On the reactivity of the Melanocarpus albomyces laccase and formation of coniferyl alcohol dehydropolymer (DHP) in the presence of ionic liquid 1-allyl-3-methylimidazolium chloride

Article abstract of DOI:10.1016/j.molcatb.2012.09.005

Some ionic liquids are able to dissolve wood, including lignin and lignocellulose, and thus they provide an efficient reaction media for modification of globally abundant wood-based polymers. Lignin can be modified with laccases (EC 1.10.3.2), multicopper

Full text of DOI:10.1016/j.molcatb.2012.09.005

Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 169–177
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Journal of Molecular Catalysis B: Enzymatic
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On the reactivity of the Melanocarpus albomyces laccase and formation of
coniferyl alcohol dehydropolymer (DHP) in the presence of ionic liquid
1-allyl-3-methylimidazolium chloride
Maarit Lahtinena,∗, Liisa Viikarib, Pirkko Karhunena, Janne Asikkalaa, Kristiina Kruusc,
Ilkka Kilpeläinena
a Department of Chemistry, University of Helsinki, P. O. Box 55, FIN-00014 HU, Finland
b Department of Applied Chemistry and Microbiology, University of Helsinki, P. O. Box 27, FIN-00014 HU, Finland
c VTT, Technical Research Centre of Finland, P. O. Box 1000, FIN-02044 VTT, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 January 2012
Received in revised form 3 September 2012
Accepted 12 September 2012
Available online 19 September 2012
Some ionic liquids are able to dissolve wood, including lignin and lignocellulose, and thus they provide
an efficient reaction media for modification of globally abundant wood-based polymers. Lignin can be
modified with laccases (EC 1.10.3.2), multicopper oxidases, which selectively catalyze the oxidation of
phenolic hydroxyl to the phenoxy radical in lignin by using oxygen as the co-substrate and an electron
acceptor. Many enzymes, including laccases, retain their catalytic activity in the presence of ionic liq-
uids. However, the enzyme activity is usually decreased in the presence of ionic liquids, and the most
deactivating ionic liquids have been observed to be those dissolving wood most efficiently. In the present
study the activity, pH optimum and catalyzed oxidation of coniferyl alcohol by the laccase from the
ascomycete Melanocarpus albomyces was investigated in the ionic liquid 1-allyl-3-methyl-imidazolium
chloride ([Amim]Cl), known to dissolve wood and expected to affect the laccase activity. Indeed, with an
increasing concentration of [Amim]Cl, the activity of M. albomyces laccase decreased, and the pH range
of the enzyme activity was narrowed. The pH optimum, using 2,6-dimethoxyphenol as the substrate,
was shifted from 6.5 to 6.0 when the amount of [Amim]Cl was increased to 60% (m-%). It was also found
that the inhibition of laccase with NaN3 was not as severe in the ionic liquid as in water. The insoluble
fraction of the dehydropolymer (DHP) formed in the presence of [Amim]Cl had clearly higher molecular
weight compared to the one formed in water. DHPs formed in the absence and presence of [Amim]Cl
both contained ˇ-5, ˇ–ˇ, ˇ-O-4, ˛-C O/ˇ-O-4 and ˛-O-4/ˇ-O-4 structures. However, in the presence of
[Amim]Cl, less ˇ-O-4, slightly less ˇ-5 and more ˇ–ˇ structures were formed.
Keywords:
Ionic liquid
Enzyme
Lignin
Dehydropolymer (DHP)
Two-dimensional nuclear magnetic
resonance (2D NMR)
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
lignocellulose, and their main constituents (cellulose and lignin) are
highly insoluble in common solvents because of their large molec-
Lignin is a heterogenous polymer consisting of phenylpropane
units, which are coupled to form an irregular polymer network.
Wood contains approximately 30% of lignin, which is, after cellu-
lose, the second most abundant biopolymer [1,2]. Today, lignin is
mainly used for production of energy in pulp and paper processes
by burning it. Lignin, extracted in the production of pulp (cellu-
lose), is also used as dispersing agents, in concrete, textile dyes,
pesticides, batteries and ceramic products or as binding agents in
animal foods and briquettes.
ular size, the hydrophobic nature of lignin and the crystallinity of
cellulose. However, recent studies show that wood and other ligno-
cellulosic material are soluble in some ionic liquids (ILs) and thus
they provide potential media for modification of these materials
[3–7].
Swatloski et al. reported first the dissolution of cellulose
to an imidazolium-based IL, 1-butyl-3-methylimidazolium chlo-
ride ([Bmim]Cl) [8]. More recently, 1-allyl-3-methylimidazolium
chloride ([Amim]Cl) and 1-ethyl-3-methylimidazolium acetate
([Emim]Ac) have also turned out to be able to dissolve cellulose
[7,9,10]. The two most powerful solvents for lignin are 1-butyl-3-
methylimidazolium trifluoromethanesulfonate [Bmim]CF3SO3 and
1,3-dimethylimidazolium methylsulfate [Mmim]MeSO4, which on
the other hand are not able to dissolve wood flour [10,11]. Other
good ILs for dissolving lignin are [Amim]Cl and [Emim]Ac, while
[Bmim]Cl is less efficient [5,11]. According to comparative analysis
For any chemical modification, it is advantageous for the
reagents to be soluble in the reaction media, because this increases
the probability of molecules to react with each other. Wood and
∗
Corresponding author. Tel.: +358 9 191 50350; fax: +358 9 191 50366.
E-mail address: maarit.lahtinen@helsinki.fi (M. Lahtinen).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.09.005

170
M. Lahtinen et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 169–177
[12], [Emim]Ac is the most efficient IL for dissolving cellulose and
[Amim]Cl for dissolving wood chips.
The aim of the present study was to investigate reactivity of
the laccase from the ascomycete Melanocarpus albomyces in the
presence of the ionic liquid, 1-allyl-3-methylimidazolium chlo-
ride ([Amim]Cl). This IL was expected to affect the laccase activity,
because it dissolves wood by breaking the hydrogen-bonding net-
work of cellulose. The M. albomyces laccase was chosen for this
study, as it is thermostable (optimum temperature 60–70 ◦C),
which is expected to improve the general stability of the enzyme.
This enzyme also has rather a broad pH optimum (e.g. pH 5.0–7.5,
determined with guaiacol, and pH 6–7 with syringaldazine) [34].
The M. albomyces laccase has also been shown to remain active
in the presence of various organic solvents [35]. Activity of the
enzyme was determined in different pH-values and different
[Amim]Cl concentrations by following the product formation of
2,6-dimethoxyphenol. Coniferyl alcohol was oxidized to dehy-
dropolymers (DHPs), and possible structural differences caused
by the IL were investigated with GPC and NMR. Formation of the
oligomeric coniferyl alcohol oxidation products were followed with
HPLC to detect possible changes compared to aqueous solvent.
The dissolution mechanism of wood and its constituents, cel-
lulose and lignin, by ILs is not yet completely understood. Both
the cation and the anion, and apparently also their combination,
play a role in the dissolution [10,13]. Dissolution of cellulose is pro-
posed to be based on the interruption of inter- and intramolecular
hydrogen-bond-network, responsible for crystalline nature of the
polymer. Thus, the cations of ILs that are able to dissolve cellulose
are mainly imidazolium-based and relatively small (e.g. [Amim]+,
[Emim]+ and [Bmim]+, and the anions are small hydrogen-bond
acceptors (e.g. Cl−, AcO−). However, even this generalization seems
to be somewhat inadequate, as a recent article shows that also
tetramethylguanidine-based ILs are able to dissolve cellulose effi-
ciently [14]. Generalizations are also difficult in the case of lignin,
because all of the existing results are not comparable [10]. It has,
however, been shown that the nature of the anion is decisive and
−
large, non-coordinating anions, such as PF4 and BF6−, are not
favorable. The allyl chain in [Amim]Cl could also interact with
lignin, since [Amim]Cl is a better solvent for lignin than [Bmim]Cl
[10].
2. Experimental
Lignin can be selectively modified with oxidative enzymes, such
as laccases [15,16]. Laccases (EC 1.10.3.2) are multicopper oxidases
that are found from plants, fungi, insects and bacteria [17,18]. In
nature, plant laccases take part in lignin biosynthesis and those
of the white-rot fungi in lignin biodegradation. Laccases use oxy-
gen as their co-substrate, which is reduced to water during the
catalytic cycle [19]. Laccase oxidizes the phenolic group of lignin
by removing one electron to form a phenoxy radical, which then
reacts further [20]. Based on electron-spin resonance spectromet-
ric studies of laccase-oxidized beech wood fibers, it seems that
lignin-derived phenoxy radicals in the solution can introduce radi-
cals further to the matrix lignin in wood fibers [21]. Studies with
syringylic ˇ-1 and ˇ-O-4 lignin model compounds have revealed
that in laccase-catalyzed oxidation, several degradation products
are formed as a result of side chain cleavages from various positions,
as well as oxidation of the benzylic hydroxyl [22]. Oxidation of ben-
zylic hydroxyl can also take place in laccase-catalyzed oxidation
of guaiacylic compounds, which however prefer more oxidative
coupling to form biphenylic- and dibenzodioxepin-type products
[23–25]. It has been suggested that the differences of product dis-
tributions observed in laccase-catalyzed oxidation of lignin model
compounds could be affected by the enzyme dosage used and the
pH of the reaction media [24,25].
2.1. Materials
All commercial reagents and solvents were used as received
unless otherwise mentioned.
The M. albomyces laccase was overproduced in T. reesei and puri-
fied as described earlier [36].
[Amim]Cl was synthesized according to Zhang et al. [9] with
slight modification to the procedure; both allyl chloride (Aldrich)
and 3-methylimidazole (Aldrich) were distilled prior to use. To
remove traces of color, [Amim]Cl was further purified by dissolving
the crude [Amim]Cl in water and refluxing with activated charcoal
for 18 h. The solution was filtered through silica plug and water
was removed by distillation and drying for 2 days in vacuum to
yield [Amim]Cl as a pale yellow crystalline solid, with a melting
point of 52 ◦C.
From the used substrates 2,6-dimethoxyphenol (Aldrich) was
commercially available and coniferyl alcohol was synthesized
according to a well-known method [37].
2.2. Determination of laccase activity
The
laccase
activity
was
determined
using
2,6-
dimethoxyphenol (1 mmol kg−1) as substrate in different buffers
at room temperature. The buffers used were 50 mmol kg−1 sodium
citrate, pH 5.0–7.0; McIlvaine buffer (0.2 mol kg−1 disodium
hydrogen phosphate, 0.1 mol kg−1 citric acid), pH 3.0–8.0 and
25 mmol kg−1 sodium succinate, pH 4.5. The measured solution
contained also 0.5 ␮l g−1 ethanol from the substrate stock solution.
Oxidation of the substrate was monitored for 5 min by
absorbance measurements, and the activity was determined from
the slope of the linear curve. The determined absorbance maxima of
the 2,6-dimethoxyphenol oxidation product at different [Amim]Cl
concentrations were ([Amim]Cl m-% – ꢀmax): 0% – 469 nm, 20%
– 474 nm, 40% – 479 nm, 60% – 480 nm, 80% – 475 nm. The fil-
tered oxidation product was not soluble in water and thus, the
same molar absorption coefficient (ε = 49 600 kg mol−1 cm−1) was
used for activity calculations in all concentrations [38], different
wavelengths.
The number of reports concerning reactions catalyzed by
different enzymes in ILs is still limited, but based on the knowl-
edge acquired some generalizations can be made [26–28]. Many
enzymes, such as lipases, proteases, glycosidases and oxidoreduc-
tases retain some of their activity in the presence of ILs, even in very
low water contents. It has been observed that the anion of an IL has
a significant influence on the enzyme activity. Enzymes are most
active in ILs that have large, hydrophobic and non-coordinating
−
anions such as BF4 and PF6−. The most deactivating ILs contain
anions (like Cl−), which are capable of breaking hydrogen bonds.
For example, cellulases from Trichoderma reesei are inactive in
[Bmim]Cl [29]. On the other hand, these types of anions are required
for efficient dissolution of wood components.
The enzyme activities of laccases are usually decreased with
increasing IL concentration [30–33]. The decrease of enzyme activ-
ity is caused by decreasing maximum reaction velocity (Vmax) and
usually with decreased substrate affinity (increased KM). When
the anion of the IL is of alkylsulfate or alkylsulfonate type, KM is
slightly decreased, but nevertheless the observed enzyme activity
is decreased because the presence of the IL affects more to the Vmax
[32]. When the IL cation is imidazolium-based, laccase deactivation
increases with the length of the alkyl chain of the ring [33].
2.3. Determination of pH optima in different [Amim]Cl
concentrations
The optimum pH for M. albomyces was determined in solu-
tions containing 0%, 20% or 60% (m-%) [Amim]Cl by activity

M. Lahtinen et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 169–177
171
determinations in buffers with different pH values. The buffer pH
was determined before [Amim]Cl was added, because addition of
the [Amim]Cl salt did not significantly change the pH. McIlvaine
buffer was used at a pH range 3.0–8.0 with the 20% [Amim]Cl.
Because of the low solubility of phosphate at pH values above
6.0 in 60% [Amim]Cl, Na-citrate buffer was used at the pH range
5.0–7.0. Activity determinations were done in both buffers with-
out [Amim]Cl. To produce linear absorbance curves, enzyme stock
solution was added to reaction mixtures in different amounts:
0% [Amim]Cl 0.33 ␮l g−1, 20% [Amim]Cl 0.67 ␮l g−1, 60% [Amim]Cl
1.33 ␮l g−1. Thus, the highest activity of each solvent system (buffer
– [Amim]Cl concentration pair) was set to 100% and other activities
in the same solvent system were calculated as percentages related
to this highest activity.
Dimers 1–3 (ˇ-5, ˇ–ˇ and ˇ-O-4) were identified with authentic
compounds. The dimers used for identification were isolated from
oxidation mixture of coniferyl alcohol and structures verified with
NMR [40].
The structure of trimer 5 [41] was confirmed by analyzing
a sample collected from HPLC using high-resolution electro-
spray ionization mass spectrometry (HR-ESI-MS). HR-ESI-MS was
acquired with Bruker Daltonics microTOF ESI-TOF and Agilent
ES Tuning Mix (G2421A) was used for internal calibration. HR-
ESI-MS (positive) m/z 561.2088 [M+Na]+ (C30H34O9Na requires
561.2095).
The structure of dimer 4 was deduced based on HPLC retention
times, characteristic carbonyl absorbance in the UV spectrum and
chemical structure of the polymerized DHP (Section 3.4).
HPLC retention times and UV absorption maxima (ꢀmax
)
2.4. Determination of laccase activity in different [Amim]Cl
concentrations
for compounds: CA 3.89 min, 221 nm, 265 nm; 5.84 min,
1
230 nm, 277 nm; 2 9.65 min, 230 nm, 277 nm; 3 3.25 min, 221 nm,
268 nm; 4 4.64 min, 230 nm, 279 nm, 310 nm [1-(4-hydroxy-3,5-
dimethoxyphenyl)-2-(2ꢀ-methoxyphenoxy)-1-ethanone 218 nm,
314 nm] [24]; 5 8.90 min, 230 nm, 268 nm.
M. albomyces laccase activity was measured at pH 4.5 (Na-
succinate) and pH 6.0 (Na-citrate) using [Amim]Cl concentrations
(m-%) 0%, 20%, 40%, 60% and 80%. To produce a linear absorbance
curve, the reaction mixture contained the used enzyme stock
solution 0.67 ␮l g−1 at pH 4.5 and 0.33 ␮l g−1 at the opti-
mum pH 6.0. Because the used enzyme dosages were different,
the results were expressed as the activities of the enzyme
stock solution as nanokatals (nmol of substrate oxidized per
second).
2.6. Oxidation of coniferyl alcohol and analysis of the formed
dehydropolymer
Coniferyl alcohol was oxidized to dehydropolymer (DHP) in
0% and 40% [Amim]Cl concentrations. The high-molecular-weight
fraction of the polymer (high-fraction) from both experiments was
isolated, acetylated and analyzed with GPC and NMR.
2.5. Oxidation of coniferyl alcohol and analysis of the oligomeric
products as a function of time
For the oxidation of coniferyl alcohol in 0% [Amim]Cl, coniferyl
alcohol (100 mg) was first dissolved in dioxane (1 ml, distilled
over sodium). To the resulting solution, 400 mM Na-citrate, pH 6.0
(1.25 ml) was added, followed by water (7716.5 ␮l) and laccase
stock solution (33.5 ␮l). The final laccase activity in the reaction
mixture was 2 nkat g−1. The reaction mixture was stirred for 20 h
and centrifuged (2500 rpm, 20 min) to separate the high-fraction
DHP. The polymer was washed with water, dried under an argon
flow and acetylated with pyridine and acetic anhydride (1:1). Yield
of the fine structured, oily and light brown acetylated DHP was
80 mg.
In the case of 40% [Amim]Cl, coniferyl alcohol (100 mg),
[Amim]Cl (4 g), 400 mM Na-citrate (1.25 ml), water (4605 ␮l) and
laccase stock solution (145 ␮l) for the reaction were added. The lac-
case dosage was 4.3 times compared to 0% [Amim]Cl to compensate
the decrease in activity due to the ionic liquid. The high-fraction
DHP was isolated as with 0% [Amim]Cl, except that water was added
prior to centrifugation to dilute the [Amim]Cl. Yield of the dense and
dark brown acetylated DHP was 80 mg.
Oxidation of coniferyl alcohol (CA) was followed as a function
of time with [Amim]Cl concentrations 0% and 40% by stopping the
reactions with NaN3 (Riedel-de Haën), an inhibitor of laccase [39]
after 0.5, 1, 2 and 4 h.
In reference experiments without the IL ([Amim]Cl 0%), CA
(10 mg) was dissolved in 100 ␮l dioxane (synthesis grade, distilled
over sodium). To the resulting solution, 125 ␮l of 400 mM Na-
citrate, pH 6.0, 765 ␮l of water and M. albomyces laccase in 10 ␮l
of 50 mM Na-citrate buffer was added. The final laccase activity of
the resulting solution was 2 nkat g−1. After the appropriate reaction
time, 2 ml of 10 mM NaN3 was added to stop the reaction, followed
by 0.4 g [Amim]Cl and 1 ml acetonitrile (ACN, HPLC grade). The
HPLC sample was prepared by adding, for internal HPLC standard,
10 ␮l of guaiacol (Aldrich) in ACN (100 mg ml−1) to 990 ␮l of the
reaction mixture. The sample was filtered using a syringe filter
(PTFE, 0.45 ␮m pore size) and analyzed immediately.
In experiments with [Amim]Cl concentration of 40%, CA (10 mg),
[Amim]Cl (0.4 g), 400 mM Na-citrate, pH 6.0 (125 ␮l), water
(460.5 ␮l) and M. albomyces laccase stock solution (14.5 ␮l) were
added to the reaction mixture. The decrease in laccase activity
compared with 0% [Amim]Cl was compensated by increasing the
enzyme dosage 4.3 times. The reaction was stopped by adding 2 ml
of 10 mM NaN3. After that, 100 ␮l of dioxane, 300 ␮l of water and
1 ml of ACN were added to make the samples identical with 0%
[Amim]Cl samples. The HPLC sample with internal HPLC standard
was prepared as in the case of 0% [Amim]Cl.
HPLC analyses were performed with Waters HPLC, including
600 Pump, 600 Controller, 996 Photodiode Array Detector and
717plus Autosampler. The used column was Waters Symmetry C18
5 ␮m (4.6 mm × 150 mm) and a wavelength of 280 nm was used for
detection. The DAD (diode array detector) UV spectra of the com-
pounds were acquired. ACN-water was used as the eluent and the
flow rate was 0.8 ml min−1. Isocratic flow (ACN-H2O, 30:70) was
continued for 11 min, followed by 9 min gradient phase (ACN-H2O
to 100:0).
Molar masses of the obtained DHP samples were determined
by using organic gel permeation chromatography (GPC). Samples
were dissolved (1 mg ml−1) in THF (HPLC grade, without stabilator)
and filtered using syringe filter (PTFE, 0.45 ␮m pore size). After fil-
tration samples were injected to GPC system. GPC system included
degasser, pump, autosampler and diode array UV detector (Waters
1050 series). Mobile phase was THF, flow 0.5 ml min−1. Columns
were Waters Styragel guard, HR-5E and HR-1 (7.8 mm × 300 mm)
connected in series. GPC system was calibrated using polystyrene
standards (500, 890, 1000, 4000, 9000, 42,300, 177,000, 434,000,
1,270,000 Da) using UV detection at multiple wavelengths (220,
254, 280, 350 nm). Molar masses of the samples were calculated
using calibration. GPC system was run under Agilent Chemstation,
which had GPC add-on software.
Varian Inova 500 MHz spectrometer was used for NMR mea-
surements, which were performed at 27 ◦C. Deuterated dimethyl
sulfoxide (d6-DMSO) was used as a solvent and internal refer-
ence in analysis of the acetylated high-fraction DHPs. The selected
NMR (500 MHz) data for structures 1–5 from acetylated DHPs are

172
M. Lahtinen et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 169–177
Table 1
Selected NMR data (500 MHz) from analysis of acetylated high-fraction DHPs (in d6-DMSO).
Structure
1H assignments
13C assignments
H˛
Hˇ
Hꢁ
C˛
Cˇ
C␥
64.7
1
2
3
4
5
5.60 (5.46–5.63)
4.70 (4.64–4.76)
5.96 (5.92–6.00)
–
3.74 (3.71–3.77)
4.30, 4.36 (4.25–4.41)
86.9 (86.6–87.2)
84.7
73.2 (71.6–74.8)
–
49.3 (49.1–49.5)
3.06 (3.04–3.08)
3.97, 4.04 (3.79–4.21)
53.6 (53.5–53.7)
71.2 (71.0–71.3)
a
a
a
a
5.52 (5.45–5.58)
4.87
4.64, 4.86
4.07, 4.22
78.7
79.5
∼64.7b
5.62
79.5
62.5
a
Could not be assigned because of overlapping correlations.
Estimated value, because of overlapping correlations.
b
presented in Table 1. The quantitative Q-HSQC spectra were mea-
sured according to Heikkinen et al. [42].
At 60% [Amim]Cl concentration the pH optimum was shifted to
6.0. Also, the pH scale at which the laccase remained active was fur-
ther narrowed compared to 20% [Amim]Cl. The activity decreased
more at the acidic end of the scale, even though the pH optimum
shifted to a more acidic value.
3. Results and discussion
The activity and reactivity of laccase M. albomyces in the pres-
ence of the ionic liquid, 1-allyl-3-methylimidazolium chloride
([Amim]Cl) was investigated with two monomeric lignin model
compounds. First, 2,6-dimethoxyphenol was used as a substrate to
investigate the effect of [Amim]Cl to the optimum pH and enzyme
activity in different [Amim]Cl concentrations. Coniferyl alcohol was
then used to study polymerization reactions in the presence and
absence of [Amim]Cl.
3.2. Effect of [Amim]Cl concentration on the laccase activity
The activity of M. albomyces laccase was determined at
five different [Amim]Cl concentrations (0–80%) with 2,6-
dimethoxyphenol as substrate. Activity measurements were
carried out in two different buffers, Na-citrate (pH 6.0) and
Na-succinate (pH 4.5), i.e. at optimal and suboptimal pH values.
It can be clearly seen from Table 2, that increasing the amount
of [Amim]Cl decreased the laccase activity, as could be expected.
When the [Amim]Cl concentration increased from 40% to 60%, lac-
case activity at the optimum pH (6.0) decreased most clearly, from
72% to 23%, as compared to the activity in the absence of [Amim]Cl.
At 80% [Amim]Cl concentration there was still 13% of the activity
left. At pH 4.5, the most remarkable decrease in activity, from 100%
to 41%, occurred already when the [Amim]Cl concentration was
increased from 0% to 20% and at 80% [Amim]Cl concentration there
was only 2% of the activity left.
3.1. Effect of [Amim]Cl concentration on the pH optimum of the
laccase
The optimum pH of laccase M. albomyces was determined in the
absence and in the presence of 20% and 60% [Amim]Cl with 2,6-
dimethoxyphenol as the substrate (Fig. 1). Full, 100% activity refers
to the highest activity in that particular solvent system (buffer –
[Amim]Cl concentration pair). Measurements were done in two dif-
ferent buffering systems, McIlvaine and Na-citrate buffers, because
phosphate was insoluble at higher [Amim]Cl concentrations above
pH 6.0.
In the absence of [Amim]Cl, the optimum pH was 6.5 for 2,6-
dimethoxyphenol. The obtained value is very similar to the pH
optima of M. albomyces laccase determined for other phenolic sub-
strates, guaiacol (5–7.5) and syringaldazine (6–7) [34].
When the concentration of [Amim]Cl was increased to 20%,
the pH optimum remained the same. However, the activity of
the enzyme at the acidic and the basic ends of the pH scale was
markedly reduced, as compared to 0% [Amim]Cl. At pH values 4.0
and 8.0 there were only 7% and 17% left of the maximum activity.
3.3. Oxidation of coniferyl alcohol as a function of time and
analysis of the oligomeric products
Coniferyl alcohol is oxidized by different oxidants first to the
dimeric products (ˇ-5, ˇ–ˇ and ˇ-O-4, 1–3, Fig. 2) and then further
to oligomeric and polymeric products. Laccases from Rhus verni-
cifera and Pycnoporus coccineus also catalyze the formation of these
dimers from coniferyl alcohol [43]. Coniferyl alcohol was oxidized
at [Amim]Cl concentrations of 0% and 40%. Reactions were stopped
with the known laccase inhibitor, NaN3, at 0.5, 1, 2 and 4 h and
the products were monitored with HPLC immediately after sample
preparation.
120
100
80
60
40
20
0
First of all, the inhibitory effect of NaN3 was interestingly grad-
ually decreased when the ionic liquid was present, but in the
absence of other efficient ways to inhibit laccase, this inhibitor
was, however, used to terminate the reactions, and the samples
Table 2
Activity of the M. albomyces laccase, determined as a function of [Amim]Cl concen-
tration at pH 4.5 (Na-succinate buffer) and pH 6 (Na-citrate buffer). Activity was
determined using 2,6-dimethoxyphenol as the substrate. The used enzyme dosage
was twice as high at pH 4.5 compared to the dosage used at pH 6.0.
[Amim]Cl (m-%)
Activity at pH 4.5
nkat ml−1
Activity at pH 6.0
nkat ml−1
%
%
3
3.5
4
4.5
5
5.5
pH
6
6.5
7
7.5
8
0
20
40
60
80
700
288
93
31
11
100
41
13
4
1189
987
853
273
160
100
83
72
23
13
Fig. 1. Relative activity of the laccase M. albomyces at different pH values and differ-
ent [Amim]Cl concentrations. Straight line: McIlvaine buffer, dotted line: Na-citrate
buffer, [Amim]Cl concentrations: square 0%, triangle 20% and circle 60%.
2

M. Lahtinen et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 169–177
173
O
O
O
HO
O
R
O
OH
O
OH
O
2
OH
1
R = -CH=CH-CH2OH
R
OH
R
OH
OH
R
OH
O
O
O
O
O
O
O
O
O
R
O
O
O
OH
OH
OH
4
5
3
Fig. 2. Dimeric and trimeric products and structures identified from coniferyl alcohol oxidation catalyzed by laccase: ˇ-5 (1), ˇ–ˇ (2), ˇ-O-4 (3), ˛-C O/ˇ-O-4 (4), ˛-O-4/ˇ-O-4
(5).
were analyzed immediately. Although the laccase is relatively ther-
mostable [34], attempts to deactivate the M. albomyces laccase by
boiling surprisingly also failed. In a previous work, the same HPLC
sample preparation method, where the enzyme was first inhibited
by adding NaN3, after which guaiacol was added for internal HPLC
standard, was used without any difficulties [24]. As previously
shown, after inhibition by NaN3 the laccase is not able to react
with the phenolic guaiacol. Without inhibitor, guaiacol is oxidized
by laccase to a red-colored product in a timescale of seconds. Sur-
prisingly it was observed, that in the presence of IL and NaN3,
guaiacol was not oxidized immediately, but the typical red color
appeared after a couple of hours. It was thus concluded that NaN3
first inhibited laccase, but upon prolonged incubation in the pres-
ence of IL, the inhibitory effect was gradually decreased. Possibly,
the N3− anion was first normally bound to laccase, but the presence
of ions from the IL gradually weakened the interaction of laccase
with the inhibitor.
The HPLC-chromatograms of the samples, in which practically
all coniferyl alcohol was oxidized (0% [Amim]Cl at 2 h and 40% at
4 h), are shown in Fig. 3. Authentic compounds were used to iden-
tify ˇ-5, ˇ–ˇ and ˇ-O-4 dimers (1–3). The structure of trimer 5 [41]
was confirmed with HR-ESI-MS. It is also known, that near neutral
pH, addition of phenol to ˇ-O-4 type quinone methide to form a
˛-O-4/ˇ-O-4 product is dominating, and at more acidic pH, water
is added to the quinone methide to form a ˇ-O-4 product [44]. The
low concentration of ˛-C O/ˇ-O-4 dimer (4) prevented MS anal-
ysis of this compound. Thus, the structure was deduced based on
the characteristic UV absorbance maximum of ketone and the cor-
responding structure in the high-polymerized DHP (Section 3.4). In
addition, based on previous studies, laccases typically catalyze the
oxidation of benzylic hydroxyl groups of lignin model compounds
[24].
The amounts of coniferyl alcohol and oxidation products, rela-
tive to the internal standard guaiacol, are presented in Table 3. The
amounts are not quantitative, but the results based on HPLC peak
areas can be examined to compare the consumption of coniferyl
alcohol and formation of each product in the two different sol-
vent systems. The oxidation rate of coniferyl alcohol was clearly
slower in 40% [Amim]Cl compared to 0% [Amim]Cl, even though
the reduced laccase activity in the presence of [Amim]Cl was com-
pensated by increasing the enzyme dosage accordingly. Obviously,
the enzyme was inactivated more in the IL reaction than esti-
mated in the short term activity determination. Possibly, prolonged
incubation of laccase in the presence of the IL, caused some addi-
tional inactivating effect. However, without understanding how
and with which structures of the enzyme the ions of the IL interact
on a molecular level, and what the mechanism of the inacti-
vation is, it is impossible to explain these observations at this
stage.
Formation of ˇ-5, ˇ–ˇ and ˇ-O-4 dimers (1–3) was very simi-
lar in both solvent systems in the beginning (0.5 h) of the reaction.
The formation rate of ˛-O-4/ˇ-O-4 trimer (5) was, however, faster
in 0% [Amim]Cl already during the first 0.5 h of the reaction. The
˛-C O/ˇ-O-4 dimer (4) was first detected after 2 h in 0% and 4 h
in 40% [Amim]Cl. As the reaction proceeded, all products were
formed faster in the absence of [Amim]Cl, until their amount was
reduced, as they started to react further. There were also differ-
ences in the starting point of the further reactions of the products:
the amounts of ˇ-5 and ˇ-O-4 dimers (1, 3) started to decrease first
in 0% [Amim]Cl, while the amounts of ˇ–ˇ dimer (2) and ˛-O-4/ˇ-
O-4 trimer (5) started to decrease at the same time in both solvent
systems. There may be several reasons for these differences in the
reactivities. The IL may have changed the properties of different
substrates due to increased solubility and viscosity. The IL may also
have affected to which nucleophiles the quinone methides, formed
by the oxidation, would have reacted with. In addition, it is possi-
ble that the structure and/or activity of the laccase were affected
by the presence of IL, having an impact on the substrate preference
or specificity. These different reactivities of dimeric and trimeric
products in further reactions, between the two solvent systems,
most probably explain also the differences observed in structures
of the analyzed DHPs (Section 3.4).
3.4. Oxidation of coniferyl alchohol and analysis of the formed
dehydropolymer (DHP)
Coniferyl alcohol was oxidized for a longer period (20 h) in
[Amim]Cl concentrations of 0% and 40% in order to investigate the
possible structural differences in the formed DHPs. The reduced
laccase activity due to the presence of [Amim]Cl was compensated
by increasing the enzyme dosage based on the activity determina-
tions in the presence of the IL. Polymers (DHP high-fraction) from
both solvent systems were analyzed with GPC and NMR.
The visual appearance of the DHPs was different, as the DHP
material formed in 40% [Amim]Cl was more dense and had a darker
color, compared to the one formed in without [Amim]Cl. The aver-
age molar masses of both DHPs, formed in the absence and in the
presence of [Amim]Cl, are shown in Table 4. The DHP formed in
the presence of [Amim]Cl, had clearly a higher average molar mass,
compared to the DHP formed in water. Apparently, the increased

174
M. Lahtinen et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 169–177
1
guaiacol
5
2
CA
4
3
1
guaiacol
2
5
3
4
Fig. 3. HPLC chromatograms of products formed from laccase-catalyzed coniferyl alcohol oxidation in 0% [Amim]Cl in 2 h (above) and in 40% [Amim]Cl in 4 h (below).
Table 3
Formation of oligomeric products (1–5 from Fig. 2) in laccase-catalyzed oxidation of coniferyl alcohol (CA), in 0% and 40% [Amim]Cl as a function of reaction time by HPLC
analysis. The ratio of integrated peak areas of coniferyl alcohol and guaiacol (internal reference) was set to 100, and the other values were calculated accordingly.
Coniferyl alcohol (CA) and the oligomeric products
Time (h)
CA
0%
CA
40%
1
0%
1
40%
2
0%
2
40%
3
0%
3
40%
4
0%
4
40%
5
0%
5
40%
0
0.5
1
2
4
100.0
79.0
68.1
4.7
100.0
85.0
80.7
72.7
0.0
0.0
0.0
28.8
32.7
50.9
68.7
0.0
0.0
13.1
15.3
22.2
16.4
0.0
2.3
3.3
6.4
1.9
0.0
2.1
2.4
3.2
4.7
0.0
0.0
0.0
1.3
3.4
0.0
0.0
0.0
0.0
2.2
0.0
0.0
14.7
15.1
20.3
16.6
28.7
45.6
64.5
42.6
12.5
18.6
14.0
10.0
33.7
52.6
45.8
48.3
0.4

M. Lahtinen et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 169–177
175
1β
2β
-OMe
3γ, 5γ
4γ
coumaryl γ
1γ
3α
2γ
4β, 5α
1α
3β, 5β
2α
1β
-OMe
IL
2β
3γ, 5γ
4γ
coumaryl γ
1γ
3α
4β, 5α
1α
2γ
3β, 5β
2α
5α, 5γ
5β, 5γ
5γ, 5γ
1α, 1γ
1γ, 1γ
2γ ’, 2γ
2α, 2γ
4β, 4β
5α, 5α
4γ, 4β
5β, 5α
5γ, 5α
Fig. 4. 2D HSQC spectra (500 MHz, d6-DMSO) of acetylated DHPs (high-fraction) formed from laccase-catalyzed oxidation of coniferyl alcohol. Top: HSQC spectrum of DHP
formed in 0% [Amim]Cl, middle: HSQC spectrum of DHP formed in 40% [Amim]Cl, bottom: HSQC-TOCSY spectrum of DHP formed in 0% [Amim]Cl. Numbering used in Fig. 2
was also used in numbering of the DHP structures.

176
M. Lahtinen et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 169–177
Table 4
˛-O-4/ˇ-O-4. When reactions were terminated with NaN3 to exam-
ine the oxidation products of coniferyl alcohol as a function of time,
this laccase inhibitor was observed not to be as effective in the IL
as in water solutions. Consumption of coniferyl alcohol and for-
mation of oxidation products were clearly slower in the presence
of [Amim]Cl at later stages of the reaction, although the reduction
of laccase activity caused by the [Amim]Cl was at least partially
compensated by increasing the enzyme dosage.
Average molar masses of high-fraction DHPs from laccase-catalyzed oxidation of
coniferyl alcohol in 0% and 40% [Amim]Cl.
DHP
Mn (103 g/mol)
Mw (103 g/mol)
Mz (103 g/mol)
Mw/Mn
0% [Amim]Cl
40% [Amim]Cl
1.1
2.1
1.4
4.1
2.2
6.9
1.3
2.0
Table 5
The DHPs formed from coniferyl alcohol in 0% or 40% [Amim]Cl
differed already in their visual appearance, the one formed in the
presence of IL being denser and having a darker color. The 2D NMR
analysis revealed that the DHP formed in the presence of [Amim]Cl
contained less ˇ-O-4, slightly less ˇ-5 structures and more ˇ–ˇ
structures. Obviously, the DHP formed in the presence of [Amim]Cl
contained more 5–5 bonds, than the DHP formed in the absence of
[Amim]Cl.
Clearly, polymerization reactions catalyzed by laccases can be
performed in the presence of ionic liquids, such as [Amim]Cl, to
enhance the solubility of the substrate and the product, and to form
more highly polymerized lignin-based materials. Interestingly, the
chemical structure of the formed polymer was also structurally
different from that formed in the absence of the ionic liquid.
Relative amounts of structural units in DHPs formed in 0% and 40% [Amim]Cl from
Q-HSQC spectra [42]. The amounts were acquired, assuming each structural unit
contains one methoxyl (OMe) group, from HSQC-NMR H/C correlation volumes
using the formula: V(Hx/Cx)/[3/V(HOMe/COMe)]. The slight inaccuracy of the inte-
gration due to overlapping signals was found insignificant for the final results.
Structural unit
Correlation/ppm (H/C)
0% [Amim]Cl
40% [Amim]Cl
1
2
3
4
5
5.60/86.9 (H˛/C˛)
4.70/84.7 (H˛/C˛)
5.96/73.2 (H˛/C˛)
5.52/78.7 (Hˇ/Cˇ)a
4.87/79.5 (Hˇ/Cˇ)b
0.29
0.53
0.03
0.02
0.30
0.20
0.73
0.02
0.02
0.08
a
Volume of 5 Hˇ/Cˇ subtracted from total volume at 5.52/78.7 ppm to acquire
volume of 4 Hˇ/Cˇ.
b
Volume of 3 H˛/C˛ subtracted from total volume at 4.87/79.5 ppm to acquire
volume of 5 Hˇ/Cˇ.
Acknowledgements
solubility caused by the IL, enhanced the formation of further poly-
merized material.
Financial support from the Academy of Finland (grants 122534
and 132150) and Forestcluster Ltd. are gratefully acknowledged.
Expansions of the HSQC spectra of both DHPs, and expansion
of the HSQC-TOCSY spectrum of DHP formed in 0% [Amim]Cl are
shown in Fig. 4. The NMR assignments are based on the values found
in literature [45]. The H˛/C˛, Hˇ/Cˇ and Hꢁ/Cꢁ correlations of ˇ-5
(1) and ˇ–ˇ (2) structures separated well in the HSQC spectrum.
Also, ˇ-O-4 structure (3) could be assigned based on correlation
at 5.96/73.2 ppm (H˛/C˛) in HSQC. Correlations of ˛-C O/ˇ-O-4
(4) and ˛-O-4/ˇ-O-4 (5) structures overlapped in the HSQC spec-
trum (Hˇ/Cˇ of structure 4 with H˛/C˛ of structure 5, Hˇ/Cˇ of
structure 5 with Hˇ/Cˇ of structure 3), and these were assigned
based on patterns in the HSQC-TOCSY spectrum. The characteris-
tic Hˇ/Cˇ correlation at 4.14/82.5 ppm indicating trans-isomer of
dibenzodioxocin structure [46] was not observed.
Based on the quantitative Q-HSQC spectra (Fig. 4, and inte-
gration results Table 5), there were clear structural differences
between the DHPs formed in 0% [Amim]Cl and 40% [Amim]Cl. The
DHP formed in 40% [Amim]Cl contained less ˇ-O-4 structures (3–5)
and slightly less ˇ-5 structures (1) than DHP formed in 0% [Amim]Cl.
Also, the DHP formed in 40% [Amim]Cl, contained more ˇ–ˇ struc-
tures (2) than the one formed in 0% [Amim]Cl. Obviously, based
on higher molecular weight, the DHP formed in the presence of
[Amim]Cl contained more biphenylic (5–5) cross-linkages. Several
different variables are known to affect the frequencies of different
bond-types in DHPs: the rate at which monomers are supplied and
radicals are formed, the pH, the presence of polysaccharides and the
size of the growing polymer [2,44]. The analysis of oligomeric prod-
ucts revealed that the structural differences were formed already
at the early stages of oxidation (Section 3.3), but the formation of a
larger polymer in the presence of the IL probably also affected the
structural differences.
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