doi:10.1046/j.1432-1033.2002.03256.x
Eur. J. Biochem. 269, 5330–5335 (2002) (cid:2) FEBS 2002
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` (cid:1)La Sapienza(cid:2), 00185 Roma, Italy
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 (cid:1)oxidation shuttles(cid:2) 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.
laccase; phenols;
lignin degradation; HPI;
Keywords: TEMPO.
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
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).
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 (cid:1)non-natural(cid:2) 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
Fig. 1. Catalytic cycle of a laccase-mediator oxidation system.
(cid:1)La Sapienza(cid:2),
A conceivable role of the mediator could be that of a sort of (cid:1)electron shuttle(cid:2) 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 (cid:1)natural(cid:2) 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
Correspondence to C. Galli, Dipartimento di Chimica and Centro CNR Meccanismi di Reazione, Universita` 00185 Roma, Italy. Fax: + 39 06 490421, E-mail: carlo.galli@uniroma1.it Abbreviations: HPI, 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)
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radical role of the oxidized HPI would be comparable to the (cid:1)natural(cid:2) 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 (cid:1)physiological(cid:2) 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].
suitable 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].
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.
Fig. 2. Oligomeric compounds 1–5.
M A T E R I A L S A N D M E T H O D S
Laccase
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
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 A280/A610 of 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 by 1H NMR and HPLC.
Spectrophotometric determination of laccase activity
M
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 )1Æcm)1. ABTS (Aldrich) was recrystallized 3.15 · 10)4
the O2-laccase-HPI (A) and O2-laccase- Fig. 3. Mechanisms of TEMPO (B) oxidation of substrates 1–5 (Fig. 2).
5332 F. d’Acunzo et al. (Eur. J. Biochem. 269)
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diluted in the mobile phase and filtered through 0.2 lm (Superchrom Varisep) prior to Teflon syringe filters analysis.
Product analysis: liquid mass spectrometry (LC-MS)
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 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
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-mM stock 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 M citrate 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.
Solubility of substrates
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 0.1 M citrate 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.
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 lM can 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.
HPLC determination of substrate conversion
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
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)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
Table 1. Percent of substrate metabolized by laccase or laccase/mediator systems with and without an organic cosolvent. Substrate recovery was determined by HPLC.
No mediator HPI TEMPO
Substrate Buffer Buffer/dioxane Buffer/dioxane Buffer Buffer/dioxane Buffer
100 33 18 0 0 100 96 55 0 0 – – 20 0 0 – 100 60 0 0 – – 32 6 0 – 96 95 87 40 1 2 3 4 5
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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].
the formation of
(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- the TEMPO-substrate adduct ular, (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.
a M stands for molecular ion; A, B and C are the fragments rep- resented in the Product column.
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
Table 2. LC-MS data for the main product detected in the reaction mixture from the laccase-catalysed oxidation of substrates 1 and 2.
Fig. 5. Oxidation of substrates 1–5 by O2-laccase and O2-laccase-HPI. Phenol coupling with loss of formaldehyde (A) and ring-opening (B). Fig. 4. Expansion of 1H-NMR spectra run in dimethylsulfoxide-d6 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.
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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 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, the 1H- 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.
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 HPI, 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.
R E F E R E N C E S
1. Schoemaker, H.E. (1990) On the chemistry of lignin biodegrada- tion. Trav. Chim. Pays-Bas 109, 255–272.
(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 (by 1H-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].
2. Tien, M. & Kirk, T.K. (1983) Lignin-degrading enzyme from 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 H2O2- dependent oxidases from ligninolytic cultures of Phanerochaete chrysosporium. FEBS Lett. 169, 247–250. 4. Messerschmidt, A. (1997) Multi-Copper Oxidases, World Scienti-
C O N C L U S I O N S
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.
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
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 methoxyhydroquinones by Laccase from Pleurotus eryngii. Appl. Environ. Microb. 66, 170–175.
Oxidation of phenols (Eur. J. Biochem. 269) 5335
(cid:2) FEBS 2002
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. 8. Kersten, P.J., Kalyanaraman, B., Hammel, K.E., Reinhammar, B. & Kirk, T.K. (1990) Comparison of lignin peroxidase, horseradish peroxidase and laccase in the oxidation of methoxybenzenes. Biochem. J. 268, 475–480.
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. 9. Bourbonnais, R. & Paice, M.G. (1990) Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation. FEBS Lett. 267, 99–102.
21. Banci, L., Ciofi-Baffoni, S. & Tien, M. (1999) Lignin and Mn peroxidase-catalysed oxidation of phenolic lignin oligomers. Bio- chemistry 38, 3205–3210. 10. Bourbonnais, R. & Paice, M.G. (1992) Demethylation and delignification of kraft pulp by Trametes versicolor laccase in the presence of ABTS. Appl. Microbiol. Biot. 36, 823–827.
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. 11. Bourbonnais, R., Paice, M.G., Freiermuth, B., Bodie, E. & Borneman, S. (1997) Reactivities of various mediators and laccases with kraft pulp and lignin model compounds. Appl. Environ. Microb. 63, 4627–4632.
23. Sealey, J., Ragauskas, A.J. & Elder, T.J. (1999) Investigations into laccase-mediator delignification of kraft pulps. Holzforschung 53, 498–502. 12. Crestini, C. & Argyropoulos, D.S. (1998) The early oxidative biodegradation steps of residual kraft lignin models with laccase. Bioorgan. Med. Chem. 6, 2161–2169.
24. Ueda, C., Noyama, M., Ohmori, H. & Masui, M. (1987) Reactivity of phthalimide-N-oxyl: a kinetic study. Chem. Pharm. Bull. 35, 1372–1377.
13. Hilde´ n, L., Johansson, G., Pettersson, G., Li, J., Ljungquist, P. & Henriksson, G. (2000) Do the extracellular enzymes cellobiose dehydrogenase and manganese peroxidase form a pathway in lignin biodegradation? FEBS Lett. 477, 79–83. 25. de Nooy, A.E.J., Besemer, A.C. & van Bekkum, H. (1996) On the use of stable organic nitroxyl radicals for the oxidation of primary and secondary alcohols. Synthesis 1153–1174. in, e.g.
26. Cecchetto, A., Fontana, F., Minisci, F. & Recupero, F. (2001) Efficient Mn-Cu and Mn-Co-TEMPO catalysed oxidation of alcohols into aldehydes and ketones by oxygen under mild con- ditions. Tetrahedron Lett. 42, 6651–6654. Enzymatic (2000) 14. Deussen, H.-J. & Johansen, C. antimicrobial composition, useful laundries, cosmetic products, and contact lenses, comprises a phenol oxidising enzyme system and an N-hydroxyanilide enhancing agent. PCT Interna- tional Patent WO 0027204, A1. European Patent Office, Munich, Germany.
27. De Mico, A., Margarita, R., Parlanti, L., Vescovi, A. & Pianca- telli, G. (1997) A versatile and highly selective hypervalent iodine (III)/TEMPO-mediated oxidation of alcohols to carbonyl com- pounds. J. Org. Chem. 62, 6974–6977. 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. 28. Wolfenden, B.S. & Willson, R.L. (1982) Radical cations as reference chromogens in kinetic studies of one-electron transfer reactions: pulse radiolysis studies of ABTS. J. Chem. Soc., Perkin Trans. 2, 805–812. 29. Dahwan, B. & Gutsche, C.D. (1983) Calixarenes. 10. Oxacalix- arenes. J. Org. Chem. 48, 1536–1539. 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.
30. Minisci, F., Punta, C., Recupero, F., Fontana, F. & Pedulli, G.F. (2002) Aerobic oxidation of N-alkylamides catalysed by N-hydroxyphthalimide under mild conditions. J. Org. Chem. 67, 2671–2676. 18. Fritz-Langhals, E. & Kunath, B. (1998) Synthesis of aromatic aldehydes by laccase-mediator assisted oxidation. Tetrahedron Lett. 39, 5955–5956.
