transformation of halogenated pesticides by versatile peroxidase from bjerkandera adusta

Enzyme and Microbial Technology 36 (2005) 223–231

Transformation of halogenated pesticides by versatile peroxidasefromBjerkandera adusta

Gustavo Davila-Vazqueza, Raunel Tinocoa, Michael A. Pickardb, Rafael Vazquez-Duhalta,∗a Instituto de Biotecnologia-UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62250, Mexico

b Department of Biological Sciences, CW 405 Biological Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

Received 9 January 2004; received in revised form 8 July 2004; accepted 14 July 2004

Abstract

Purified versatile peroxidase (VP) from the white rot fungusBjerkandera adustaUAMH 8258 was used to study the transformation ofseveral pesticides, including some as highly halogenated as the wood preservative pentachlorophenol (PCP). From the 13 pesticides assayed,dichlorophen, bromoxynil and PCP were transformed by VP in the presence and in the absence of manganese(II). For all the pesticidestransformed, the activity was higher in the absence of Mn(II) at pH 3 than in the presence of Mn(II) at pH 4. Catalytic constants (k )i d3 ed thep hlorophenw ases, wef formation oft©

K

1

tcitgstbi

rc

andand

edoly-latedition,hy-

paperoc-restedi-

da-an-

eak-aveotics,CBs,

0d

cat

n the absence of Mn(II) at pH 4 were 194 and 409 min−1 for dichlorophen and bromoxynil, respectively. TheKM values were 32 an1�M for the pesticides and 26 and 19�M for the hydrogen peroxide, respectively. Analysis of reaction products by GC–MS showresence of 2,3,5,6-tetrachloroquinone among the products from pentachlorophenol oxidation, while the main product from dicas 4-chlorophenol-2,2′-methylenequinone. Several polymers were obtained from the peroxidase oxidation of bromoxynil. In all c

ound dehalogenation reactions mediated by the versatile peroxidase. The importance and potential uses of the enzymatic transhese halogenated toxic compounds is discussed.

2004 Elsevier Inc. All rights reserved.

eywords: Bjerkandera adusta; Bromoxynil; Dehalogenation; Dichlorophen; Pentachlorophenol; Versatile peroxidase

. Introduction

Pesticides are the only chemicals deliberately made to beoxic and introduced directly into the environment. Pesticidesan be absorbed through the skin, swallowed or inhaled. Dur-ng application pesticides drift and settle on ponds, laundry,oys, pools and furniture. Only 5% of pesticides reach the tar-et organism. The rest runs off into water or dissipates in theoil or air. Drift from landscape application ranges from 12 fto 14.5 miles[1]. More serious effects appear to be producedy direct inhalation of pesticide sprays than by absorption or

ngestion of toxins[2].Large amounts of halogenated phenols are produced and

epresent a public health risk. The world production of theseompounds was estimated in 1986 to 100 thousands tonnes

∗ Corresponding author. Tel.: +52 777 329 1655; fax: +52 777 317 2388.E-mail address:[email protected] (R. Vazquez-Duhalt).

[3]. They are currently used as herbicides, insecticidesfungicides, and in addition as pharmaceuticals, biocidesanthraquinonic dyes[4]. Pentachlorophenol, which was usas a wood preservative either with or without the pcyclic aromatic hydrocarbons in creosote, has accumuin the environment as a result of past practices. In addchlorophenols, chloroguaiacols, chlorolignins and chlorodrocarbons are produced from the bleaching process ofpulp[5]. Remediation of these relatively toxic compoundscurs only slowly under natural conditions and there is intein enhancing the removal of these compounds by bioremation using white rot fungi.

White rot fungi produce a number of extracellular oxitive enzymes including laccase, lignin peroxidase and mganese peroxidase, which are normally involved in the brdown of the plant structural material lignin. These fungi halso been shown to metabolize a large number of xenobiincluding PAHs, chlorinated phenols and pesticides, P

141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2004.07.015

224 G. Davila-Vazquez et al. / Enzyme and Microbial Technology 36 (2005) 223–231

dioxins, organophosphorus compounds, nitrotoluenes, chlo-ranilines, dyes and other compounds of environmental con-cern[5,6]. Manganese peroxidase (MnP) is a heme glycopro-tein enzyme first described fromPhanerochaete chrysospo-rium to require manganese(II) for its activity. Like ligninperoxidase, MnP is produced by many ligninolytic fungi in-cludingP. chrysosporium[7], Phlebia radiata[8], Ceripo-riopsis subvermispora[9], Nematoloma frowardii[10,11],Pleurotus eryngii, Pleurotus ostreatus, Pleurotus pulmonar-ius [12] andBjerkandera adusta[13–15]. Manganese perox-idases from white rot fungi have been used in the studies ofbiodegradation of lignin[16], polycyclic aromatic hydrocar-bons[15,17,18], humic acids[19] and synthetic dyes[20].The MnP isoenzymes fromP. chrysosporium[21] mediatedtheir activity through the production of freely diffusible man-ganic ions, which act as the ultimate oxidant: no activitywas ascribed to this enzyme in the absence of manganousions.

However, lignin-degrading strains ofP. eryngiihave beenshown to produce a peroxidase that can both oxidize Mn(II)to Mn(III), but can also carry out Mn(II)-independent ac-tivity on some aromatic substrates[22]. A similar “hybrid”manganese–lignin peroxidase or versatile peroxidase (VP)was also described inBjerkanderasp. BOS55 able also tooxidize various phenolic and nonphenolic substrates such as2 hol,i lsobP

e2 tedgdc2p ,4-q ymef o-m ab-s ceo prod-u on ofp

2

2

-t ainedepa ) of1 -t se

(LiP) of 7.1 U/mg protein measured as veratryl alcohol oxi-dation (see “enzyme assays” for details).

The halogenated pesticides: 3,5-dibromo-4-hydroxy-benzonitrile (Bromoxynil); 4-(2,4-dichlorophenoxy)butyricacid (2,4-DB); 3,6-dichloro-o-anisic acid (Dicamba);2,2′-methylenebis[4-chlorophenol] (Dichlorophen); 2-tert-butyl-4,6-dinitrophenol (Dinoterb); 3-(3,4-dichlorophenyl)-1,1-dimethylurea (Diuron); 2-methyl-4,6-dinitrophenol(DNOC); 3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea(Linuron); 4-amino-3,5,6-trichloropyridine-2-carboxylate(Picloram); 3′-4′-dichloropropion anilide (Propanil) werepurchased from Sigma–Aldrich Co. (St. Louis, MO).5-Chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxy benza-mide (Niclosamide) and pentachlorophenol (PCP) wereobtained from Chem Service (West Chester, PA).O-(2,4-dichlorophenyl)-O,O-diethyl phosphorothioate (Dichlofen-thion) was from Ultra Scientific (North Kingstown, RI). Allthese pesticides (Fig. 1) were purchased with the highestavailable purity. Malonic acid was obtained from SigmaChemical Co. (St. Louis, MO). Hydrogen peroxide wasfrom Aldrich Chemical Co. (Milwaukee, WI). HPLC-gradeorganic solvents were from Fisher Scientific (Springfield,NJ). All the other chemicals were obtained from J.T. Baker(Phillipsburg, NJ) as reagent grade.

2

t m-p d0 (pH4a e-t aseaf oltm ci-nt

2

d in4 ned3 ce-te y, thep mMM fferp per-f ogenp resso ys-t lumn( ).

,6-dimethoxyphenol, guaiacol, ABTS and veratryl alcon the absence of Mn(II)[13]. Versatile peroxidases have aeen reported inP. eryngii[20,23–25], P. pulmonarius[26],. ostreatus[27], as well as inB. adusta[13–15,20].

Cultures of P. chrysosporiumare able to metaboliz,4-dichlorophenol, 2,4,6-trichlorophenol, polychlorinauaiacols and different chlorinated vainillins[28]. In ad-ition, extracellular peroxidases, LiP and MnP, fromP.hrysosporiumoxidized in vitro 2,4-dichlorophenol[29],,4,5-trichlorophenol[30], 2,4,6-trichlorophenol[31] andentachlorophenol[32], producing their corresponding 1uinones. Here, we demonstrate the ability of the VP enz

rom B. adustaUAMH 8258 to oxidize dichlorophen, broxynil and pentachlorophenol optimally at pH 4 in the

ence of Mn(II) with lower activity at pH 3 in the presenf manganese. The chemical analysis of the reactioncts showed that VP catalyze an oxidative dehalogenatiesticide molecules.

. Materials and methods

.1. Chemicals

Versatile peroxidase fromB. adustaUAMH 8258 was obained and purified to a single band on a Coomassie-stlectrophoresis gel as described previously[15]. The purifiedreparation showed Reinheitzahl ratio (Rz) (A403/A280) of 2.5,nd specific activities for manganese peroxidase (MnP05 U/mg protein measured by the H2O2-dependent forma

ion of Mn(III)–malonate complex, and for lignin peroxida

.2. Enzyme assays

Manganese peroxidase activity was measured at 25◦C byhe H2O2-dependent formation of Mn(III)–malonate colex at 270 nm (ε = 11,590 M−1 cm−1). Reactions containe.5 mM manganous sulfate in 50 mM malonate buffer.5), and the reaction was started by the addition of H2O2 tofinal concentration of 0.1 mM[33]. Protein content was d

ermined with the Bio-Rad protein reagent. Lignin peroxidctivity was estimated by the method of Tien and Kirk[34]

ollowing the H2O2-dependent oxidation of veratryl alcoho veratraldehyde (ε310= 9300 M−1 cm−1) at 25◦C. Reactionixtures contained 4 mM veratryl alcohol in 40 mM sucate buffer pH 3, and were initialized by the addition of H2O2

o a final concentration of 0.4 mM.

.3. Pesticide transformations

Enzymatic pesticide transformations were performe-mL glass vials. The reaction mixture (1 mL) contai0–50�M pesticide in a medium containing 10% (v/v) a

onitrile in malonate buffer 50 mM, and from 6 nM to 2�Mnzyme preparation. For manganese peroxidase activitH of malonate buffer was adjusted to 4.0 and 0.5nSO4 was added. For lignin peroxidase activity, the buH was 3.0 in the absence of Mn(II). Reactions were

ormed at room temperature and started by adding hydreroxide to a final concentration of 0.1 mM. The progf the reaction (5–10 min) was followed by a HPLC s

em (Perkin-Elmer) equipped with a reverse phase co100 mm× 2.1 mm) Hypersil ODS 5�m (Hewlett-Packard

G. Davila-Vazquez et al. / Enzyme and Microbial Technology 36 (2005) 223–231 225

Fig. 1. Chemical structure o

UV detection of the eluted compounds was performed with adiode array UV-detector (Perkin-Elmer 235C). Mobile phaseconsisted of acetonitrile:water mixtures from 30 to 50% (v/v)according the pesticide analyzed. The pesticide transforma-tion rates were estimated by the decrease in their peak area a

t kin-E thes sub-sw tions

f halogenated pesticides.

t

heir maximum UV absorbances with a Turbochrom (Perlmer) workstation after calibration with standards, andpecific activity was expressed as moles of transformedtrate per mol of peroxidase per min (min−1). All reactionsere done in triplicate, and the mean and standard devia


are reported. Controls without enzyme and/or without hy-drogen peroxide were carried out under the same incubationconditions.

The enzymatic oxidations of dichlorophen, bromoxyniland PCP by the Mn(II)-dependent and independent activitiesof VP were evaluated in a pH range from 2 to 7 in a 50 mMsodium acetate buffer. Kinetic constants,kcat andKM, weredetermined in the reaction system without Mn(II) (pH 4.0),in substrate saturation conditions. The experimental data wasplotted and the constants calculated by using the EnzFittersoftware (Biosoft, Cambridge, UK).

2.4. Products identification

In order to have enough material for product identification10-mL reactions were carried out in a 10% (v/v) acetonitrile-50 mM acetate buffer (pH 4.0) containing 400�M pesti-cide as substrate, 0.1 mM hydrogen peroxide and in the ab-sence of Mn(II). Pesticide transformation was monitoredby HPLC and three successive additions of both enzymeand hydrogen peroxide were performed to assure the com-plete substrate transformation. Then, the reaction mixturewas lyophilized and extracted three times with 2 mL ofmethanol. The extract was concentrated under N2 and theproducts purified with a HPLC system equipped with a re-v1 rt-i for5 ol-v ingt andpcd etryt er-v pec-t teda

3

ides( othl e ofM (pH4 let cidesa enol.S thet l andb atp enolss fM

absence of Mn(II) and 3.9± 0.18 min−1 in the presenceof Mn(II). On the other hand, dichlorophen transforma-tion rate did not change with manganese (173.1± 4.5 min−1

in the absence and 184.7± 6.7 min−1 in the presence ofmanganese). In control experiments, in the absence of hy-drogen peroxide or enzyme no transformation could bedetected.

Activity pH profiles were determined with and withoutMn(II) for the three pesticides and two different behav-iors were found (Fig. 2). Bromoxynil and pentachlorophe-nol showed the maximal specific activity at pH 4.0 in theabsence of Mn(II) (181.2 and 17.9 min−1, respectively). Inthe presence of manganese, the maximal activity was, inboth cases, significantly lower (12.6 min−1 for bromoxyniland 6.9 min−1 for pentachlorophenol) showing an optimalpH at 2.5. On the other hand, dichlorophen transformationshowed no differences in the maximal activity with and with-out Mn(II) (198 min−1), and the optimal pH showed a smallshift of 0.5 pH units (from pH 3.5 to 4.0).

The kinetic constants were determined at pH 4.0 and inthe absence of Mn(II). Bromoxynil was the best substrate forthe versatile peroxidase with akcat of 409 min−1 and aKMof 31�M for the pesticide and 19�M for the hydrogen per-oxide. Dichlorophen showed a 50% lower activity constant,with a kcat= 194 min−1 and Michaelis–Menten constants of3 -i

ceo aseso e ofd ithb An in-h i-b ug-g rentp

rma-t pec-t s andtt atilep

en-zm )i COm 248a pica thesei enol-2 thee n, ass e de-p nots

erse phase preparative column (300 mm× 47 mm) RP-C185–20�m (Vydac). Elution was performed at 2 mL/min sta

ng with 100% solvent A (acetonitrile:water, 40:60 v/v)min, followed by a linear gradient to obtain 100% sent B (50:50, acetonitrile:water) over 10 min and keephis solvent for 25 min. Product peaks were collectedooled after each injection. Pools were dried over Na2SO4oncentrated under N2 to dryness and dissolved in 100�Lichloromethane prior to analysis by mass spectrom

hrough direct insertion probe (Scientific Instrument Sices, model 73DIP-1) into the ion source of the mass srometry detector (Agilent, model 5973 Network) operat 70 eV.

. Results

Enzymatic transformation of 13 halogenated pesticFig. 1) was assayed with this purified peroxidase, in bignin peroxidase conditions (pH 3 and in the absenc

n) [34] and under manganese peroxidase conditions.5 and 0.5 mM MnSO4) [33]. Versatile peroxidase was ab

o transform only three of the thirteen halogenated pestissayed, bromoxynil, dichlorophen and pentachlorophignificant differences of transformation rates between

wo reaction conditions were found. Pentachlorophenoromoxynil transformations were significantly slowerH 4.5 and in the presence of Mn(II). Pentachlorophhowed a specific activity of 10.9± 0.9 min−1 in the ab-ence of Mn(II) and 0.23± 0.05 min−1 in the presence on(II), while bromoxynil showed 68.1± 0.7 min−1 in the

2�M for the pesticide and 26�M for the hydrogen peroxde.

From data shown inFig. 2, it appears that the presenf manganese inhibits pesticide transformation in the cf bromoxynil and pentachlorophenol, and not in the casichlorophen. Inhibition experiments were performed wromoxynil at pH 4 with different amounts of Mn(II).oncompetitive inhibition behavior was found with anibition constant (Kin) of 646�M for manganese. No inhition on the dichlorophen transformation was found, sesting different transformation mechanisms for the diffeesticides.

Products from versatile peroxidase enzymatic transfoion were purified by HPLC and then analyzed by mass srometry. The mass spectral data of the identified productheir proposed chemical structures are shown inFigs. 3–5. In-erestingly, for the three transformed pesticides by verseroxidase an oxidative dehalogenation was observed.

The mass spectrum of the main product from theymatic transformation of dichlorophen (Fig. 3) shows aolecular ion of 248m/zand M-17, M-35 and M-(35 + 28

on fragments, indicating the loss of OH, Cl and Cl +oieties, respectively. In addition, the ions 231, 233,nd 250m/zshow an isotopic distribution and an isotobundances that indicate the presence of chlorine in

ons. This compound has been identified as 4-chloroph,2′-methylene-1,4-benzoquinone. This product is notnd product from the versatile peroxidase transformatiouccessive additions of enzyme and hydrogen peroxidleted this first product to a non-identified product (datahown).


Fig. 2. pH profile for versatile peroxidase activity on pentachlorophenol, dichlorophen and bromoxynil, in absence of manganese (©) and in the presence (�)of 0.5 mM MnSO4. Standard deviations were estimated from three independent replicates. Reactions were carried out in a 10% acetonitrile in 50 mM malonatebuffer and containing 20�M pesticide at 25◦C, and the reaction was started by the addition of 0.1 mM hydrogen peroxide and monitored by HPLC.

Fig. 3. Mass spectrum and proposed chemical structure of the product from the dichlorophen transformation by versatile peroxidase.


Fig. 4. Mass spectrum and proposed chemical structure of the dimer product from the bromoxynil transformation by versatile peroxidase.

Fig. 5. Mass spectrum and proposed chemical structure of the trimer product from the bromoxynil transformation by versatile peroxidase.

A dimer and a trimer are the main products from the per-oxidase reaction on bromoxynil (Figs. 4 and 5). Accordingto mass-spectroscopic analysis, the dimer shows a molecularmass of 472 and main fragments at 392, 314, 285 and 231m/zunits indicating the loss of three bromine atoms and onehydroxyl group (Fig. 4). In the case of the trimer (Fig. 5), themolecular ion was found at 669m/z, and their main fragmentsat 589, 507, 427, 392, 349m/zrepresenting the losses of thefour bromine atoms present in the molecule. The mass spec-tral patterns of these compounds are consistent with those ofcompounds containing multiple halogen atoms.

Finally, the pentachlorophenol product from the versatileperoxidase transformation was unequivocally identified as2,3,5,6-tertachloro-1,4-benzoquinone (chloranil). The iden-tification was confirmed by HPLC retention time, UV–visiblespectra and mass spectrometry analysis.

4. Discussion

White rot fungi are well known as xenobiotic degraders[5,35]. Their extracellular multi-enzymatic system is mainlybased on free radical oxidative reactions, which representan unspecific and efficient way to reach recalcitrant com-pounds. Versatile peroxidase was able to oxidize only 3of 13 halogenated pesticides tested. From these tested pes-ticides, only six contain phenolic hydroxyl groups: bro-moxynil, dichlorophen, dinoterb, DNOC, niclosamide, andpentachlorophenol, and from this list, the three non trans-formed (dinoterb, DNOC and niclosamide) contain−NO2and−Cl groups (electron-withdrawing groups) in theparaposition of their phenolic ring, which could increase the ion-ization potential of the molecule, making it harder to get thephenoxyl-free radical. However, both dichlorophen and pen-


tachlorophenol, containing−Cl groups in thepara positionof the phenolic group, are substrates for the versatile per-oxidase. It was demonstrated that the peroxidase specificityon phenols is controlled by the reorganization energy of theelectron-transfer step[36]. The reaction rates of peroxidase-mediated transformations of substituted phenols followed atypical Marcus curve, leading to the conclusion that the rate-limiting step is the transfer of an electron[37]. Thus, thecapacity of versatile peroxidase to transform the differentpesticides could be limited by the ionization potential of thesubstrate, as reported for polycyclic aromatic hydrocarbons[15]. Peroxidase-mediated reactions seem to be limited bythe substrate ionization potential. PAHs with IP lower than7.55 eV were oxidized by lignin peroxidase[38,39], man-ganese peroxidase oxidized PAHs with IP up to 8.0 eV[17],the non enzymatic proteins, hemoglobin and cytochrome c,oxidized PAHs with IP lower than 8.0 eV[40,41], while thechloroperoxidase fromCaldariomyces fumagohalogenatedPAH’s with an ionization potential up to 8.15 eV[42].

The pH profiles for activity (Fig. 2) are similar to thosefound by Wang et al.[15] for the VP-mediated oxidation ofdifferent PAH’s. For these substrates, the optimal pH was 3.0in the presence of Mn and between 3.5 and 4.0 in the absenceof Mn.

The catalytic constants (kcat) obtained from pesticidet wert , sucha lco-h undi ons[

pes-t n-g oxi-d al-u s( ibedfd ns-f

These two different catalytic sites, one for Mn and anotherfor veratryl alcohol, have been demonstrated in versatile per-oxidase fromP. eryngii[44]. In addition, the existence of twodifferent binding sites, one for the manganese ion and anotherfor the pesticide, could contribute to the differences in the pHprofiles in the presence and in the absence of manganese inthe reaction media.

The transformation capacity of fungal cultures on di-,tri-, tetra- and penta-chlorinated phenols has been docu-mented[29–32]. The first step in the metabolization pro-cess seems to be an oxidative dehalogenation mediated byextracellular peroxidases to form 1,4-benzoquinones. Theseextracellular peroxidases are able to catalyze a oxidative de-halogenation only on the carbon in theparaposition, thus anintracellular enzymatic systems have been proposed for thefull dehalogenation of pentachlorophenol after the action ofLiP and MnP[32].

Based on the mechanism studies of Samokyszyn et al.[45]and Osman et al.[46] on pentachlorophenol, the oxidative de-halogenation of dichlorophen mediated by VP seems to startwith the free radical production from the pesticide (Fig. 6).This radical is then delocalized from the phenolic oxygen tothepara carbon of aromatic ring. A second enzymatic elec-tron extraction may occurs to form a carbocation. Then, ahydroxyl group is added by nucleophilic attack to form ap mer-i ofa hichl ulesl r-t

fromB ol,d thep , pro-d bilityo y thep rtieso elop-m nated

dichlo

ransformation, were one or two order of magnitude lohan those obtained with common peroxidase substratess ABTS, guaiacol, 2,6-dimethoxyphenol and veratryl aol [43], but in the same order of magnitude that those fo

n the transformation of polycyclic aromatic hydrocarb15].

Manganese acted as a noncompetitive inhibitor foricide transformation. Noncompetitive inhibition by maanese on the VP activity has been found also in theation of azo dyes[25], and these inhibition constant ves are similar for pesticides (646�M) and for the azo dye500–600�M). A manganese-binding site has been descror manganese peroxidases (MnP) fromPleurotus, Bjerkan-eraandP. chrysosporium, and a large-range electron tra

er has been proposed for organic substrate oxidations[44].

Fig. 6. Proposed mechanism for the oxidative dehalogenation of

ara-benzoquinone moiety. On the other hand, the polyzation of bromoxynil also could start with the formation

free radical, delocalization of an unpaired electron, weads to an oxidative coupling of two pesticide molecoosing a bromine atom (Fig. 7). The polymerization ceainly continues to form trimers (Fig. 5).

We can conclude so far, that the versatile peroxidase. adustais able to transform in vitro pentachlorophenichlorophen and bromoxynil. The chemical nature ofroducts shows that an oxidative dehalogenation occursucing quinones and polymeric products. The susceptif other halogenated pesticides seems to be limited bresence of phenoxyl groups and by the electronic propef the substituents on the aromatic ring. Since the devent of pesticides, large quantities of man-made haloge

rophen catalyzed by versatile peroxidase to form apara-quinone derivative.


Fig. 7. Proposed mechanism for the polymerization of bromoxynil catalyzed by versatile peroxidase.

organic compounds have been introduced into the environ-ment at concentrations that cause ecologically undesirableeffects. Many of these compounds, as PCP, are highly resis-tant to biotic and abiotic degradation and as result remain inthe environment at toxic levels. Doubtless, enzymatic sys-tems able to transform the halogenated phenolic pesticidesand able to reduce the halogenation level of these pollutantsare biocatalytic systems with environmental interest.

Acknowledgments

We thank Rosa Roman for her technical help in the enzymeproduction and purification. This work was supported by aGrant from the National Council of Science and Technologyof Mexico (SEMARNAT-CONACYT 2002-C01-1307).

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