Báo cáo khoa học: First evidence of catalytic mediation by phenolic compounds in the laccase-induced oxidation of lignin models ppt

First evidence of catalytic mediation by phenolic compoundsin the laccase-induced oxidation of lignin models Francesca d’Acunzo and Carlo Galli Dipartimento di Chimica, Universita` ‘La S

First evidence of catalytic mediation by phenolic compounds

in the laccase-induced oxidation of lignin models

Francesca d’Acunzo and Carlo Galli

Dipartimento di Chimica, Universita` ‘La Sapienza’, Roma, Italy; IMC-CNR Sezione Meccanismi di Reazione

The sulfonephthalein indicator, phenol red, exhibits an

unusually slow rate of oxidation by laccase from

Polipo-rus pinsitus, in spite of the fact that it is a phenol and

therefore a natural substrate for this phenoloxidase enzyme

Nevertheless, after prolonged exposure to laccase (24 h)

phenol red is oxidized by more than 90% We found that

phenol red, which can be oxidatively converted into a

reso-nance-stabilized phenoxy radical, performs as a mediator in

the laccase-catalyzed oxidation of a nonphenolic substrate

(4-methoxybenzyl alcohol) and also of a hindered phenol

(2,4,6-tri-tert-butylphenol) In particular, phenol red was

found to be at least 10 times more efficient than

3-hydr-oxyanthranilate (a reported natural phenolic mediator of

laccase) in the oxidation of 4-methoxybenzyl alcohol Other

phenols, which do not bear structural analogies to phenol

red, underwent rapid degradation and did not perform as

laccase mediators On the other hand, several variously substituted sulfonephthaleins, of different pK2 values, mediated the laccase catalysis, the most efficient being dichlorophenol red, which has the lowest pK2of the series The mediating efficiency of phenol red and dichlorophenol red was found to be pH dependent, as was their oxidation Ep value (determined by cyclic voltammetry) We argue that the relative abundance of the phenoxy anion, which is easier to oxidize than the protonated phenol, may be one of the factors determining the efficiency of a phenolic mediator, together with its ability to form relatively stable oxidized intermediates that react with the desired substrate before being depleted in undesired routes

Keywords: laccase; phenolic mediators; lignin models; radi-cals; acidity

Laccases (EC 1.10.3.2) are multicopper oxidases, produced

by micro-organisms and plants, which participate in nature

in both the biosynthesis and degradation of lignin [1] In

the latter case, laccase operates in conjunction with other

ligninolitic enzymes, such as lignin peroxidase and

man-ganese peroxidase The latter two enzymes, which are

stronger oxidants than laccase, are able to oxidize most

aromatic constituents of lignin, whereas laccase oxidizes

directly only the phenolic subunits, which are easier to

oxidize but relatively less abundant (15%) It has been

speculated [1], however, that laccase may react indirectly

with nonphenolic lignin components through mediation by

phenolic species present in its natural environment These

natural mediators could be metabolites [2], or even lignin

fragments [3] generated by the other ligninolitic enzymes,

and would open up alternative, possibly radical, routes to

the oxidation of nonphenolic components [1] It is quite

reasonable to argue that natural mediators may participate

in the laccase-catalyzed oxidation of nonphenolic lignin

subunits in those micro-organisms that only rely on laccase

for their ligninolitic action [4,5], and a number of sub-stances, such as phenolic acids [6,7] and 3-hydroxyanthrani-late (HAA) [8], have indeed been proposed as natural laccase mediators However, in order to prime laccase towards the oxidation of nonphenolic substrates, a large excess of these mediators is often needed [7,9] or, at least, a stoichiometric amount is required in the most favourable cases [10] This might be because phenolic mediators are, themselves,
good substrates for the enzyme Laccase would oxidize them to short-lived reactive species that undergo further reactions (noncatalytic undesired routes in Fig 1: radical coupling, fragmentation, etc.), preventing their reduction to the original state by the nonphenolic substrate they are supposed to oxidize (Fig 1, catalytic cycle)

As an example, dimerization of the phenolic compound, HAA, once oxidized by laccase, is so fast and quantitative that we exploited the formation rate of this dimer in a new spectrophotometric assay of laccase activity in mixed solvents [11]

In the context of our studies [12–15] on laccase-mediator systems, and of the speculations on the natural role of phenolic mediators [1], we were intrigued by the results of Li

et al [9] on the violuric acid-mediated oxidation of phenol red by laccase It was reported, in fact, that some fungal laccases oxidize phenol red (Fig 2; an advocated phenolic lignin model) at a negligible rate, but that this rate increases substantially in the presence of violuric acid when it is added

in up to a 100· molar excess with respect to phenol red Why is phenol red oxidized so slowly, by laccase, that a mediator (a large excess of violuric acid, in this case) is needed in order to speed up its metabolization? By studying

Correspondence to C Galli, Dipartimento di Chimica, Universita`

La Sapienza, 00185 Roma, Italy.

Fax: + 39 06 490 421, Tel.: + 39 06 4991 3386,

E-mail: carlo.galli@uniroma1.it

Abbreviations: ABTS, 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic

acid); HAA, 3-hydroxyanthranilic acid; HBT,

1-hydroxybenzo-triazole.

Enzyme: laccases (EC 1.10.3.2).

(Received 19May 2003, revised 10 July 2003, accepted 14 July 2003)

the peculiar behaviour of phenol red and other

phenol-sulfonephthaleins we aimed to gain a better insight into the

interaction between laccase and phenols in general More

specifically, we wished to identify the characteristics that a

phenol should have in order to be a good laccase mediator,

i.e one that follows the catalytic cycle shown in Fig 1

without being rapidly metabolized by the enzyme (undesired

routes) In this study, we first investigated the kinetics of

oxidation of phenol red by Poliporus pinsitus laccase in the

absence of any mediator, and then evaluated the ability of

phenol red to act as a laccase mediator in the oxidation of

a hindered phenol and of a nonphenolic compound We

extended this study to other sulfonephthalein indicators,

characterized by different pK2values, and investigated the

effect of solution pH on the mediating ability of phenol red

and dichlorophenol red

Materials and methods

Laccase

Laccase from P pinsitus was kindly donated by Novo

Nordisk Biotech; it was purified by ion-exchange

chroma-tography on Q-Sepharose by elution with phosphate buffer;

laccase fractions with an absorption (A)280/A610 ratio of

20–30 were considered sufficiently pure [16] The collected

fractions were concentrated by dialysis in cellulose

mem-brane tubing (Sigma) against poly(ethylene glycol) to a final

activity of 9000 UÆmL)1, as determined

spectrophotometri-cally by the standard reaction with

2,2¢-azinobis(3-ethyl-benzo-6-thiazolinesulfonic acid) (ABTS) [17]

Reagents

4-Methoxybenzylalcohol, HAA, 1-hydroxybenzotriazole

(HBT) and all phenols employed (Aldrich) were used as

received from the manufacturer Buffers were prepared using ultrapure water obtained from a MilliQ apparatus

Kinetics of laccase-catalyzed oxidation of phenol red and 2,4,6-trichlorophenol

In a 10-mm quarz UV-Vis cell, the reagent (phenol red or 2,4,6-trichlorophenol; 40 lMin 0.1-Mcitrate buffer, pH 5.0) was allowed to react with 0.3 UÆmL)1of laccase at 25C Phenol red consumption was monitored at 420 nm, while dichloroquinone formation was monitored at 273 nm, using

an HP 8453 diode array UV-Vis spectrophotometer run with

HPUV-VIS CHEMSTATIONsoftware The rate of phenol red oxidation is expressed as the initial rate of depletion of the absorption at the wavelength followed (e.g DA420Æ min)1), in keeping with the literature [9] The complexity of the kinetic trend, i.e a biphasic profile (Fig 3), and the possibility that the absorption at 420 nm may be also attributed to products other than phenol red, prevented us from converting data from absorbance units to concentration units

Laccase-catalyzed oxidation of phenol red Phenol red (20 lMin 0.1-Msodium citrate buffer, pH 5.0), was incubated with 4 UÆmL)1of purified laccase for 24 h at

25C The amount of residual phenol red, after metaboli-zation by the enzyme, was determined by HPLC The internal standard (3,4-dimethoxybenzaldehyde) was added

to the reaction mix, which was then diluted in the mobile

Fig 2 Structure of phenol red and related molecules.

Fig 3 Degree of conversion of phenol red (thick line) and 2,4,6-tri-chlorophenol (thin line) as a function of time The kinetics of laccase-catalyzed oxidation of phenol red and 2,4,6-dichlorophenol were monitored spectrophotometrically through the decrease in absorption

at 420 nm and the increase in absorption at 273 nm, respectively Reaction conditions: phenol red or 2,4,6-trichlorophenol, 40 l M in 0.1 M citrate buffer, pH 5.0; laccase, 0.3 UÆmL)1 Calculation of the degree of conversion:

C ¼ 100AðtÞ  Aðt0Þ Aðt f Þ  Aðt 0 Þ where t f ¼ 20 h for phenol red (>90% conversion by HPLC), but

t f ¼ 4 h for trichlorophenol [the time at which A(t) has already reached its plateau value] The inset shows the spectrum of phenol red

at t ¼ 0 and t ¼ 20 h; residual absorption at 420 nm after 20 h cannot

be entirely attributed to phenol red (according to HPLC data), but rather to some unidentified product(s).

Fig 1 The catalytic cycle of a laccase-mediator system, where the

oxidized mediator partitions between a significant undesired side-reaction

and a catalytic cycle of oxidation of a nonphenolic substrate.

phase and filtered through 0.2-lm Teflon syringe filters

(Superchrom Varisep) prior to analysis We used an Agilent

Technologies HPLC system (pump, degasser, UV-Vis

detector and solvent delivery system) equipped with a

Zorbax Agilent Eclipse XDB-C815 cm· 4.6 mm column,

and run with AgilentCHEMSTATION FOR LCsoftware The

elution was carried out with an MeOH/H2O (40 : 60) mobile

phase containing 0.3& trifluoroacetic acid, at a flow rate of

0.8 mLÆmin)1, and products were detected at 265 nm

Oxidation of lignin model compounds

4-Methoxybenzyl alcohol or 2,4,6-tri-t-butylphenol

(Ald-rich) (20 lMin 0.1Mcitrate buffer, pH 5.0), was incubated

for 24 h at 25C with 4 UÆmL)1of purified laccase and a

mediator (HAA, HBT, or one of the phenolic derivatives

listed in Table 1; 6.7 lM) Reaction products were identified

by GC-MS analysis, run on a HP 5892 GC equipped with a

12-m· 0.2 mm methyl silicone gum capillary column,

coupled to an HP 5962 MSD instrument operating at

70 eV The yields of oxidation (yield of

4-methoxybenzal-dehyde or consumption of 2,4,6-tri-t-butylphenol) were

determined by GC analysis with respect to

p-methoxy-acetophenone as the internal standard, using a Varian 3400

Star instrument fitted with a 20 m· 0.2 mm methyl silicone

gum capillary column coupled to an FID detector

Cyclic voltammetry

The electrochemical equipment consisted of a

computer-controlled home-made potentiostat with a Vernier

Soft-wareMULTI PURPOSE LABORATORY INTERFACE (MPLI) pro-gram forWINDOWS The three-electrode system consisted of

a glassy-carbon disc (of 1.5 or 3 mm diameter) working electrode, an aqueous Hg/HgCl2/saturated KCl reference electrode (E vs NHE ¼ E vs SCE + 0.242 V), and a Pt reference electrode (1 cm2) Prior to recording each scan, the working electrode was polished using a Cypress Systems polishing kit, sonicated for 1 min, and rinsed with distilled water All scans were obtained at room temperature NaH2PO4/Na2HPO4 buffers (0.5M) at pH 4.0 and

pH 7.4, respectively, were prepared by carefully weighing appropriate amounts of monobasic and dibasic phosphates, which were dissolved in water previously filtered through a MilliQ apparatus (Millipore, France) The cyclic voltam-metry scans of 0.5-mMphenol red or dichlorophenol red in solutions of different pH were run at a rate of 0.5 VÆs)1 The cyclic voltammetry scans of 0.5 mM phenol red in the presence of 2.5–30 mM4-methoxybenzyl alcohol were run

at a rate of 5 mVÆs)1

Results and discussion

Kinetics of the laccase-catalyzed oxidation of phenol red

In a previous study, on the oxidation of oligomeric phenols,

we unambiguously pinpointed poor substrate solubility and steric hindrance as two factors that make the use of a mediator necessary for the laccase-catalyzed oxidation of otherwise recalcitrant phenolic substrates [18] Phenol red, however, does not seem to present any steric problem, and its solubility is not limited in aqueous solutions Therefore, its reported [9] lack of reactivity with laccase is puzzling

By following the reaction spectrophotometrically at

420 nm, we found that the initial oxidation rate of phenol red (3· 10)3 DAÆmin)1 for a 1-h reaction time) in the absence of a mediator was significantly slower than that of 2,4,6-trichlorophenol (Fig 3), a structurally simple phenol that laccase easily oxidizes to dichlorobenzoquinones (most probably a mixture of o- and p-benzoquinone isomers, the latter being the most abundant: see Fig 4 for product identification by MS [19]) However, the decrease in absorption at 420 nm proceeded slowly throughout the 20-h time span during which the reaction was monitored (the rate at 10–20 h was 3· 10)5DAÆmin)1)

For a direct comparison of the kinetic data of phenol red and 2,4,6-trichlorophenol, the degree of conversion of the two phenols, as a function of time, was plotted (see the equation in the legend to Fig 3) Figure 3 clearly shows that, while 2,4,6-trichlorophenol was quantitatively meta-bolized within 2 h, the conversion of phenol red was only

 80% after 5 h HPLC analysis (where phenol red is chromatographically separated from other components of the reaction mixture that may absorb at 420 nm) indicated that <10% residual phenol red was present after 20 h of exposure to laccase In Fig 3 therefore the A420(at 20 h) was taken as an approximation of the residual absorption at

zero phenol red concentration This residual absorption may be attributed to some product(s) of phenol red metabolization, which, in turn, could react slowly with laccase As a result, the kinetic curve is biphasic and the data cannot be expressed simply in terms of phenol red concentration

Table 1 Phenolic structures investigated as laccase mediators in the

oxidation of 4-methoxybenzyl alcohol at pH 5.0, unless stated otherwise.

Reaction conditions: substrate, 20 m M in 0.1 M citrate buffer, pH 5.0;

mediator concentration, 6.7 m M ; laccase concentration, 4 UÆmL)1;

reaction time, 24 h pK ArOH values obtained from refs [25], [26], [29]

and [30].

Entry Mediators investigated

Yield (%) of 4-methoxy-benzaldehyde pK ArOH

2 Phenol red, 24 h 35

3 Phenol red, pH 3.5 <5

4 Phenol red, pH 5.5 20

5 Phenol red, pH 6.0 15

6 Dichlorophenol red 55 5.74

7 Dichlorophenol red, pH 3.5 10

8 Dichlorophenol red, pH 5.5 30

9Dichlorophenol red, pH 6.0 10

10 Cresol red 20 7.87

11 m-Cresol red 15 8.04

12 Phenolphthalein <5 9.4

13 Aurintricarboxylic acid <5 >7.2

14 Chrome azurol S <5

15 Resveratrol <5

16 Alizarin red <5

17 Quercetin <5

18 2,6-Dimethoxy-4-allylphenol <5

192,6-Dichloroindophenol <5

Oxidation of lignin model compounds

by the laccase/phenol red system

In the previous paragraph we presented evidence to suggest

that the laccase/phenol red couple is a
long-lasting reacting

system, compared, for example, with the

laccase/2,4,6-trichlorophenol couple Phenol red is probably oxidatively

converted into a phenoxy radical [3], like any other

laccase-oxidizable phenol If its phenoxy radical, or secondary

products thereof, were sufficiently long-lived, phenol red

might behave as a laccase mediator in radical oxidation

routes towards recalcitrant substrates [20] We tested this

hypothesis in the oxidation of the nonphenolic

4-meth-oxybenzyl alcohol, and also of the hindered

2,4,6-tri-t-butylphenol The former was not oxidized by laccase in the

absence of a mediator, whereas the latter yielded only a trace

of di-t-butylbenzoquinone (predominantly the

p-benzoqui-none isomer See Fig 4 for product identification by MS)

Oxidation of 4-methoxybenzyl alcohol In Table 2 we

report the amount of substrate converted with phenol red

acting as the mediator, compared to the laccase/HAA

(a phenolic mediator) and laccase/HBT systems, HBT being

a well-known mediator of laccase, having an >N-OH

structure [15] The mediator is always in defect with respect

to the substrate, unlike many phenolic mediators reported in

the literature [7,9] The only product detected in the

medi-ated oxidations of 4-methoxybenzyl alcohol is

4-methoxy-benzaldehyde, and recovery of the unreacted alcohol

complements the amount of product quantitatively

Table 2 shows that, while the conversion of 4-meth-oxybenzyl alcohol is much higher with HBT, phenol red is

at least 10 times more efficient than HAA, a reported naturally occurring phenolic laccase mediator [8] The laccase/phenol red system maintains its oxidizing activity even during the second day of reaction, when phenol red is

no longer present Therefore, part of the mediation efficiency of phenol red, compared with other structurally simple phenols, is the result of by-products that are still reactive towards the substrate

Oxidation of 2,4,6-tri-t-butylphenol Phenol red per-formed significantly better than HBT as a mediator of laccase in the oxidation of 2,4,6-tri-t-butylphenol to di-t-butylbenzoquinone (predominantly p-benzoquinone isomer: see Fig 4 for product identification) The laccase-generated phenoxy radical of phenol red, in this case, would remove the H-atom from the OH group of the hindered phenol in an almost thermoneutral step; the resulting 2,4,6-tri-t-butylphenoxyl radical drives the reaction towards the loss of isobutene and results in the observed di-t-butyl-benzoquinones Two moles of substrate are oxidized per mole of mediator, indicating that phenol red participates in

a catalytic cycle such as the one described in Fig 1

A mechanistic parallel between N-hydroxy and phenolic mediators

The laccase mediator, HBT, is oxidatively converted by the enzyme into an >N–O• reactive intermediate [21,22] Electrochemical data [23] show that the >N–O•radical of HBT is sufficiently long-lived to be able to abstract a benzylic hydrogen atom from a benzylic alcohol, converting

it into the aldehyde through the intervention of dioxygen (Fig 5) This route, via a radical, circumvents the low tendency of a benzyl alcohol to be involved in an electron-transfer route with laccase, and makes its oxidation possible [13,21] This hydrogen abstraction route is thermodynami-cally feasible because the energy of the O–H bond that N–O•forms is comparable to that of the benzylic C–H bond that is cleaved from the substrate [12,21]

It can be suggested that, if the laccase-generated phenoxy radical from phenol red is resonance-stabilized [24], and therefore as long-lived as the >N–O• radical from HBT

Fig 4 Fragmentation patterns that are diagnostic for o- and

p-benzo-quinone isomerism [19] R ¼ Cl, m/z=52 (amenable to

2,6-dichloro-benzoquinone) is the base peak; m/z=110 is 52% of the base peak.

R ¼ t-Bu, the abundance of m/z ¼ 52, 53, 67 (amenable to

2,6-di-t-butylbenzoquinone) altogether is five times that of m/z ¼ 164; the

base peak is (M-43) ¼ 177.

Table 2 Oxidation (%) of 4-methoxybenzyl alcohol and 2,4,6-tri-t-butylphenol by laccase/phenol red, compared with two other laccase-mediator systems The reaction conditions were as follows: substrate,

20 m M in 0.1 M citrate buffer, pH 5.0; mediator concentration, 6.7 m M ; laccase concentration, 4 UÆmL)1; reaction time, 24 h HAA, 3-hydroxyanthranilic acid; HBT, 1-hydroxybenzotriazole.

Substrate

Mediator Phenol red HAA HBT 4-Methoxybenzyl alcohol 20 a 2 a [15] 75 a [15]

35 a,b – – 2,4,6-tri-t-butylphenol 55c – 30c

a Yield of aldehyde b Reaction time: 48 h c Yields of di-t-butyl-benzoquinone were determined by gas chromatography.

[22], it has time to follow an analogous H-abstraction route

of oxidation of the nonphenolic substrate In fact, phenol

red is more acidic (pK2¼ 7.42) [25] than simple phenols

(pKa¼ 9–10) [26] This is a result of the delocalization of

the negative charge of the anion onto the adjacent quinoid

ring (see Fig 2) Analogously, delocalization of the

un-paired electron of the corresponding phenoxy radical onto

the quinoid ring would enhance the survival time of the

phenoxy radical intermediate from phenol red, giving it a

greater chance to take part in catalytic cycles (Fig 1) before

being consumed in undesired routes Because O–H bond

energies of phenols (approximately 82–88 kcalÆmol)1) are

similar to those of the C–H benzylic bonds of the alcohol

[21], the radical route of the phenoxy radical is as

thermodynamically feasible as that of the > N–O•species

from a >N–OH mediator This suggested route bears

strong analogies to the reported mechanism of oxidation of

primary alcohols to aldehydes by the enzyme galactose

oxidase [27], where the phenoxy radical of a tyrosine residue

abstracts an H-atom from the alcoholic substrate [28]

Our hypothesis, that part of the mediating ability of

phenol red (i.e besides the reactivity of its secondary

oxidation products) may be attributed to its phenoxy

radical, finds support in the following results obtained by

cyclic voltammetry Figure 6 shows the irreversible

vol-tammogram corresponding to the oxidation of phenol red

to its phenoxy radical More precisely, at the pH of the experiment (7.4), phenol red (pK2¼ 7.42) is  50% deprotonated, so that the pH-dependent Ep results from both the oxidation of the phenol to the corresponding radical cation (which rapidly releases a proton to yield the phenoxy radical) and to the oxidation of the phenolate ion

to the phenoxy radical directly [29] The scans run at pH 7.4,

in the presence of an increasing excess of 4-methoxybenzyl alcohol, showed some increase in current intensity ( 10% increase with a fivefold excess of 4-methoxybenzyl alcohol; 50% increase with a 60-fold excess; we were unable to achieve a larger excess of alcohol because of its limited solubility in the buffer) This indicates that phenol red is, at least in part, regenerated from its phenoxy radical through the abstraction of a benzylic hydrogen from the substrate,

so that it can be oxidized once again at the electrode, this resulting in an increase of charge transport and therefore of current intensity

Effect of the pK2of the mediator and of solution pH

on the laccase-catalyzed oxidation of 4-methoxybenzylalcohol

So far we have shown that the laccase/phenol red couple is

a long-lasting reacting system that is able to perform as a laccase-mediator system In our search for other types of phenolic mediators, we tested a number of other phenolic structures, mostly antioxidants, by means of the benchmark oxidation of 4-methoxybenzylalcohol Most turned out to

be unable to perform as mediators (Table 1, entries 15–19), because they undergo rapid degradation We believe that the potential for the oxidative formation of highly conju-gated (and, hence, stabilized) oxyradicals in structures akin

to phenol red may be responsible for their peculiar behavior

We have provided some partial support to this view through the electrochemical experiment depicted in Fig 6 and described above, in the previous paragraph These consid-erations, combined with the fact that sulfonephthaleins, phenolphthaleins and related structures (Fig 2) cover a wide range of pK values for the dissociation of their phenolic moiety, and that the oxidation potential of phenols

is modulated by solution pH (at pH < pK) [29], led us

to expand on the subject of phenol red-like mediators All sulfonephthaleins exhibit some mediating effect, while aurintricarboxylic acid and chrome azurol S, which are the only structures carrying an electron-withdrawing group on the phenolic ring (Fig 2), are not even oxidized by laccase (as determined by UV-Vis spectrometry) The most suc-cessful mediator is dichlorophenol red, which is the most acidic (with a pK2of 5.74) [25] among the sulfonephthalein structures tested In fact, in Table 1, a correlation is observed between 4-methoxybenzyl alcohol conversion and the pK2 of the sulfonephthalein mediator It can be argued that at the reaction pH of 5.0, the lower the pK2of the mediator, the easier it is for laccase to oxidize it, as the fraction of sulfonephthalein that is present in its phenolate form (which is expected to be a better one-electron reductant with respect to laccase, and consequently to be more easily converted into the phenoxy radical by electron transfer) is greater In short, deprotonation of the phenolic moiety seems to be the factor that turns a phenol red-like structure from a somewhat slowly reacting laccase substrate

Fig 6 Cyclic voltammetry scans (m = 5 mVÆs)1) for 0.5 m M phenol red

at pH 7.4 in 0.5 M NaH 2 PO 4 /Na 2 HPO 4 buffer in the presence of 0 m M

(m), 2.5 m (d) or 30 m (j) 4-methoxybenzyl alcohol.

Fig 5 The radical mechanism of oxidation of a nonphenolic substrate

by a laccase->N–OH mediator system.

into an efficient phenolic laccase mediator Once again, we

resorted to electrochemical determinations in order to verify

whether there was any pH dependence of the Ep of

sulfonephthaleins, and, if any, whether it proceeded in the

same direction as the pH dependence of the

sulfonephtha-lein mediators in Table 1 By increasing the pH (from 4.0 to

7.4) at which the cyclic voltammetry scans were run (see the

Materials and methods), we found a decrease of Epfrom

0.84 to 0.68 V vs SCE for phenol red (compared with a

calculated increase in the phenolate/phenol ratio from

4· 10)4to 1), and from 0.81 to 0.71 V for dichlorophenol

red (phenolate/phenol ratio from 2· 10)2 to 45) These

increments are in keeping with those reported by Li &

Hoffman [29] for phenol and 2-chlorophenol, and support

the idea that a slow-reacting phenolic mediator, which by

virtue of its lower pK is present in solution in a more easily

oxidizable form, should be a better laccase mediator

Further support of this idea is provided by the pH

dependence of the laccase-mediating effect of phenol red

and dichlorophenol red (Table 1) Both sulfonephthaleins

are poor mediators at pH 3.5 compared with pH 5.0 On a

further increase of the pH solution (to a pH of‡6) the

conversion of 4-methoxybenzyl alcohol again decreases,

because P pinsitus laccase starts to lose its activity Hence,

at pH 5.0, a compromise is reached between activity of

the enzyme and partial (or substantial) deprotonation of the

mediator into a more oxidizable species

Conclusions

Phenol red is a nonhindered, highly soluble phenol that

gives rise to a long-lasting laccase-phenolic mediator

system It is an efficient mediator, compared with other

phenolic molecules [7], in that it does not need to be

present in large excess in order to obtain significant

conversion of laccase-resistant substrates This favourable

feature of phenol red is shared by other sulfonephthalein

phenols, whereas other phenols tested, which do not have

this structural motif, were rapidly degraded by laccase and

failed to mediate The peculiar behaviour of phenol red

(and related structures) may be the result of a combination

of several factors, such as (a) its phenoxy radical may be

longer lived than that of a simple phenol, because of

resonance stabilization, (b) this longer-living phenoxy

radical may give dimerization (a reaction generally

respon-sible for the instability of phenoxy radicals) less extensively

than other reactions, such as H-abstraction from a

substrate [this is supported by the observed regeneration

of phenol red, following its oxidation at the electrode, by

the presence of 4-methoxybenzyl alcohol (cf Figure 6)] and

(c) this phenoxy radical, as suggested by

spectrophoto-metric determinations and HPLC data, may, in turn, form

secondary species (which we did not attempt to identify)

that can also act as mediators; it is possible that the latter

phenomenon occurs to an even greater extent with those

mediators that need to be used in large excess with respect

to their substrate [7,9] In this study we also showed that

other structures based on the phenol red template can

mediate laccase catalysis towards recalcitrant substrates,

and that a correlation exists between their efficiency and

the acidity of their phenolic group In particular,

dichloro-phenol red, the most acidic of the series we tested, was the

most efficient at mediating the oxidation of 4-methox-benzyl alcohol This correlation can be rationalized, based

on electrochemical evidence and pH dependence of mediation efficiency, in terms of the larger fraction of the more easily oxidizable phenolate vs the slowly reacting phenol form of these sulfonephthaleins Clearly, phenol red

is only a modest model of the phenolic subunits of lignin Nevertheless, the new biogenic hypotheses that ascribe the reported ligninolytic activity of some fungi to the laccase-catalyzed formation of phenoxy radical species [1] from suitable phenolic fragments, begin to receive support here

Acknowledgements

Thanks are due to Novo Nordisk Biotech (Denmark) for their generous gift of laccase We also thank the EU project OXYDELIGN (grant QLK5-CT-1999-01277) for financial support.

References

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