activation of lignin peroxidase in organic media by reversed micelles

Activation of Lignin Peroxidase in OrganicMedia by Reversed Micelles

Masayuki Kimura,1 Junji Michizoe,1 Shin-ya Oakazaki,1 Shintaro Furusaki,1

Masahiro Goto,1,2 Hiroo Tanaka,3 HiroyukiWariishi 3

1Department of Chemical Systems and Engineering, Graduate School ofEngineering, Kyushu University, 6-10-1, Hakozaki, Fukuoka 812-8581, Japan;telephone/fax:+81-92-642-3575; e-mail:[email protected] of Chemical Systems and Engineering, Kyushu University, JST,PRESTO, Fukuoka 812-8581, Japan3Department of Bioresources and Bioenvironmental Sciences, KyushuUniversity, Fukuoka 812-8581, Japan

Received 19 February 2004; accepted 16 July 2004

Published online 30 September 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20277

Abstract: Activation of lignin peroxidase (LIP) in an organicsolvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used asa surfactant to form a reversed micelle. Lyophilized LIPfrom an optimized aqueous solution exhibited no enzymaticactivity in any organic solvents examined in this study;however, LIP was catalytically active by being entrapped inthe AOT reversed micellar solution. LIP activity in thereversed micelle was enhanced by optimizing either thepreparation or the operation conditions, such as watercontent and pH in water pools of the reversed micelle andthe reaction temperature. Stable activity was obtained inisooctane because of the stability of the reversed micelle.The optimal pH was 5 in the reversed micellar system,which shifted from pH 3 in the aqueous solution. Thedegradation reaction of several environmental pollutantswas attempted using LIP hosted in the AOT reversedmicelle. Degradation achieved after a 1-h reaction reached81%, 50%, and 22% for p-nonylphenol, bisphenol A, and2,4-dichlorophenol, respectively. This is the first report onthe utilization of LIP in organic media. B 2004 WileyPeriodicals, Inc.

Keywords: lignin peroxidase; reversed micellar system;organic solvent; biodegradation

INTRODUCTION

Lignin is the most abundant renewable aromatic polymer

and is known as one of the most recalcitrant biomaterials

on Earth (Crawford, 1981; Sarkanen and Ludwig, 1971).

Its degradation plays a key role in the carbon cycle of

the biosphere (Crawford, 1981; Gold et al., 1989; Kirk

and Farrell, 1987). Only white-rot basidiomycetes are re-

sponsible for the complete mineralization of this polymer.

Phanerochaete chrysosporium, the best-studied white-rot

fungus, secretes two heme peroxidases, lignin peroxidase

(LIP) and manganese peroxidase, under ligninolytic condi-

tions (Gold et al., 1989; Kirk and Farrell, 1987; Tien, 1987).

Thus, these enzymes are believed to be involved in trig-

gering lignin biodegradation.

Nucleotide sequences of a number of LIP cDNAs and

genomic clones (Gold and Alic, 1993; Tien and Tu, 1987)

and crystal structures of LIP (Edwards et al., 1993; Piontek

et al., 1993; Poulos et al., 1993) have demonstrated that

important peroxidase catalytic residues are all conserved.

Therefore, LIP shares many structural and mechanistic

features with other peroxidases, yet it has several unique

properties (Dunford and Stillman, 1976; Gold et al., 1989).

The enzyme catalyzes the one-electron oxidation of non-

phenolic aromatic compounds with high redox potentials

via the formation of a substrate cation radical (Gold et al.,

1989; Kirk and Farrell, 1987; Tien, 1987). It has also been

reported that LIP oxidizes a wide range of environmentally

persistent aromatic compounds, such as polyaromatic hy-

drocarbons and polychlorinated dibenzo-p-dioxins as well

as lignin (Bumpus et al., 1985; Gold et al., 1993). Never-

theless, these pollutants are hydrophobic; the enzymatic

treatment has been attempted in aqueous media with a

limited concentration of pollutants, because the enzymes

including LIP are inactivated in organic media. If LIP is

utilized in organic media, the enzyme system could be ap-

plied to concentrated pollutant solutions. Therefore, the ac-

tivation of LIP in organic media is of great interest.

In this study, the activation of LIP in organic solvents was

investigated by using a reversed micellar solution. Reversed

micelles are a thermodynamically stable molecular assem-

bly, which are formed by surfactant molecules in a hy-

drophobic organic solvent (Shield et al., 1986). Enzymes

entrapped in reversed micelles have recently been reported

to be active and stable (Ly et al., 1998; Okazaki et al., 2002;

Ono and Goto, 1997). However, there has been no report

describing the application of the reversed micellar system to

B 2004 Wiley Periodicals, Inc.

Correspondence to: M. Goto

Contract grant sponsors: Ministry of Education, Science, Sports and

Culture of Japan; Kyushu University; New Energy and Industrial Tech-

nology Development Organization (NEDO) of Japan

Contract grant number: B 12450322

biodegradation reactions to date. In the present study,

important parameters affecting the efficiency of the LIP

reaction were examined in detail, showing the optimal

conditions for activating LIP in organic solvents. Finally,

the oxidation of p-nonylphenol, bisphenol A, and 2,4-

dichlorophenol by LIP in organic solvents was performed.

MATERIALS AND METHODS

Chemicals

Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was

purchased from Kishida Chemical Ltd. (Osaka, Japan).

Veratryl alcohol was obtained from Aldrich (St. Louis,

MO) and purified by vacuum distillation before use. p-

Nonylphenol, bisphenol A, and 2,4-dichlorophenol were

purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo,

Japan). All organic solvents used in this work were of

analytical grade. Sequentially distilled and deionized water

was employed throughout the experiments.

Lignin Peroxidase

LIP (isozyme 2) was isolated from the extracellular culture

medium of P. chrysosporium (ATCC 34541) and purified as

previously described (Johjima et al., 1999; Wariishi et al.,

1990). The concentration of LIP was determined by the

absorbance at 408 nm by using the extinction coefficient of

133 mM�1 cm�1 (Gold et al., 1989). The enzyme was

electrophoretically homogenous and has an RZ (A408/A280)

value >4.6. LIP activity in the aqueous solution was es-

timated from veratryl alcohol (VA) oxidation activity as

previously described (Gold et al., 1989; Kirk and Far-

rell, 1987).

Preparation of Reversed Micelles Containing LIP

Reversed micelles containing LIP were prepared by direct

injection of an aliquot of the LIP stock solution in 20 mM

potassium phosphate buffer, pH 2–8, into 100 mM AOT in

isooctane. The concentration of the LIP stock solution

varied in order to adjust the water content (Wo; [H2O]/

[Surfactant]) to 10–70 in the reversed micelles but the

final concentration of LIP was constant at 3 AM or other-

wise indicated.

To optimize the preparation conditions, the effect of the

concentration of AOT and the LIP final concentration on the

activity in isooctane were also examined as follows. AOT

concentration was varied from 50 to 400 mM at the Wo

value of 50, and the final LIP concentration of 3 AM. The

final concentration of LIP in the reversed micelles was also

varied from 0.3 to 5 AM at the Wo value of 50 and AOT

concentrations at either 100 or 400 mM. All the concen-

trations are based on the total volume of the reversed

micellar solution.

Reaction Conditions for LIP encapsuledin Reversed Micelles

To optimize either preparation or operation conditions, VA

was utilized as the reducing substrate. VA (100 mM toluene

stock solution) was added to the reaction system at the final

concentration of 1.0 mM. H2O2 reversed micellar solution

prepared separately in AOT/isooctane was added to initiate

reactions at the final H2O2 concentration of 0.01–1.0 mM.

The reactions were conducted at 20–50jC and monitored

by the formation of veratraldehyde using e310 = 9,470 M�1

cm�1 with a JASCO V-570 UV spectrophotometer (JASCO,

Tokyo, Japan). The extinction coefficient was separately

obtained in the present study. The enzyme reactions were

carried out at least three times under the same experimental

conditions, and the initial rates were plotted using the

average value.

Oxidation of Aromatic Pollutants by LIPin Organic Media

The oxidations of p-nonylphenol, bisphenol A, and 2,4-

dichlorophenol were employed in the reversed micellar

solution. The decrease of substrates was monitored by a

HPLC equipped with an ODS-3 column (GL Science,

Tokyo, Japan) eluted with 30% acetonitrile in water (0.1%

phosphoric acid) (0–10 min), followed by a linear gradient

from 30% to 90% acetonitrile (10–20 min) and then by

90% acetonitrile (20–25 min) at a flow rate of 1.0 mL/min

using a UV detector at 268 nm (p-nonylphenol), 276 nm

(bisphenol A), and 294 nm (2,4-dichlorophenol), respec-

tively. The experiments were repeated at least three times,

and we evaluated the degree of degradation for the

pollutants as the average value.

RESULTS AND DISCUSSION

Although it has been reported that LIP can oxidize aromatic

substrates in aqueous solutions containing water-miscible

organic solvents (Yoshida et al., 1997), there is no

successful report on LIP-catalyzed reactions in organic

media, especially hydrophobic solvents. Recently, laccase

and manganese peroxidase, well-characterized fungal lig-

ninolytic enzymes, were reported to be active in organic

solvents when coated with surfactant molecules (Okazaki

et al., 2000, 2002). The lyophilized enzymes covered with

surfactant molecules were soluble and active in various or-

ganic solvents (Kamiya et al., 2000; Maruyama et al., 2002,

2003; Okazaki et al., 2000, 2002). We then applied this

surfactant-coating method to LIP as previously reported;

however, surfactant–LIP complex showed no activity in

organic solvents (data not shown). Because the substrate-

binding site of LIP is known to exist on the surface of

the protein, which is one of very unique properties of this

enzyme (Doyle et al., 1998; Johjima et al., 1999; Wariishi

et al., 1994), the interaction of surfactant molecules and LIP

substrate oxidation site(s) may interfere with the catalytic

496 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 88, NO. 4, NOVEMBER 20, 2004

action. Thus another technique was required to activate LIP

in organic media. A reversed micellar system seemed to be

advantageous because enzyme molecules are entrapped in

water pools of the reversed micelles. We can readily control

the size of reverse micelles (3–10 nm) by changing the

water content in the reversed micellar solution, which is the

same meaning as changing the Wo values. The longest

dimension of LIP is around 4 nm, therefore, the size of

reversed micelles is large enough for entrapping LIP.

Activation of LIP in Organic Media

VA oxidation by LIP hosted in the reversed micelles and

lyophilized LIP was performed, clearly showing that LIP in

the reversed micelles was active, while no activity was

observed with lyophilized LIP in isooctane (Fig. 1). LIP was

thought to be entrapped in water pools inside the reversed

micelles; therefore, the enzyme maintained a high activity

in isooctane. This is the first report indicating that LIP

catalyzes a reaction in organic media.

Effect of pH During Reversed Micelle Preparation

One of the most important factors affecting the enzymatic

activity is the pH in the reaction medium. The pH values of

the reaction mixtures in an aqueous system and the stock

solutions for reversed micelle preparation were varied. The

latter value was thought to be the pH of water pools in the

reversed micelles. The initial reaction rates were then

plotted against pH values (Fig. 2). The optimal pH was

observed at 3 for the aqueous system as previously reported

(Gold et al., 1989; Kirk and Farrell, 1987; Tien, 1987). On

the other hand, in the reversed micellar system, the enzyme

activity became maximal at around pH 5. The shift of

optimal pH might be explained by the negative charge of the

surfactant. AOT has an anionic sulfo group at the hydro-

philic head; therefore, the microenvironment for enzyme

surface becomes acidic due to the condensation of protons at

the oil–water interface (Barbaric and Luisi, 1981; Douzou

et al., 1979; Michizoe et al., 2001, 2003; Shield et al., 1986).

As a result, in the reversed micellar system, the optimal pH

shifts toward a more alkaline value than that of LIP in the

aqueous solution. The water content in the reversed micelle

(Wo value) did not seem to affect the optimal pH.

Effect of Water Content and Sizeof Reversed Micelles

As described above, the interaction of the protein with

surfactant molecules should be considered to effectively

activate LIP in organic media. The Wo value defined by

[H2O]/[Surfactant] was used as the indicator of water

content in the reversed micelles. The Wo value has also been

known to correlate linearly with the size of the reversed

micelles (Pileni et al., 1985). The effect of the Wo value on

the initial rates of VA oxidation is shown (Fig. 3). The

enzymatic activity increased as the Wo value was increased,

and the activity attained a maximum at the Wo value of 50.

It is well known that the relationship between the Wo value

and enzymatic activity shows a bell-shaped tendency. An

entrapped enzyme often exhibits the highest activity when

Figure 1. LIP reaction in isooctane. Reactions were conducted at 25jC.

The reversed micelle was prepared by 100 mM AOT in isooctane and

3 AM LIP in 20 mM phosphate buffer, pH 3.0. Lyophilized LIP was pre-

pared from LIP in 20 mM succinate buffer (pH 3.0) and used in the

absence of AOT. The reaction was initiated by adding 0.1 mM H2O2.

Figure 2. pH dependency of LIP activity in aqueous system (o) and in

reversed micelle with Wo value of 50 (5) and 20 (D). The reactions were

conducted at 30jC.

Figure 3. Effect of Wo value on catalytic property of LIP in re-

versed micelle.

KIMURA ET AL.: ACTIVATION OF LIGNIN PEROXIDASE BY REVERSED MICELLES 497

the size of water pools coincides with that of an enzyme

(Khmelnitsky et al., 1989). The optimal Wo value for LIP

was substantially higher than the smaller protein a-

chymotrypsin and near that of the larger protein (laccase)

(Michizoe et al., 2001; Shield et al., 1986).

Effect of Reaction Temperature on the Activityof LIP in Reversed Micelles

Figure 4 shows the effect of the temperature on the reaction

rate in the reversed micellar system. Maximal activity was

observed at 40jC. The optimal temperature of LIP did not

depend on the water content in the reversed micelle. The

lignin peroxidase activity at the lower Wo dropped off more

quickly with temperature than that at the higher Wo. This

suggests that temperature has a larger effect on the stabil-

ity of smaller micelles. The tendency of the temperature

dependency for the reversed micellar system was similar to

that in an aqueous solution (Kirk and Farrell, 1987; Tien,

1987). It has been thought that thermal stability of enzymes

is enhanced by use in a dry organic solvent where the en-

zyme structure becomes rigid (or loose flexibility) (Zaks

and Klibanov, 1988). Because the enzyme exists in water

pools in the present system, the conformation of LIP is not

rigid in the reversed micellar solution. Therefore, when the

sizes of reversed micelles were large enough, the Wo values

exhibited little effect on the temperature dependency

(Fig. 4).

Effect of AOT Concentration in Isooctane

The effect of AOT concentration on the enzymatic activity

of LIP in the reversed micelles was examined and indicated

that the activity reached a maximum at 100 mM (Fig. 5).

The reversed micelles are a kinetic molecular assembly;

thus water molecules between the reversed micelles

mutually fuse and collapse at every collision. An exchange

rate constant (kex) in the AOT reversed micellar system has

been reported to be 106–108 M�1 s�1 at 10–30jC. The kex

has also been known to be increased upon increasing the

concentration of surfactant and temperature (Fletcher et al.,

1987). The catalytic rate of LIP prepared with 100 mM AOT

was improved compared to that with 50 mM AOT (Fig. 5).

In the system utilized in the present study, H2O2 was added

as the AOT reversed micelle solution. Therefore, the

exchange rate of water molecules in the LIP micelle and

the H2O2 micelle might be increased at a higher concen-

tration of AOT, which is reflected in a better activity with

100 mM AOT than with 50 mM.

However, beyond the AOT concentration of 100 mM, the

catalytic activity decreased (Fig. 5). The viscosity of the

reversed micelle solution drastically increased at an AOT

concentration of over 200 mM (Table I). This may result in

lowering the exchange rate of water molecules of LIP– and

H2O2–reversed micelles, which instantly causes a decrease

in enzymatic activity at a higher surfactant concentration.

However, we cannot explain the decrease in activity only by

the viscosity change because the decline of activity does not

correspond to the increase in viscosity with surfactant

concentration. This means that the rate-limiting process in

the enzyme reaction is not necessarily the exchange rate of

reversed micelles.

Effect of Enzyme Concentration in Reversed Micelles

Figure 6 shows the effect of LIP concentration in the

reversed micelles on the reaction rate. At the optimal AOT

concentration (100 mM), the enzymatic activity linearly

increased in the low enzyme concentration range and

attained a maximum value at the concentration of 3 AM. It

was also found that with a higher concentration of AOT

(400 mM), where the number of micelles increased, no

decrease of LIP activity occurred at the higher enzyme

concentration of 3 AM (Fig. 6). The addition of more LIP

into the reversed micelle may cause stronger repulsion

between the negatively charged enzyme (pI = 3.2) and the

Figure 4. Effect of reaction temperature on LIP activity in reversed

micellar system at Wo of 20 (D) and 50 (5).

Figure 5. Effect of AOT concentration during reversed micelle prep-

aration on LIP activity. Wo and pH were adjusted to 50 and 5.0, respec-

tively. The reaction was conducted at 40jC.

Table I. Viscosity of AOT isooctane solutions at 40jC.

AOT concentration (mM) 100 200 300 400

Viscosity (cP) 1.24 1.33 2.85 6.84


anionic surfactant AOT. As a result, the reversed micelle

becomes unstable. Therefore, using more AOT molecules

helps maintain the LIP activity with a higher enzyme

concentration. As far as examined, the optimal activity was

seen with 100 mM AOT and 3 AM LIP.

Effect of H2O2 Concentration on LIP Activity

To oxidize an organic substrate by peroxidase, the resting

enzyme should be oxidized by two electrons coupled with

the reduction of H2O2 to H2O to form the oxidized

intermediate, compound I; compound I, in turn, is reduced

back to the resting state via the intermediate formation of

compound II, coupled with two successive one-electron

oxidations of organic substrates (Gold et al., 1989; Kirk and

Farrell, 1987; Tien, 1987). LIP compound II has also been

reported to readily react with H2O2 to form a catalytically

inactive intermediate, compound III, which further reacts

with H2O2, causing an irreversible heme bleach (Wariishi

and Gold, 1989, 1990). Therefore, the initial concentration

of H2O2 is crucial to optimize the LIP reaction. Figure 7

clearly indicates that the activity of LIP in the reversed

micellar system strongly depends on the H2O2 concen-

tration. The dependency of LIP activity with hydrogen

peroxide concentration shows a bell-shaped curve with the

optimal [H2O2] at 0.1 mM. This behavior is very similar to

the LIP reaction in the aqueous system (Kirk and Farrell,

1987; Tien, 1987), suggesting that the LIP reactivity with

H2O2 in water pools of the reversed micelles is almost

identical to that in aqueous solutions and that collision

efficiency of LIP–micelle and H2O2–micelle might not be a

rate-determining step under the conditions utilized.

Effect of Solvents on LIP Activity

The reversed micellar solutions were prepared using various

organic solvents and the enzymatic activity was measured in

each organic solvent (Fig. 8). When decane was used for the

solvent, the initial rate was increased about 4-fold compared

to that in isooctane. However, when the reaction temper-

ature increased to 50jC, the phase separation was observed

in the decane system. Furthermore, when the preparation

temperature was at 30jC or more, it was difficult to generate

a stable reversed micelle in decane. All the solvents used,

except for isooctane, are straight-chain hydrocarbons. The

phase separation observed when using these solvents may

suggest alignment of the linear chains with the hydrophobic

moiety of AOT, thereby disrupting the reversed micelles.

On the basis of the results in Figure 8, we conclude that

isooctane is a more suitable solvent to prepare stable

reversed micelles for the LIP reaction system.

Oxidation of Organic Pollutants

The oxidation of aromatic pollutants was conducted using

LIP hosted in the AOT reversed micelle under the optimized

conditions described above. A decrease in substrates was

observed for all three pollutants, p-nonylphenol, bisphenol

A, and 2,4-dichlorophenol, utilized in this study (Fig. 9). On

Figure 7. H2O2 dependency of LIP activity in reversed micelle. Prep-

aration and operation conditions are the same as described in Figure 6,

but 100 mM AOT was utilized.

Figure 6. Effect of LIP concentration in water pools of the reversed

micelle on reactivity. Wo and pH were adjusted to 50 and 5.0, respectively.

The micelles were prepared using 100 mM (o) or 400 mM (5) AOT. The

reaction was conducted at 40jC.

Figure 8. LIP activity in several organic solvents. LIP – reversed

micelles were prepared in the solvents listed, and the activity was

examined in the same solvent. Preparation and operation conditions are the

same as described for Figure 7, except for the solvents. The activity in

isooctane was defined as one unit.


the other hand, no reaction was observed with the

lyophilized LIP in isooctane. The decrease of bisphenol A

and 2,4-dichlorophenol was less compared to that of p-

nonylphenol; however, these reactions were confirmed to be

reactivated by adding H2O2 as the reversed micellar solution

(data not shown). Although a large amount of H2O2 allows

LIP denaturation, intermittent addition of H2O2 is effective

to improve the degradation efficiency of pollutants. We

investigated the solubility of the substrates into the reaction

solvent isooctane. 2,4-Dichlorophenol was shown to be

more soluble in isooctane than p-nonylphenol, which was

most completely degraded. This result means that the

solubility of substrates could not be an indicator for

evaluating the degree of degradation of the pollutants by

lignin peroxidase. When the substrate has a surface-active

property, the degree of degradation tends to be high. The

development of an effective procedure using the LIP–

reversed micellar system to degrade environmentally per-

sistent hydrophobic pollutants, including the hydrophobic

phenols used in this study, is now under way as well as

product identification.

CONCLUSIONS

The activation of LIP in an organic solvent was achieved by

using a reversed micellar system. LIP exhibited a high and

stable peroxidative activity in isooctane by being entrapped

in the anionic AOT reversed micelle. This is the first report

that LIP shows a high activity in organic media. The

reversed micellar system was actually very efficient to

activate LIP in organic media, since LIP in the reversed

micelle exhibited only one-tenth of the LIP activity in a

physiological aqueous solution. The enzymatic performance

of LIP in the reversed micelles was influenced by the water

content in the micelle and by pH and the LIP concentration

of water pools of the micelles. The size of the water pool

was also suggested to be larger than the LIP protein for

better performance. Some unique properties for optimizing

LIP catalytic action in organic media were derived from the

unique catalytic mechanism of the enzyme; that is, the

substrate binding and oxidizing site exists at the surface of

the LIP protein. Finally, a possible application of LIP for

degrading environmentally persistent hydrophobic pollu-

tants in organic media is described for the first time.

This research was supported by a Grant-in-Aid for Scientific

Research (B 12450322) from the Ministry of Education, Science,

Sports and Culture of Japan, Kyushu University Interdisciplinary

Programs in Education and Projects in Research Development (to

M.G. and H.W.), and the Proposal-Based New Industry Creative

Type Technology R&D Promotion Program from the New Energy

and Industrial Technology Development Organization (NEDO) of

Japan (to M.G., S.F., H.W., and H.T.).

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activation of lignin peroxidase in organic media by reversed micelles

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