Structure/Redox Potential Relationship of Simple Organic Compounds as

Potential Precursors of Dyes for Laccase-Mediated Transformation

Jolanta Polak and Anna Jarosz-WilkolazkaBiochemistry Dept., Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland

DOI 10.1002/btpr.713Published online October 11, 2011 in Wiley Online Library (wileyonlinelibrary.com).

The aim of this study was to examine the ability of an extracellular fungal laccase (LAC)to form colored products from simple non-colored organic precursors. Thirty different phe-nolic and non-phenolic precursors (o-, m-, and p-methoxy-, hydroxy-, sulfonic-, and amino-substituted) were tested as single and coupled substrates in a LAC-catalyzed oxidation. Thefindings show that LAC catalyzes the formation of colored products (from yellow/brown tored and blue) by oxidation of single substrates that are benzene derivatives containing atleast two substituents comprised of amino, hydroxy, and methoxy groups. All precursorswere tested by cyclic voltammetry and the correlation between their structure and redoxpotential, and the possibility of their transformation into colored products by fungal LACwas found. Colored products were yielded from single substrates possessing a value of theoxidation peak (Eo) lower than 1,150 mV vs. normal hydrogen electrode (NHE). Substrateswith an oxidation peak higher than 1,150 mV vs. NHE were transformed by LAC into co-lored compounds only in the presence of an additional precursor characterized by a lowvalue of Eo and the presence of reactive substituents such as methoxy, hydroxy, and aminogroups. Therefore, additional hydroxylation, methoxylation, and amination of phenolic andnon-phenolic substrates may represent a strategy to increase the range of these compoundsas potential dyes precursors. VVC 2011 American Institute of Chemical Engineers Biotechnol.Prog., 28: 93–102, 2012Keywords: laccase, redox potential, phenolic compounds, coupling reactions, dyes

Introduction

It is well known that fungi are able to synthesize coloredcompounds in their natural environment. Amongst the pro-duced molecules are carotenoids, melanins, flavonoids, qui-nones, and more specifically monascins, violacein, orindigo.1,2 However, there is a long way from Petri dishes inthe laboratory to the market place as only few syntheses areoperated at an industrial scale.3 An isolation of natural pig-ments and/or bioconversions of precursors to obtain naturalpigments are alternative biotechnological techniques thatcould achieve the environmentally friendly synthesis of dif-ferent and commercially valuable products.4

A promising candidate for this kind of enzyme-assistedbiotransformation is laccase (LAC).5–9 LAC (EC 1.10.3.2) isa copper-containing oxidase, which is widely distributed infungi,8,10,11 higher plants,12 some bacteria,13 and insects.14 Itis able to catalyze the oxidation of many different substrateswith the concomitant reduction of dioxygen to water and itsspecific affinity for oxygen as an electron acceptor is veryhigh.10 The most important factors limiting the efficiency ofLAC-mediated oxidation process is the value of redox poten-tial of copper in the T1 site of LACs active centre and thedifference between this potential and the redox potential ofreducing substrate.15,16 The high redox potential of the T1

copper in the active centre makes fungal LAC of special inter-

est in biotransformation processes.17 The specificity of LAC

towards its reducing substrates is rather low and therefore it

has the ability to oxidize many phenolic and non-phenolic

compounds.18 The possible use of this enzyme has been sug-

gested for biotransformation and decolorization of dyes,19

pulp delignification,20 oxidation of organic pollutants,21 micro-

bial transformation of natural products,22 and the development

of biosensors23–25 or biofuel cells.26,27

One of the main constraints in LAC-catalyzed bioconver-sions is the formation of oligomeric by-products.28 Neverthe-

less, some reports concerning the use of fungal LACs for thebioconversion of simple organic compounds into active com-

pounds imply the suitability of these enzymes as regioselec-tive reagents. A number of specific or selective reactions

have been reported, where LACs have been used to synthe-size products of commercial importance7 such as synthesis

of actinocin from 3-methyl-4-hydroxyanthranilic acid,29 con-version of alkaloids,30 or production of mithramicin.31

Lacccase has also been used to transform substrates intointensely colored products, which in some cases were cha-racterized by good dyeing properties. Dyes have beenformed from LAC-mediated transformations of substratesbelonging to the methoxy- and amino-phenolic derivatives orto the anthraquinone compounds.32–37

The aim of this study was to examine the ability of anextracellular LAC from the ligninolytic fungus Cerrena

Correspondence concerning this article should be addressed toJ. Polak at jpolak@poczta.umcs.lublin.pl.

VVC 2011 American Institute of Chemical Engineers 93

unicolor to form colored compounds from simple precursors.Screening of 30 different phenolic and non-phenolic deriva-tives such as o-, m-, and p-methoxy-, hydroxy-, sulfonic-,and amino-substituted were studied in the presence of LACin liquid media. The findings show that LAC catalyzes theoxidative coupling reactions between selected substrates pro-ducing colored compounds (from yellow by brown to redand blue) and the structures of these substrates and theirredox potentials highly affected those oxidation processes.The results of our work are useful because there are a limi-ted number of reports containing numerical data about influ-ence of oxidation potential of substrates on the possibility oftheir oxidation by LAC.16,38 Additionally, we investigatedand report here on the outcome of combining substrates.

Materials and Methods

Catalyst

Extracellular LAC from the white rot fungus C. unicolor(Fungal Collection of the Department of Biochemistry,UMCS, Lublin, Poland) was purified using a previouslydescribed procedure.11 LAC was stored frozen at a tempera-ture �18�C prior to use. After thawing, LAC was dilutedwith a tartrate buffer solution to prepare a working LACsolution with known activity. LAC activity was determinedby following the oxidation of 2,20-azino-bis(3-ethylbenzo-thiazoline-6-sulphonic acid) (ABTS).33 The reaction mixturecontained LAC in 100 mM Na-tartrate buffer pH 3 and

2.5 mM ABTS. Oxidation of ABTS was monitored spectro-photomerically at 414 nm (e414 ¼ 36,048 M�1 cm�1) andLAC activity was expressed in U L�1. One unit of LAC ac-tivity (U) corresponds to the amount of enzyme which oxi-dizes 1 micromole of ABTS per 1 min at 25�C.

Chemicals

All chemicals were of analytical grade and used withoutfurther purification (Table 1). Catechol, pyrogallol, 4-hydroxy-benzoic acid, 4-methoxyphenol, 4,5-dihydroxy-1,3-benzene-disulfonic acid, epinephrine, norepinephrine, N-(1-naphtyl)ethylendiamine, D-catechin, ABTS, and tartaric acid werepurchased from Sigma (USA, Germany); 4-aminophenol, 3-amino-4-hydroxybenzenesulfonic acid, anthranilic acid, 2-amino-3-methoxybenzoic acid, 4-methylaminobenzoic acid,anthranilamide, acetovanillone, 2-amino-4-methoxy-6-meth-ylpyrimidine, and 2-amino-4-methoxybenzothiazole werepurchased from Aldrich (USA, Germany, UK); hydroqui-none, 2-aminophenol, 2-methoxyhydroquinone, 3,4-dihy-droxybenzoic acid, 3-aminobenzoic acid, 4-aminobenzoicacid, acetosyringone, 3-(3,4-dihydroxyphenyl)-L-alanine,sodium-1-naphtyl phosphate, and anthraquinone 2-sulfonicacid sodium salt were obtained from Fluka (France, Swe-den, Italy); 3-amino-1,2,4-triazole was obtained from ICNBiomedical Inc. (USA).

All substrate solutions were prepared from 100 mM or 20mM stock solutions in proper solvent. The stock solutions of

Table 1. The List of Precursors Used in Micro-Plate Screening for LAC-Mediated Transformations into Colored Compounds and their

Electrochemical Properties

Chemicals Acronym Substituents

Potential (mV) KM

Eo Er (mM) k (nm)

Compounds very fast oxidized by LAC to colour products2-Amino-3-methoxybenzoic acid AMxBA 2-NH2, 3-OCH3, 1-COOH 552, 922 nf 1.73 4752-Methoxyhydroquinone MeHQ 4-OH, 2-OCH3 573 292 0.22 3704-Aminophenol 4Aph 1-OH, 4-NH2 599 535, 296 13.54 5104-Methylaminobenzoic acid PMAB 4-NHCH3, 1-COOH 611, 1062 nf 12.33 5122-Aminophenol 2Aph 1-OH, 2-NH2 675 nf 3.12 4203-Amino-4-hydroxybenzenesulfonic acid AHBS 4-OH, 3-NH2, 1-SO3H 717 96 0.18 436Pyrogallol Pyr 1,2,3-(OH)3 735, 1024* nf 0.28 417Catechol Cat 1,2-(OH)2 747 484 1.59 3923,4-Dihydroxybenzoic acid 3,4DHB 3,4-(OH)2, 1-COOH 756 484 2.13 400Norepinephrine Neph 2-OH, 4-CHOHCH2NH2 773 539 0.96 3872,20-Azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) ABTS 6,6‘-(SO3NH4)2 799, 1190 633 0.02 4153-(3,4-Dihydroxyphenyl)-L-alanine DHPAla 2-OH, 5-CH2CHNH2COOH 803 458 0.54 4004-Methoxyphenol 4MxPh 1-OH, 4-OCH3 897 365 0.66 360Epinephrine Eph 2-OH, 4-CHOHCH2NHCH3 914 204 0.43 487n-(1-Naphthyl) ethylendiamine NEDA 1-NHCH2CH2NH2 947 nf 0.12 460Acetosyringone ACS 2,6-(OCH3)2, 4-COCH3 960, 1194 nf 0.49 392D-Catechin CatN 3,30,40,5,7-(OH)5 1016 nf 1.04 3904,5-Dihydroxy-1,3-benzene-disulfonic acid dHBdSA 1,6-(OH)2, 2,4 -(SO2Na)2 1113† nf 14.39 372

Compounds very slow oxidized or non oxidized by LACHydroquinone HQ 1,4-(OH)2 731, 1067 136 ndAcetovanillone ACV 2-OCH3,4-COCH3 1139 nf ndAnthranilic acid AntAcid 2-NH2, 1-COOH 1161‡ 485‡ 234.8 4584-Aminobenzoic acid PABA 4-NH2, 1-COOH 1169 nf 21.21 4803-Aminobenzoic acid MABA 3-NH2, 1-COOH 1182 nf 4.37 380Anthranilamide AntAmi 2-NH2, 1-CONH2 1203 nf 34.54 3722-Amino-4-methoxybenzothiazole AMxBT 2-NH2, 4-OCH3 1245, 1637 nf nd4-Hydroxybenzoic acid PHBA 4-COOH 1302‡ 329‡ nd3-Amino-1,2,4-triazole 3ATZ 3-NH2 1390 458* ndAnthraquinone-2-sulfonic acid ASAS 2-SO3Na 135 �116 nd2-Amino-4-hydroxy-6-methylpyrimidine AHMP 4-OH, 2-NH2, 6-CH3 nf nf ndSodium-1-naphtyl phosphate SNP -OPO3HNa nf nf 307.2 505

*Very small peak. † The scan rate 25 mV s�1. ‡ The scan rate 100 mV s�1.nf, not found; nd, not determined.Oxidation peak (Eo) and reduction peak (Er) were obtained for the first scan at the speed 50 mV s�1 except of dHBdSA, AntAcid, and PHBA.

94 Biotechnol. Prog., 2012, Vol. 28, No. 1

2Aph and 4Aph were made in methanol. Ethanol was used asthe solvent in the case of Cat, HQ, Pyr, PHBA, 4MxPh,3,4DHB, AntAcid, MABA, PABA, AmxBA, PMAB, AntAmi,ACV, ACS, 3ATZ, NEDA, ASAS, and CatN. The final con-centration of alcohols in the reaction mixture did not exceed10% in micro-plate screening and 2% in a large volume syn-thesis. The stock solutions of MeHQ, dHBdSA, Neph, SNP,and ABTS were prepared using water purified with Milli-Qsystem (Millipore, USA). HCl or NaOH were added toimprove the solubilization of AHBS, Eph, DHP-Ala, orAHMP. The structures and acronyms of all compounds withfunctional substituents are summarized in Table 1.

Electrochemical measurements of redox potential

Cyclic voltammetry of the precursors were carried out for1 mM substrates in sodium tartrate buffer 0.1 M, pH 4.5,using M161 electrochemical analyzer (MTM-Anko, Poland).A one-compartment cell of 20 ml was used in all voltammet-ric studies and this cell consisted of a saturated Ag/AgCl/KClsat reference electrode, a platinum wire counter electrode,and 3 mm diameter graphite working electrode (all pur-chased from MTM-Anko, Poland). The potential wasscanned from –400 to 1500 mV vs. Ag/AgCl/KClsat afterholding the electrochemical system at the initial potential for10 s; the scan speed was varied from 25 to 100 mV s�1. Thegraphite working electrode was always thoroughly polishedwith alumina and rinsed with distilled water. All measure-ments were done in triplicate at room temperature.The measured potentials recorded vs. Ag/AgCl electrodewere corrected by 199 mV to the normal hydrogen electrode(NHE).

Determination of KM

The apparent binding constants (KM) for the precursor’soxidation by LAC were determined in Na-tartrate buffer pH4.5. Experimental data were analyzed according to theMichaelis–Menten equation by Lineweaver–Burk plots andnon-linear regression analysis with the program Origin 6.1.All measurements were done in triplicate.

Micro-plate screening

The experiments were conducted using microplates (96-well) with the reaction mixtures containing 1 U mL�1 ofLAC in 100 mM Na-tartrate buffer pH 4.5 and 1 mM ofeach substrate per well. The pH of 4.5 was chosen in thisstudy as the best for LAC-mediated transformation of chemi-cals (data not shown) based on the protonation of the phe-noxy and other substituted groups of the tested substrates. Acontrol without LAC was prepared to check for the autoxida-tion of substrates. Plates were incubated at temperature 28�Cunder shaken conditions (140 rpm) for 24 h. The evolutionof color formation as a function of time was recorded by vi-sual observation and using UV–vis spectrophotometer. Allmeasurements were done in triplicate.

Results and Discussion

Cyclic voltammetry of substrates

It was found (Table 1) that most of the tested substrateswere effective electron donors in the LAC-catalyzed reduc-tion of dioxygen to water and the electrochemical potential

of these phenolic and non-phenolic molecules was tested atthe graphite electrode. All the compounds generated well-shaped reversible, quasi-reversible, or irreversible signals. Inmost cases, they exhibited well-defined, irreversible oxida-tion peaks (Eo). Irreversible reduction peaks (Er) were alsoobserved for some compounds.

Table 1 presents Eo and Er values measured vs. an Ag/AgCl/KClsat electrode corrected to the NHE by the additionof 199 mV. Most precursors displayed the oxidation peak inthe potential range from 135 mV (for ASAS) to 1,637 mV(for AMxBT) and the reduction peak ranged from�116 mV (for ASAS) to 633 mV (for ABTS) for a scan rateof 50 mV s�1. Oxidized products formed during LAC-medi-ated oxidation of phenolic and non-phenolic compounds canpolymerize rapidly and this polymerization reaction com-petes with the reduction process.28 Therefore, in most of thecases the oxidation reaction was irreversible; hence, duringcyclic voltammetry measurements only the oxidation peakswere detected (Table 1).

In the case of all tested amino-substituted non-phenolicderivatives, one or two irreversible anodic peaks were gene-rated. Only for AntAcid and 3ATZ was an additional verysmall reversible cathodic peak observed. On the voltammo-gram of ABTS, two anodic peaks and only one cathodicpeak were recorded at the scan rate of 50 mV s�1, whichwas previously reported by Burbonnais et al.39 and Fernan-dez-Sanchez et al.40 At the scan rate of 250 mV s�1 thiscompound generates reversible signals with two anodic andtwo cathodic peaks. Similar quasi reversible signals at thescan rate of 50 mV s�1 with two anodic peaks and onecathodic peak were generated in the case of hydroquinone(Table 1).

Most substrates, especially those which were convertedinto colored products, were characterized in terms of theirapparent binding constant (KM) (Table 1). It was possible toobserve that the value of oxidation potential was correlatedwith the KM values of the precursors during LAC-mediatedoxidation. Compounds with the oxidation potential valuesbelow 1,113 mV (dHBdSA) were characterized by low val-ues of KM ranging from 0.18 mM (AHBS) to 14.39 mM(dHBdSA). These precursors contained at least two reactivesubstituents (hydroxy, amino, and methoxy groups), whichconfirms the participation of these substituents in the forma-tion of colored products. In this range of potential values itwas not possible to determine the value of KM only for HQdue to the lack of a colored product. The lowest KM valuewas determined for ABTS (0.02 mM) and this compoundhas been commonly used as a substrate in a LAC activityassay.33 Precursors for which the oxidation potentials werehigher than 1,139 mV (ACV) displayed very high valuesof KM or in some cases it was impossible to determine theKM value due to the formation of colorless products. Anexception was the precursor MABA for which the KM was4.37 mM.

LAC-catalyzed transformation of single compounds(homomolecular transformation)

Microplate-screenings of several different phenolic andnon-phenolic derivatives such as methoxy-, hydroxy-, car-boxy-, sulfonic-, and amino-substituted compounds wereconducted in the presence of LAC under buffered conditionsto assess their potential as dye precursors. In the literaturethere are some examples of colored products yielded from

Biotechnol. Prog., 2012, Vol. 28, No. 1 95

the LAC-mediated transformation of substrates with differentchemical structures. Amongst them are methoxy- and amino-substituted phenolic derivatives or anthraquinone com-pounds, which are also transformed to textile dyes.35–37

In this part of our work, selected substances were testedand the colored products resulting from the LAC-mediatedhomomolecular transformations were analyzed. During incu-bation with LAC most of the tested precursors were oxidized

by the enzyme in a very short time yielding reaction pro-

ducts that were endowed with intense colors. In particular,

the oxidation products of Pyr, dHBdSA, and CatN were

yellow while these from Neph and Eph were red (Table 2).

The oxidation of NEDA, AHBS, and AMxBA resulted in the

formation of orange-colored products. Brown products were

obtained during the oxidation of Cat and DHP-Ala, and

green products were formed from ABTS oxidation (Table 2).

A significant dependence was found between the structureof the precursor, the value of its oxidation potential (Eo),and the ability of LAC to transform the precursor into a co-lored product. Most of transformed precursors were phenolicderivatives and possessed at least one of the highly reactivesubstituents: -NH2, -OH, or -OCH3 (Table 1). The presenceof an additional -OH group in the aromatic ring of a precur-sor increased the possibility of its transformation into

Table 2. The Examples of Homotransformation of Single Precursor Which Gave Large Amounts of Stable Product About Interesting Color

During LAC-Catalyzed Transformation with the Maximum Wavelength Characterizing Each Product (in the brackets)

Products Coloration Precursors of Homomolecular Transformations

Yellow

CatN (390 nm) dHBdSA (430 nm) Pyr (414 nm)

Orange

NEDA (450 nm) AMxBA (436 nm) AHBS (436 nm)

Red

Eph (487 nm) Neph (387 nm)

Light rose

ACS (390 nm)

Green-blue

ABTS (635 nm)

Brown

DHP-Ala (450 nm) Cat (392 nm)

96 Biotechnol. Prog., 2012, Vol. 28, No. 1

colored product. For example, PHBA (4-OH) did not trans-form to a colored product whereas 3,4DHB (3,4-(OH)2)yielded a colored product. It should be noted here that in thecase of a LAC-modified electrode used as a biosensor,increased sensitivity was observed for diphenolic compoundsin comparison to their respective monophenols.23 An addi-tional -OH group, with at least one single pair of electronson the atom adjacent to the aromatic ring, allows the -OHgroup to be oxidized efficiently by LAC. Various LACs ca-talyze the removal of a hydrogen atom from the hydroxylgroup of ortho- and para-substituted mono- and polyphenolicsubstrates.23,41 In the present study, all compounds possess-ing at least two hydroxyl substituents displayed the oxidationpeak in the range from 731 mV (HQ) to 1,113 mV(dHBdSA) endowing them as accessible substrates for LAC-mediated transformations.

All tested NH2-substituted phenols were transformed byLAC to colored products. Among them were precursors thatdisplayed an oxidation peak that ranged from 599 mV to914 mV such as 4Aph, 2Aph, AHBS, DHPAla, Eph, andNeph. One of them, namely AHBS, was transformed byLAC into a phenoxazinone compound, which has good dye-ing properties.33

The absence of hydroxyl substituents highly affected the

possibilities of transformation of non-phenolic NH2-substi-

tuted compounds by LAC. In most cases, these compounds

lacking a -OH group were not oxidized by LAC to colored

products in spite of the presence of additional functional

groups. Amino-substituted precursors with ACOOH group

(PABA, MABA, and AntAcid) were transformed to slightly

colored products over a long oxidation time. Moreover

these compounds are characterized by very high values of

redox potentials such as 1,161 mV for AntAcid. Non-phe-

nolic precursors with a low-value of redox potential

(AMxBA, PMAB, and NEDA) were transformed by LAC

into colored products, probably because they possess addi-

tional reactive substituents such as methoxy (AMxBA),

amino (NEDA), and methylamino (PMAB) groups. The

presence of a -OCH3 group allows LAC to transform

methoxylated phenolic precursors more effectively, where

4MxPh (4-OCH3), MeHQ (2-OCH3), ACV (1-OCH3,

3-OCH3), and ACS (1-OCH3, 3,5-(OCH3)2) provide exam-

ples. This is in agreement with the oxidation of 2,6-dime-

thoxyphenol by LAC, which is the best substrate for many

LACs of different origin.23

The values of Eo for substrates efficiently transformedby LAC to colored compounds ranged from 552 mV(AMxBA) to 1,113 mV (dHBdSA), which is comparableto the values of redox potentials of typical fungal LACs(from 460 to 790 mV)18 and also with the redox potentialof C. unicolor LAC (750 mV).42 Substrates with a valueof Eo higher than 1,203 mV (AntAmi, AMxBT, PHBA,3ATZ) and substrates not displaying an oxidation peak(AHMP, SNP) or with very low value of Eo (ASAS) werenot oxidized by LAC into colored products. Our resultsconfirm the relationship between the redox potential of thesubstrates and the efficiency of their transformations byLAC, which has been previously reported by Zille et al.(2004).43 They found a linear relationship between the re-dox potential of the azo dyes and the efficiency of theirdecolorization by LAC. Dyes with a less positive anodicpeak were more prone to oxidative degradation by fungalLAC.43

Only in the case of a few precursors (CatN, NEDA, andDHP-Ala) was the production of colored product residuesobserved after a longer time of incubation with LAC (morethan 24 h). Two precursors (2Aph and 4Aph) autoxidizedunder the test conditions without the addition of LAC;hence, they were rejected from the next step of our study.

Laccase-catalyzed transformation of coupled substrates(heteromolecular transformation)

Based on the results from the first step of our study (homo-molecular transformations), 28 different phenolic and non-phe-nolic derivatives were tested in terms of their combinatorialpossibilities through microplate-screening. The colored pro-ducts formed by the LAC-mediated oxidatively heteromoleculartransformations were then analyzed. Six combinations of pro-duct colorations obtained after the LAC-mediated transforma-tion of colorless precursors are possible (Table 3). Within thisset, three combinations (IV, V, and VI) seem to be the mostpreferable as possibly new dyes. The combination IV relates tothe possibility of forming a new colored products from high-value redox potential precursors (not oxidized by LAC alone)in the presence of easily oxidized additional precursor such asABTS and Cat. In the combination V are two different precur-sors that are transformed into a colored product both duringthe homomolecular and heteromolecular transformationsmediated by LAC, but different colors of products wereobserved in these two transformations. In this group, veryinteresting results were obtained for precursors containingANH2, AOH, and AOCH3 substituents such as AMxBA, Cat,PMAB, NEDA, and 4MxPh. In the combination VI twodifferent precursors are not transformed into a colored productduring homomolecular transformation mediated by LAC, but itis possible to obtain colored products during their LAC-media-ted heteromolecular transformations (Table 3). In our study,we did not find any precursors transformed in this type ofcombination.

Table 4 presents combinations of uncolored precursorsthat yielded products with interesting colors after a hetero-molecular transformation mediated by LAC. For example, inthe presence of LAC the mixture of AMxBA and dHBdSAresulted in a red product in comparison to orange and yellowproducts that were yielded by the single homotransforma-tions of the respective single precursors (Tables 2 and 4).Two other precursors, AMxBA (1-COOH, 2-NH2, 3-OCH3)and PMAB (1-COOH, 4-NHCH3), which possess three andtwo different substituents, respectively, were transformed tointeresting colored products after the addition of many otherdifferent phenolic and non-phenolic compounds as co-sub-strates (Table 4). Dark red and violet products were obtainedwhen PMAB was heterotransformed in a mixture withAntAmi, 4MxPh, and 3,4DHB. Furthermore, the addition ofCat (-OH group in ortho position) to a mixture containingprecursors with ANH2 or ANHR groups such as MABA(3-NH2), PABA (4-NH2), and PMAB (4-NHCH3) resulted inthe appearance of red colored products, clearly differentfrom the color of the products obtained during homotransfor-mation of these precursors without addition of Cat. The redcolored product could be the result of the coupling reactionbetween radicals formed from catechol by the action of LACand non-transforming aminobenzoic compounds having ahigh oxidation potential value (MABA, PABA). Theseresults indicate that catechol can be involved in couplingreactions with aromatic amines, which was previously

Biotechnol. Prog., 2012, Vol. 28, No. 1 97

reported by Zille et al.44 It is noteworthy that precursorscharacterized by a high value of oxidation peak (AntAmi,AntAcid) and not transformed to colored products by LACduring homomolecular transformations gave intensivelycolored products in the presence of additional substratescontaining hydroxyl substituents (e.g., 4MxPh and MeHQ;Table 4).

Overall, the LAC-mediated oxidation of substrates in thepresence of co-substrates yielded colored products that were

significantly different from those obtained when oxidized indi-

vidually by LAC (Table 3, Figure 1). Only in a few cases

were the colors of products formed from two precursors

approximately the same (but sometimes more intense) as the

colors of products produced in the control variants (LAC-

Table 3. Possible Combination of Products Coloration After LAC-Catalyzed Homo- and Heteromolecular Transformations of Precursors A and B

Combination

Coloration of Product

Homomolecular Transforma-tion of Precursor A Homomolecular Transformation of Precursor B

Heteromolecular Transformation of Mixture ofPrecursor A and Precursor B

I

Colorless A Colorless B Colorless A/B

AMxBT AHMP

II

Colorless A Colored B Colored B

AntAmi AHBS

III

Colored A Colored B Colored A/B

dHBdSA AHBS

IV

Colorless A Colored B Colored C

PHBA ABTS

V

Colored A Colored B Colored C

Cat NEDA

VI Colorless A Colorless B Colored C Not found

98 Biotechnol. Prog., 2012, Vol. 28, No. 1

mediated oxidation of single substrates). This type of transfor-

mation (combinations II, III; Table 3) is represented by var-

ious combinations of precursors like 3,4DHB and dHBdSA,

dHBdSA and AntAcid, HQ and PABA, 3,4DHB and MeHQ.

LAC mediated transformation of substrates in the presenceof ABTS

In the case of ABTS, a well-known mediator in LAC-mediator systems,45,46 the characteristic green-blue color ofABTS radicals (ABTS.þ) was observed in the mixture withother co-substrates during the first one or two hours of incu-bation with LAC (Table 5). After this time, the green-bluecolor of some transformation mixtures disappeared andchanged into a light pink or violet color in the presence of-NH2 derivatives (AntAcid, AntAmi, and MABA). Alternateoutcomes were a change to a yellow color when AMxBT

was present or to a brown color in the presence of AHMP,Cat, D-CatN, DHP-Ala, 3,4 DHB, 4MxPh, PABA, PMAB,and Pyr. During the oxidation of the remaining precursors byLAC in the presence of ABTS (such as ACS, ACV, 3ATZ,and AMxBA), a very stable green-blue color of transforma-tion mixtures were observed (Figure 1). In the case of thesetransformation mixtures, LAC probably did not transform theaforementioned precursors using ABTS as mediator, or elsethe products of these transformations were also green-blue.

The developed colors of products were relatively stable overone week (data not shown). The observed colors and spectralpeaks (Table 3, Combination IV) from the substrate mixtureslikely correspond to the presence of a new product obtainedfrom ABTS as the first substrate and from other phenolic com-pounds used as the second substrate. Such an outcome has alsobeen described by Bourbonnais et al.47 Campos and Lissi48

described the kinetics of the reaction between ABTS derived

Table 4. Coupling Reaction of Single Precursors During LAC-Catalyzed Heteromolecular Transformations (Combination IV and V) with the

Maximum Wavelength Characterizing Each Product (in the brackets)

Products Coloration Precursors of Heteromolecular Transformation

Orange

4MxPh þ PABA (440 nm) 4MxPh þ AntAmi (440 nm) AMxBA þ AntAmi (435 nm)

Red

AMxBA þ dHBdSA (490 nm) Cat þ PABA (495 nm) Cat þ PMAB (520 nm) AntAmi þ PMAB (520 nm)

Violet

PMAB þ 4MxPh (540 nm) PMAB þ 3,4 DHB (535 nm)

Green-grey

Cat þ NEDA (650 nm)

Brown

NEDA þ MABA (450 nm) NEDA þ DHP-Ala (450 nm)

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radical cations and phenols and they suggested that incompleterecovery of ABTS radicals occurs. Our results suggest that insome cases, ABTS radicals could undergo coupling reactionswith radical forms of simple phenolic compounds like SNP,MABA, and PHBA (used as co-substrates) to produce newmolecules with interesting colors such as violet, violet andblue, respectively, (Table 5, Figure 1), which is consistent withthe results described by Matsumura et al.49 In these cases,ABTS can be engaged in LAC catalyzed reactions both as amediator and as co-substrate. In the present screening, ABTSwas involved in the coupling reaction with two substrates,AMxBT and PHBA, characterized by high values of Eo (1245mV and 1302 mV, respectively), which indicates the ABTS-mediated oxidation of compounds with high redox potentials.The reactions of ABTS-derived radicals with simple phenols,polyphenols, and amino acids exhibit very complex kinetic pro-files and there is a lack of a relationship between the rate lawand stoichiometric factors.48,50 Recently, Osman et al.51 foundthat ABTS-derived radicals were not as stable as usuallyclaimed because they could be degraded upon interaction withpolyphenols such as (þ)-catechin and (�)-epicatechin. Theidentification of these degradation products could explain thepuzzling aspects of the kinetic data regarding the reaction ofABTS radicals with simple phenols and polyphenols reportedin the literature.48,50 ABTS can play two roles, firstly as themediator in LAC-catalyzed transformations and secondly as thesource of ABTS-derived oxidation products, which possiblyreact with the other substrates to give new molecules.45,46

Conclusions

Our work is a preliminary approach to estimate the capa-bilities of LAC from C. unicolor to be used as biocatalyst indyes synthesis. Thirty different phenolic and non-phenolicnon-colored precursors were tested as single and coupledsubstrates for oxidation by LAC. We investigated the rela-tionship between the values of anodic peak potential (Eo) ofthe substrates, their structures, and the possibilities of theirtransformations by fungal LAC into colored products. Sub-strates characterized by low values of oxidation peak weremore easily transformed. The presence of additional substitu-ents (belonging to the methoxy, hydroxy, and amino groups)in aromatic ring of substrates was enhanced the formation of

colored products during LAC-catalyzed homo- and hetero-molecular transformations. Moreover, substrates with veryhigh oxidation peaks were transformed by LAC into coloredproducts only in the presence of additional precursors, play-ing the role of mediators or co-substrates. Hydroxylation,

Table 5. Coupling Reaction of Single Precursors with ABTS During

LAC-Catalyzed Heteromolecular Transformation (Combination IV)

with the Maximum Wavelength Characterizing Each Product (in the

brackets)

Products colorationPrecursors of Heteromolecular Transforma-

tion with ABTS

Violet

SNP (545 nm)

Blue

PHBA (588 nm)

Light pink

AntAmi (490 nm) AntAcid (490 nm)

Yellow

AMxBT (390 nm)

Figure 1. Colored products obtained from biotransformations of selected precursors using LAC from Cerrena unicolor.

100 Biotechnol. Prog., 2012, Vol. 28, No. 1

methoxylation, and amination of various phenolic and non-phenolic substrates, may represent a strategy to increase therange of these compounds as potential dyes precursors.

One advantage of using LAC-mediated transformation forgenerating potentially new colored molecules is the rapidreaction results in the synthesis of generally stable products.Another advantage of this synthesis is its easiness and theuse of oxygen as the ‘‘clean oxidant.’’ Considering the smallquantities of enzyme employed and possible application ofimmobilized LAC, the LAC-mediated transformation of sim-ple compounds as potential dyes’ precursors could prove tobe very effective.

Acknowledgment

This work was partially supported by SOPHIED (NMP2-CT-2004-505899) and the National Science Centre (6330/B/P01/2011/40).

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Manuscript received Jun. 9, 2011, and revision received Aug. 29, 2011.

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