase/ TEMPO system, possibly in a mixed solvent, may prove useful for the oxidation of alcohols of limited solubility, that are not directly oxidized by laccase These results therefore provide the first experimental support to the idea that a mediator of laccase activity may act as an oxidation shuttle capable to overcome solubility and/or restricted-access problems of the substrate In general, a possible strategy
to extend the use of laccase to the oxidation of substrates that cannot react with the enzyme alone is to select mediators that can specifically interact with target func-tional groups, through reaction pathways that are different from those directly accessible to laccase A foreseeable strategy for any laccase-catalysed wood pulp bleaching is to investigate mediators that can specifically interact with functional groups present on lignin Alternatively, specific functional groups that are susceptible to oxidation from this particular mediator might be introduced on lignin in a preoxidation stage
A C K N O W L E D G E M E N T S
We thank the EU for financial support (grant QLK5-CT-1999–01277)
to the OXYDELIGN project.
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