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Enzyme and Microbial Technology 45 (2009) 150–155

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Enzyme and Microbial Technology

journa l homepage: www.e lsev ier .com/ locate /emt

nactivation of Coprinus cinereus peroxidase during the oxidation of varioushenolic compounds originated from lignin

an Sang Kima, Dae Haeng Choa, Keehoon Wonb, Yong Hwan Kima,∗

Department of Chemical Engineering, Kwangwoon University, 447-1, Wolgye-Dong, Nowon-Gu, Seoul 139-701, Republic of KoreaDepartment of Chemical and Biochemical Engineering, Dongguk University, 26 Pil-dong 3-ga, Jung-gu, Seoul 100-715, Republic of Korea

r t i c l e i n f o

rticle history:eceived 30 January 2009eceived in revised form 21 April 2009ccepted 1 May 2009

eywords:eroxidaseiPhenolicsnactivation

a b s t r a c t

In this study, the inactivation of Coprinus cinereus peroxidase (CiP) during the oxidation of various pheno-lic compounds originating from lignin was investigated. The CiP was significantly inactivated duringthe oxidation of phenolic compounds, such as vaniline, p-coumaric acid, 2,6-dimethoxy phenol, 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, p-cresol, m-cresol and phenol. Conversely, the CiP nearlymaintained its initial activity for the oxidation of syringic acid, vanillic acid and ferulic acid. Hydrogenperoxide affected the CiP inactivation, while the polymerized reaction product hardly affected the CiPinactivation. The thermodynamic parameter (��G0

f298K) and turnover capacity (�S/�E) were adapted toexplain the CiP inactivation due to covalent bonding between the enzyme and phenolic compounds. Inthe cases of syringic acid, vanillic acid and ferulic acid, which maintained high residual CiP activities after

0

urnover capacityovalent bonding

reaction, the ��Gf298K were more negative and the turnover capacities were higher than the other val-ues. This means that these compounds prefer to form a dimer rather than an enzyme–phenolics complex.Among the inactivation factors, the formation of covalent bonding between the enzyme and phenolicradicals was concluded to be the main mechanism for the inactivation of CiP. The new thermodynamicparameter (��G0

f298K) used in this study could help to quantitatively show the reaction tendency of phe-nolic compounds to form a dimer or covalent bonding with the enzyme, which could be used to predict

tion.

the degree of CiP inactiva

. Introduction

The enzyme peroxidase catalyzes the oxidation of phenolic com-ounds to generate phenoxy radicals, which react with each othero form dimeric, oligomeric and polymeric compounds. This enzy-

atic polymerization method has previously been exploited for thereatment of wastewater polluted with phenolic compounds andor the production of industrially useful polymers [1–5]. Recently,he production of biofuel from lignocelluloses, such as ethanol andutanol, results in the discharge of a huge amount of lignin by-roducts. The phenolic compounds originating from lignin coulde used as a source for the synthesis of new phenolic resin withhis enzyme [6]. Enzymatic polymerization of phenolics using per-xidase has several advantages over conventional polymerization,s follows: (i) phenolic monomers having various substituents are

olymerized to give a new class of functional polyaromatics andii) the structure and solubility of polymer can be controlled byhanging the reaction conditions [7].

∗ Corresponding author. Tel.: +82 2 940 5675; fax: +82 2 941 1785.E-mail address: metalkim@kw.ac.kr (Y.H. Kim).

141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2009.05.001

© 2009 Elsevier Inc. All rights reserved.

The peroxidases originating from plants, such as soybean per-oxidase (SBP) and horseradish peroxidase (HRP), are economicallyinefficient for industrial application. On the other hand, fungal per-oxidase can be readily produced in a large quantity in a bioreactor,which would make it economical to oxidize some phenolic com-pounds [8,9]. It has recently been reported that polycardanol andpoly (bisphenol A) can be successfully synthesized by enzymaticpolymerization using the fungal peroxidase, CiP (Coprinus cinereusperoxidase) [10,11].

However, CiP and other peroxidases significantly lose their activ-ity during the oxidation of phenolic compounds, which results in alimitation to their wide application to industrial processes. Thereare theories according to the mechanism of peroxidase inactiva-tion during phenolic compound oxidation: firstly, inactivation dueto the addition of excessive amounts of hydrogen peroxide in thereaction solution [12–14]; secondly, the inactivation by adsorptionof polymeric reaction products onto the enzyme [15,16]; thirdly,the inactivation by covalent bonding between phenolic compounds

and the amino acid residues of peroxidase as a result of the reac-tion of free phenoxyl radicals with peroxidase [17,18]. The firsttwo are well known mechanisms, which can be proved easilyusing experimental procedures, but the third remains hypotheti-cal. Experimental proof for HRP inactivation by radical attack has

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ecently been obtained through various spectroscopic analyses19].

In this study, the factors responsible for the inactivation of CiPere investigated. Phenolic compounds originating from ligninere selected as the substrates for the reaction. The effects of hydro-

en peroxide and polymerized reaction product on the inactivationf CiP were examined. A thermodynamic parameter was introducedo explain the inactivation of CiP due to covalent bonding betweeniP and the phenolic compounds. The difference in the inactiva-ion of CiP in the presence of various phenolic compounds was alsoxplained via a thermodynamic approach and turnover capacity.

. Materials and methods

.1. Chemicals

The phenolic compounds, syringic acid, vanillic acid, ferulic acid,anillin, p-coumaric acid, 2,6-dimethoxy phenol, 4-hydroxybenzoic acid, 4-ydroxybenzaldehyde, p-cresol, m-cresol, and phenol were purchased from Sigmahemicals (St. Louis, MO, USA). Water, methanol, and acetic acid of high performanceiquid chromatography (HPLC) grade were supplied by Mallinkrodt Backer Inc.Philipsburg, NJ, USA). Microbiological culture media such as yeast extract, peptone,ryptone were purchased from Becton Dickinson Co. (Sparks, MD, USA). The otherhemicals and laboratory media components were obtained from either Sigmahemicals (St. Louis, Mo, USA) or Fluka chemicals (Buchs, Switzeland), and theyere of analytical grade.

.2. Preparation of Coprinus cinereus peroxidase (CiP)

C. cinereus IFO 8371 was used as the peroxidase-producing strain. The mediumsed for the production of the peroxidase contained 30.0 g/L glucose, 5.0 g/L pep-one, and 3.0 g/L yeast extract. The details of the production and purification ofungal peroxidase have previously been reported [10]. Finally, the purified CiP wasoncentrated to a final concentration of 25,000 U/mL.

.3. CiP-catalyzed reaction

The batch reactors consisted of a vial (100 mL) containing 30 mL of 100 mMhosphate buffer and 10 mM phenolic compound. Before the addition of hydrogeneroxide, the CiP was added to the batch reactor to a final concentration of 25 U/mL.0 mM of hydrogen peroxide was added to the reactor to initiate the phenolic com-ound oxidation, with the reactor stirred strongly using a Teflon-coated magneticar at room temperature for 10 min. After initiating the phenolic oxidation, the mix-ure was centrifuged for 15 min at 4000 rpm to remove polymerized precipitates.he supernatant was analyzed for the final concentration of phenolic compound.

To investigate the effect of polymerized reaction products on the inactivation ofiP, the phenol polymer was obtained as follows: to precipitate the phenol polymerrom the reaction solution, 10 mM phenol solution was mixed with 25 U/mL of CiPnd hydrogen peroxide. After the reaction, the mixture was centrifuged, with theupernatant removed. The resultant precipitates were washed with deionised dis-illed water until no enzyme activity remained, and then added to the solution with5 U/mL of fresh CiP.

.4. Analysis

The CiP activity (U/mL) was measured as follows: 10 �L of reaction solution wasdded to 3 mL of a 0.18 mM 2,2′-azino-bis(3-ethylbenzthia-zoline-6-sulfonic acid)ABTS) solution in 50 mM phosphate-citrate buffer (pH 5.0) at room temperature.ne microliter of 15% hydrogen peroxide solution was added to initiate the coloreneration reaction. One unit of peroxidase was defined as the amount of enzymeequired to catalyze the conversion of 1 �mol of ABTS (∈ = 34,700 cm−1 M−1).

Phenolic compound concentrations were determined using an Agilent model200 liquid chromatograph with a diode-array detector working at 280 nm. Sepa-ation was carried out using a Zorbax XDB-C18 column (150 mm × 3.0 mm, 3.5�m)t 25 ◦C, with a mobile phase of 0.3% acetic acid (70%) and methanol (30%) at a flowate of 1.0 mL/min. The concentrations of the phenolic compounds were quantifiedsing calibration curves prepared from external standards.

Gibbs free energy of formation (�G0f298K

) was estimated by Joback method usinghemOffice 2004 software. Joback method is based on the assumption that each frag-ent of a molecule contributes to the value of its physical property [21]. According

o this assumption, Gibbs free energy of formation was calculated at standard state298.15 K, 1 atm, 1 mol) as follows:

G0f298K = 53.88 +

∑niGi

here ni and Gi represent the number of occurrences and a group contribution,espectively.

Technology 45 (2009) 150–155 151

The new thermodynamic parameter, ��G0f298K

, was defined as follows:

��G0f298K = �G0

f298K − �G′0f298K

where ��G0f298K

and �G′0f298K represent the Gibbs free energy of the formation of

dimer and Gibbs free energy of the formation of phenylalanine–phenolic complex(PPC) or tyrosine–phenolic complex (TPC), respectively.

The turnover capacity is expressed as the ratio of exhausted substrate (�S) perenzyme used (�E) during 10 min of reaction

3. Results and discussion

3.1. Inactivation of CiP during the oxidation of various phenoliccompounds

In this study, several phenolic compounds were selectedaccording to their structure. The tested phenolic compounds arecategorized into three main structures, these being cinnamic, ben-zoic, and phenolic derivatives, which have a different numbers ofmethoxy groups. Fig. 1 shows the structures of the phenolic com-pounds used in this experiment.

Fig. 2 shows the residual CiP activity after 10 min of reaction.The residual activity of CiP (RAC) in the presence of phenoliccompounds depended on the structure of the tested materials.The results examined can be represented as follows: (1) TheRAC was higher in the presence of benzoic and cinnamic deriva-tives than the phenolics. (2) The greater the number of methoxyside groups on the phenolic compounds, the greater the amountof RAC remained. (3) The RAC was much higher in the com-pounds in the acid than aldehydic form. With most of the benzoicand cinnamic derivatives the RAC remained above 17.0%, whilethe RAC was below 1.5% with phenol, p-cresol, and m-cresol.The difference in the RAC showed a harsh contrast in the pres-ence and absence of compounds with methoxy groups. The RACwith vanillic acid (4-hydroxy-3-methoxybenzoic acid), vanillin(4-hydroxy-3-methoxy-benzaldehyde), and 2,6-dimethoxyphenolwere 95.3%, 34.3%, and 19.9%, respectively. However, the RACwith 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, and phe-nol were 17.0%, 0.3%, and 1.2%, respectively. Regardless of thepresence of methoxy groups, the RAC with vanillic acid and 4-hydroxybenzoic acid were much higher than with vanillin and4-hydroxybenzaldehyde.

3.2. Inactivation of CiP by hydrogen peroxide

The CiP inactivation by hydrogen peroxide experiment was alsoperformed in a reaction solution without phenolic compounds. Itis well known that peroxidase is inactivated by hydrogen perox-ide [12–14]. As shown in Fig. 2, the activity of CiP decreased about50% due to hydrogen peroxide inactivation. Interestingly, the RACwith vanillic acid, syringic acid, and ferulic acid were higher thanthose of the reaction solutions containing only hydrogen perox-ide. This result shows that these compounds significantly preventthe inactivation of CiP caused by hydrogen peroxide. Except forthese compounds, most of the tested compounds caused muchhigher inactivation of CiP than the inactivation by hydrogen per-oxide. These results indicate that there are other factors involved inthe inactivation of CiP during the oxidation of phenolic compounds.

3.3. Inactivation of CiP by polymerized product

In addition to the inactivation of peroxidase by hydrogen per-

oxide, two inactivation mechanisms have been proposed. First,the peroxidase is inactivated by polymerized reaction products,which are adsorbed onto the enzyme [15,16]. Second, free radi-cals produced from the oxidation reaction cause covalent bondingbetween the enzyme and substrate, which inactivates the per-

152 H.S. Kim et al. / Enzyme and Microbial Technology 45 (2009) 150–155

F ingicd esol; (

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ig. 1. Chemical structure of the phenolic compounds used in this study. (1) Syrimethoxyphenol; (7) 4-hydroxybenzoic acid; (8) 4-hydroxybenzaldehyde; (9) p-cr

xidase [17–19]. In order to examine which factor causes thenactivation of CiP, CiP inactivation experiments were performednder various conditions. As shown in Fig. 3, the CiP activity inhe solution on the addition of the phenol polymer maintainedts initial activity, which was no different from that of the solu-ion without the phenol polymer. Conversely, in the presence ofydrogen peroxide and phenol the CiP activity was dramaticallyecreased. Therefore, the reaction product produced by oxidation ofiP was concluded to hardly affect the inactivation of CiP. It has been

eported that the polymerization product of phenol itself causes thenactivation of horseradish peroxidase (HRP) [16]. The difference inhe inactivation by phenol polymer between HRP and CiP is consid-red to be due to the difference in the adsorptivity of the polymernto the enzyme.

Fig. 2. Residual activity of CiP after the oxid

acid; (2) vanillic acid; (3) ferulic acid; (4) vanillin; (5) p-coumaric acid; (6) 2.6-10) m-cresol; and (11) phenol.

3.4. Inactivation of CiP by phenoxy radical attack

From the above results, the main factor causing the inactiva-tion of CiP together with the inactivation by hydrogen peroxidewas considered to be phenoxy radicals attacking the CiP. Actually,experimental evidence exists regarding the peroxidase inactiva-tion due to covalent bonding between the substrate and peroxidase[18,19]. As shown in Fig. 2, the inactivation of CiP was dependanton the structure of the substrate. In order to investigate the inac-

tivation of CiP by radical coupling during a variety of phenoliccompound oxidations, attempts were made to investigate the inac-tivation of CiP through a thermodynamic approach. Two possiblepathways were assumed when a radical was formed during the oxi-dation of a phenolic compound (Fig. 4). One is the formation of

ation of various phenolic compounds.

H.S. Kim et al. / Enzyme and Microbial Technology 45 (2009) 150–155 153

Foo

dnCotptlatcICw

eeTp(aoftt

Fig. 5. Correlation between the residual CiP activity and �G0f298K

. ��G0f298K

wascalculated with difference between the Gibbs free energy of the formation of dimerand Gibbs free energy of the formation of (a) phenylalanine–phenolic compoundand (b) tyrosine–phenolic compound.

ig. 3. Effect of polymerized reaction products of phenol on CiP inactivation. Activityf CiP in phosphate buffer pH 7.0 (�), in the presence of phenol polymer (�), in 10 mMf hydrogen peroxide (©), and in 10 mM hydrogen peroxide and 10 mM phenol (�).

imers through a radical–radical coupling between oxidized phe-olics, which might have a minimal affect on the inactivation ofiP. The other is a radical attack of CiP, resulting in the formationf enzyme–phenolic complexes through covalent bonding betweenhe oxidized phenolic compound and enzyme. If phenolic radicalsrefer to form the enzyme–phenolic complexes rather than a dimer,hey could easily inactivate the enzyme. The CiP contains 17 pheny-alanine and 2 tyrosine residues [20]. Phenylalanine and tyrosinere amino acids which have an aromatic ring; hence, they can formhe phenylalanine–phenolic complex (PPC) or tyrosine–phenolicomplex (TPC) through covalent bonding with phenolic radicals.t has been proposed that phenylalanine and tyrosine residues iniP have highly feasible sites that can easily form a covalent bondith 4-chloroaniline [18].

In order to investigate the ability of phenolic radicals to formither the dimer or enzyme–phenolic complex, the Gibbs freenergy was used as the criterion of spontaneity for the reaction.he relationship between ��G0

f298K and the residual activity of CiPresented in Fig. 2 is shown in Fig. 5. In the cases of syringic acid1), vanillic acid (2), and ferulic acid (3) which retained a high RAC

0

fter reaction, the ��Gf298K had more negative values than thether tested materials. This means that these compounds prefer toorm a dimer rather than the enzyme–phenolic complex; hence,hey might not cause CiP inactivation. Conversely, the majority ofhe tested phenolic compounds had low levels of RAC and positive

Fig. 4. The feasible pathways for the formation of a phenoxy radical.

Fig. 6. Correlation between turnover capacity and ��G0f298K

. ��G0f298K

was calcu-lated with difference between the Gibbs free energy of the formation of dimer andGibbs free energy of the formation of (a) phenylalanine–phenolic compound or (b)tyrosine–phenolic compound.

154 H.S. Kim et al. / Enzyme and Microbial Technology 45 (2009) 150–155

during

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ct

e�ifmhpeafcttbiCptb

iftipa

TT

P

SVFVp244pmP

Fig. 7. Inactivation pathways of CiP

�G0f298K values. From these results, it was thought there was a

orrelation between CiP inactivation and ��G0f298K. In other words,

he higher the ��G0f298K value, the greater the CiP inactivation.

In addition, the turnover capacity (�S/�E) was introduced tolucidate the relationship between the degree of inactivation and�G0

f298K. As shown in Table 1, the order of the turnover capac-ty value for the tested phenolic compounds was the same as thator the RAC presented in Fig. 2. This means that the enzyme was

arginally inactivated when it had a high turnover capacity andigh residual activity. The turnover capacity value is not directlyroportional to the degree of enzyme inactivation. However, if thenzyme has a simultaneous high turnover capacity and residualctivity, the enzyme could be considered as very stable and there-ore, will not be inactivated. From this result, the enzyme under thisondition would be expected to have a low ��G0

f298K value. In ordero verify this assumption, the relationship between ��G0

f298K andhe turnover capacity is presented in Fig. 6. The linear correlationsetween ��G0

f298K and the turnover capacity are also representedn the figure. Fig. 6 shows that the higher the turnover capacity ofiP, the lower the ��G0

f298K. This result indicates that some of thehenolic compounds require more CiP for their conversion due tohe inactivation of CiP resulting from their formation of covalentonding with CiP.

In this study, the inactivation of CiP during oxidation of var-ous phenolic compounds was investigated and the inactivationactors via several pathways were proposed. As shown in Fig. 7,

here are three mechanisms of the inactivation of CiP, as follows;nactivation by hydrogen peroxide, inactivation by adsorption ofolymerized reaction product, and inactivation due to a radicalttacking the enzyme. Among these inactivation factors, the forma-

able 1urnover capacity of CiP for various phenolic compounds.

henolic compound Turnover capacity (�S/�E) (mol/mol)

yringic acid 1.14 × 106

anillic acid 1.77 × 106

erulic acid 2.32 × 105

anillin 1.40 × 105

-Coumaric acid 9.80 × 104

,6-Dimethoxyphenol 9.25 × 104

-Hydroxybenzoic acid 8.28 × 104

-Hydroxybenzaldehyde 4.77 × 104

-Cresol 2.14 × 104

-Cresol 9.99 × 103

henol 9.13 × 103

oxidation of phenolic compounds.

tion of covalent bonding between the enzyme and phenolic radicalswas concluded to be the main mechanism for the inactivation of CiP.From the experimental result, the CiP was considered not to be inac-tivated by the polymerized reaction products. Although hydrogenperoxide affects the inactivation of CiP, its impact was thought tobe small, as the effect of hydrogen peroxide on the inactivation ofCiP could be diminished in the presence of phenolic compounds. Itis known that hydrogen peroxide based HRP inactivation is largelysuppressed in the presence of phenol [19,22].

Herein, the thermodynamic parameter (��G0f298K) has been

proposed to explain the inactivation of CiP by covalent bondingbetween the enzyme and phenolic compounds. Although no exper-imental evidence has been presented, our method satisfactorilyexplains the difference in the CiP inactivation in the presence ofvarious phenolic compounds. This method could help to quantita-tively show the reaction tendency of phenolic compounds to forma dimer or covalent bonding with the enzyme, which could be usedto predict the degree of CiP inactivation. Furthermore, the peroxi-dases containing many amino acids with phenol like residues, suchas phenylalanine and tyrosine, near its active sites were assumed tobe vulnerable to phenolic radicals and; therefore, inactivated moreeasily. Therefore, if these amino acids are changed, it could be pos-sible to protect the peroxidase from the attack by phenolic radicals.The production of a strong CiP more resistant against the inactiva-tion by phenolic radicals, through protein engineering, has been thesubject of our recent studies, the results of which will be publishedin the near future.

Acknowledgements

This work was supported by the Korea Research FoundationGrant funded by the Korean Government (MOEHRD, Basic ResearchPromotion Fund) (KRF-2008-314-D00081) and Kwangwoon Uni-versity Research Grant (2009).

References

[1] Aiken MD. Waste treatment applications of enzyme: opportunities and obsta-cles. Chem Eng J Biochem Eng J 1993;52:B49–58.

[2] Cooper VA, Nicell JA. Removal of phenols from foundry wastewater usinghorseradish peroxidase. Wat Res 1996;30:954–64.

[3] Kauffmann C, Petersen BR, Bjerrum J. Enzymatic removal of phenols fromaqueous solutions by Coprinus cinereus peroxidase and hydrogen peroxide. JBiotechnol 1999;73:71–4.

[4] Toanami H, Uyama H, Kobayashi S, Rettig K, Ritter H. Chemoenzymaticsynthesis of a poly(hydroquinone). Marcromol Chem Phys 1999;200:1998–2002.

robial

[

[

[

[

[

[

[

[

[

[

[21] Joback KG, Reid RC. Estimation of pure-component properties from group-contributions. Chem Eng Commun 1987;57:233–43.

H.S. Kim et al. / Enzyme and Mic

[5] Kadota J, Fukuoka T, Uyama H, Hasegawa K, Kobayashi S. New positive-typephotoresists based on enzymatically synthesized polyphenols. Macromol RapidCommun 2004;25:441–4.

[6] Hu TQ. Chemical modification properties and usage of lignin. New York:Springer; 2002.

[7] Uyama H, Kobayashi S. Enzyme-catalyzed polymerization to functional poly-mers. J Mol Cat B: Enzym 2002;19–20:117–27.

[8] Abelskov AK, Smith AT, Rasmussen CB, Dunford HB, Welinder KG. pHdependence and structural interpretation of the reactions of Coprinuscinereus peroxidase with hydrogen peroxide, ferulic Acid, and 2,2′-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid). Biochemistry 1997;36:9453–63.

[9] Nakayama T, Amachi T. Fungal peroxidase: its structure, function, and applica-tion. J Mol Cat B: Enzym 1999;6:185–98.

10] Kim YH, Won K, Kwon JM, Jeong HS, Park SY, An ES, Song BK. Synthesis of poly-cardanol from a renewable resource using a fungal peroxidase from Coprinuscinereus. J Mol Cat B: Enzym 2005;34:33–8.

11] Kim YH, An ES, Park SY, Lee JO, Kim JH, Song BK. Polymerization of bisphenol ausing Coprinus cinereus peroxidase and its application as a photoresist resin. JMol Cat B: Enzym 2007;44:149–54.

12] Arnao MB, Acosta M, Del Río JA, García-Cánovas F. Inactivation of peroxidaseby hydrogen peroxide and its protection by a reductant agent. Biochim Biophys

Acta 1990;1038:85–9.

13] Valderrama B, Ayala M, Vazuquez-Dehalt R. Suicide inactivation of perox-idase and the challenge of engineering more robust enzymes. Chem Biol2002;9:555–65.

14] Nakajima R, Yamazaki I. The mechanism of oxyperoxidase formation from ferrylperoxidase and hydrogen peroxide. J Biol Chem 1987;262:2576–81.

[

Technology 45 (2009) 150–155 155

15] Nakamoto S, Machida N. Phenol removal from aqueous solution by peroxidase-catalyzed reaction using additives. Wat Res 1992;26:49–54.

16] Wu Y, Taylor KE, Biswas N, Bewtra JK. A model for the protective effect of addi-tives on the activity of horseradish peroxidase in the removal of phenol. EnzymeMicrob Technol 1998;22:315–22.

[17] Ghioureliotis M, Nicell JA. Assessment of soluble products of peroxidase-catalyzed polymerization of aqueous phenol. Enzyme Microb Technol1999;25:185–93.

18] Chang HC, Holland RD, Bumpus JA. Inactivation of Coprinus cinereus per-oxidase by 4-chloroaniline during turnover: comparison with horseradishperoxidase and bovine lactoperoxidase. Chem Biol Interact 1999;123:197–217.

19] Huang Q, Huang Q, Pinto RA, Griebenow K, Schweitzer-Stenner R, Weber WJ.Inactivation of horseradish peroxidase by phenoxyl radical attack. J Am ChemSoc 2005;127:1431–7.

20] Kunishima N, Fukuyama K, Matsubara H, Hantanaka H, Shibano Y, AmachiT. Crystal structure of the fungal peroxidase from arthromyces ramosus at 1.9angstroms resolution. Structural comparisons with the lignin and cytochromec peroxidase. J Mol Biol 1994;235:331–4.

22] Choi YJ, Chae HJ, Kim EY. Steady-state oxidation model by horseradishperoxidase for the estimation of the non-inactivation zone in theenzymatic removal of pentachlorophenol. J Biosci Bioeng 1999;88:368–73.