Enzyme and Microbial Technology 27 (2000) 330 336

www.elsevier.com/locate/enzmictec

Characterization and immobilization of the laccase from

Pleurotus ostreatus and its use for the continuous
elimination of phenolic pollutants
Gerd Hublik*,1, Franz Schinner
Institute of Microbiology, Technikerstrasse 24, University of Innsbruck, 6020 Innsbruck, Austria
Received 30 March 1999; received in revised form 20 March 2000; accepted 4 April 2000

Abstract
A laccase, the only ligninolytic enzyme produced by the basidiomycete Pleurotus ostreatus strain RK 36 was purified to homogeneity
and characterized. The enzyme is a monomeric protein with a molecular weight of 67 000 Da and an isoelectric point of 3.6. Type I and
type III Cu2 centers were identified by spectrophotometry. With syringaldazine as substrate laccase showed the highest oxidation rates at
pH 5.8, 50C, and in 40 mM phosphate buffer. Among the tested stabilization parameters laccase retained most of its activity in high ionic
buffer, pH 10, 20C, in the presence of 10 mM benzoic acid and with 35% ethylene glycol respectively. Crude laccase was covalently
immobilized to EupergitC. Benzoate was found to stabilize the enzyme during the immobilization process. The activity loss of laccase
during 10 days at 25C storage was 2% on average. Continuous elimination of 2,6-dimethoxyphenol by immobilized laccase was carried
out in a packed bed reactor followed by filtration of the formed precipitate. The solubility of the polymerisates of oxidized syringaldazine,
o-dianisidine, and 2,6-dimethoxyphenol with respect to temperature, pH-value and organic solvents were examined. The precipitates were
found to be insoluble under non-extreme environmental conditions. 2000 Elsevier Science Inc. All rights reserved.
Keywords: Pleurotus ostreatus; Laccase; Enzyme immobilization; Protein characterization; Detoxification

1. Introduction
Laccase (EC 1.10.3.2) is a blue oxidase capable of oxidizing phenols and aromatic amines by reducing molecular
oxygen to water by a multicopper system. The catalytic
center consists of three types of copper with different functions: type 1 (blue copper) catalyzes the electron transfer,
type 2 activates molecular oxygen and type 3, a copper
dimer, is responsible for the oxygen uptake [1]. The molecular reaction models of Latour [2] and Yaropolov et al. [3]
can be simplified to the following equation:
2 Cu2 benzendiol 3 2 Cu quinone 2 H

(1)

2 Cu 12 O2 2 H 3 2 Cu2 H20

(2)

In most cases, the oxidation of the substrates by laccase

finally lead to polymerization of the products through oxi* Corresponding author. Tel.: 43-2527-200-140.
E-mail address: hublik@aon.at (Gerd Hublik).
1
Present address: Stadtplatz 39/1/7, 2136 Laa/Thaya, Austria.

dative coupling. Products of oxidative coupling reactions

result from C-O and C-C coupling of phenolic reactants
and from N-N and C-N coupling of aromatic amines [4].
This reaction is generally seen as a detoxification of
phenolic contaminants [57]. The application of laccase
in waste water treatment systems in immobilized form
could be of interest, besides the substrate only molecular
oxygen as electron acceptor is necessary for the enzymatic reaction.
Although the molecular properties and gene sequences of
several laccases as well as purification procedures are described [3,8,9] there is still a lack of information on the
enzymes kinetic properties and factors that influence stability. The catalytic lifetime of an enzyme can be extended
through optimization of process variables that significantly
affect enzyme activity and stability. The laccases of various
fungi show considerable heterogeneity regarding their molecular properties and substrate specificities [3,4,10]. The
purpose of this work is to present a concise study of the
laccase of Pleurotus ostreatus showing molecular characteristics and parameters that improve the activity and sta-

0141-0229/00/$ see front matter 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 1 4 1 - 0 2 2 9 ( 0 0 ) 0 0 2 2 0 - 9

G. Hublik, F. Schinner / Enzyme and Microbial Technology 27 (2000) 330 336

bility of this enzyme and a simple immobilization method

that prolonged the catalytic lifetime of laccase.

2. Materials and methods

2.1. Organism
P. ostreatus RK 36 was obtained from Biochemie
GmbH, Kundl, Austria.
2.2. Culture conditions
Maximum laccase yield was obtained by incubating P.
ostreatus RK 36 in a complex medium with ligninsulfonic
acid as inducer. It contained (per liter) 30 g of glucose, 15 g
of tryptone, 7.5 g of yeast extract, 30 mg of CuSO4 5 H2O,
and 40 mg of ligninsulfonic acid. The pH was adjusted to
5.6 with 1 M HCl. Agar cubes cut from P. ostreatus colonized agar plates made from the same medium were used to
produce an inoculum. The fungus was cultivated in indented
Erlenmeyer shake flasks at 25C and 150 rev./min. After 14
days the culture medium was separated from the mycelium
by filtration.
2.3. Assay for laccase activity
Laccase was able to oxidize syringaldazine, ABTS, guaiacol, 2,6-dimethoxyphenol, benzidine, and o-dianisidine,
but only syringaldazine and ABTS were used routinely.
1. The assay mixture contained 0.2 mM syringaldazine
(dissolved in 60% ethanol) and 40 mM citrate-buffer,
pH 5.6. Oxidation of syringaldazine was followed by
an absorbance increase at 525 nm (525 65 000
dm3 M1 cm1) at a temperature of 25C [11].
2. The assay mixture contained 0.5 mM ABTS and 40
mM citrate-buffer, pH 5.6. Oxidation of ABTS
[2,2-Azino-di-(3-ethylbenzothialozin-6-sulfonic acid)]
was followed by an absorbance increase at 420 nm
(420 36,000 dm3 M1 cm1) at a temperature of
25C [12]. Enzyme activity was expressed in international units.
2.4. Enzyme purification
Culture fluid was concentrated by fractionated precipitation between 50 and 80% (NH4)2SO4 saturation at 0C.
After centrifugation the precipitate was resuspended in 20
mM Tris buffer, pH 8.0 and extensively dialyzed against the
same buffer. Three milliliters of the solution were loaded
onto a Q-Sepharose Fast Flow column (Pharmacia LKB,
2.5 7 cm) equilibrated with 20 mM Tris buffer, pH 8.0.
Proteins were eluted with a linear gradient from 0 to 1 M
NaCl in 90 min at a flow rate of 1 ml min1. The laccase
fractions were pooled and applied to a Sephadex G-75

331

column (Pharmacia K 26/40) and eluted with 0.2 M Tris

buffer, pH 8.0 at a flow rate of 0.25 ml min1. The laccase
containing fractions were pooled and dialyzed against 10
mM sodium-phosphate-buffer, pH 7.2. 3 ml of the dialysate
were loaded onto a Econo Pac HTP Cartridge (BioRad,
Richmond, CA, USA) and eluted 10 min with 0.4 M sodium-phosphate-buffer, pH 6.8 at a flow rate of 1 ml min1
after pre-equelibration for 30 min. The laccase did not bind
to Hydroxyapatite. For routine purification the pooled samples were ultrafiltrated in a stirred Swinnex-47 cell (Millipore, Bedford, MA, USA) using a 10 kDa cellulose triacetate filter.
2.5. Electrophoresis
SDS-PAGE on 12.5% polyacrylamide gels was performed by the method of Laemmli [13]. Protein bands were
visualized by silver staining.
2.6. Enzyme characterization
The molecular weight of laccase was estimated under
denaturating conditions with SDS-PAGE using protein standards from Boehringer Mannheim (Combithek 1317474).
For estimating the Mr under native conditions, gel filtration
chromatography was carried out by using a Sephadex G 75
column (Pharmacia K 26/40) previously calibrated with
protein standards (Serva Kit MS II; 39064). Isoelectric point
estimation was carried out using a pH-dependent binding
analysis to ionic exchangers. Test tubes containing 2 ml of
swollen Q-Sepharose or S-Sepharose (Pharmacia LKB)
were washed three times with 20 mM citrate-buffer and
equilibrated with 1 ml of the same buffer with pH-values
from 1.0 to 5.0. 200 l of purified laccase solution (19.2 U
ml1 in 2 mM sodium-phosphate buffer, pH 7.2) were
added to each aliquot and gently mixed. After the gel has
settled the enzyme activity in the supernatant was determined using the assay with syringaldazine. The isoelectric
point was indicated by the pH-value where the enzyme
activities in the supernatants of Q- and S-Sepharose were
equal.
The catalytic center was characterized with purified laccase (740 mg l1) diluted in 10 mM sodium-phosphatebuffer, pH 7.2. The enzyme was reduced with 10 mM
ascorbic acid [14]. Spectrophotometric measurements were
carried out on a Ultrospec 2000 UV/Vis spectrophotometer
(Pharmacia Biotech).
Experiments to identify maximum laccase activity were
carried out using partially purified laccase (183 U mg1)
with the syringaldazine assay described above. If not otherwise mentioned the stability was determined by diluting
partially purified laccase (183 U mg1) 1:50 in 40 mM
citrate-NaOH buffer, pH 5.6 and storing the vials at 4C.
After various preincubation times the remaining activity
was measured using the syringaldazine assay.

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G. Hublik, F. Schinner / Enzyme and Microbial Technology 27 (2000) 330 336

2.7. Immobilization of laccase

EupergitC and EupergitC 250 L were donated by
Roehm Pharma, Darmstadt, Germany. Culture fluid of P.
ostreatus was dialyzed against 1 M potassium-phosphatebuffer, pH 7.5. Per gram EupergitC 5 ml of enzyme solution were added (6 ml per gram EupergitC 250 L). To
protect the catalytic center of the enzyme benzoate was
added to a final concentration of 10 mM. The suspension
was gently mixed and kept at 10 or 20C for 3 days. The
beads were then transferred into chromatography columns
(BioRad, 1 cm x 10 cm) and washed with 50 ml 0.1 M
potassium-phosphate-buffer, pH 7.5. The washing fluid was
checked for laccase activity using the assay with ABTS.
2.8. Assay for immobilized laccase activity
For the determination of the binding efficiency and activity loss laccase activity was measured using 1 mM ABTS
suspended in 40 mM potassium-phosphate-buffer, pH 5.6.
Enzyme activity was determined at 25C by measuring the
absorption of 20 ml oxidized ABTS that passed through the
column at a flow rate of 4 ml min1. The activity of free
laccase was determined by the laccase assay with ABTS as
described above. The activity of immobilized laccase was
daily measured to estimate the operational stability. Between the batch cycles the bed was stored in tap water at
25C.

phenol was extracted with diethylether 1:2 (v/v). The temperature of the gaschromatograph (HewlettPackard 5890
with DB-5 column from J&W Scientific) increased from
40C to 300C at 20C min1. The detection was performed
with a flame ionization detector.
2.11. Solubility of polymerisates
Ten millimolars syringaldazine, o-dianisidine, or 2,6dimethoxyphenol in 40 mM potassium-phosphate-buffer,
pH 5.6, were oxidized by laccase. After six hours the three
precipitates where separated from the solution by filtration
and dried overnight at room temperature. Pieces of filter
paper were cut out and incubated for 24 h (2, 3) at the
following conditions:
1. incubation in water from 20 to 100C with 10C steps
every 15 min
2. incubation in 20 mM buffer at 20C with pH-values
from 114 in; pH 1, 3, 5, 7 in citrate-buffer (adjusted
with HCl or NaOH), pH 9, 11, 13 in glycine-NaOHbuffer and pH 14 in 1 M NaOH
3. incubation in pure organic solutions of acetone, chloroform, cyclohexane, ethanol, n-hexane and toluene
at 20C.
The solubility of the polymerisates was detected by UV/
Vis photometric scanning.

2.9. Continuous elimination of 2,6-dimethoxyphenol

3. Results

The oxidation of 1 mM 2,6-dimethoxyphenol by immobilized laccase was performed in a packed bed reactor as
shown in Fig. 7 (BioRad column, 2.5 cm 10 cm).
2,6-dimethoxyphenol was suspended in 10 mM potassiumphosphate-buffer, pH 7.0, the flow rate in the column was
set to 1 ml min1. The reaction products were filtered after
3, 5, and 20 h, the residual substrate content in the liquid
was determined by gas chromatography in each case.

3.1. Laccase production

2.10. Gas chromatography

The solution was acidified with 50% sulfuric acid to a
final concentration of 1% to stop the enzymatic reaction and
for a better extraction of the substrate. Then 2,6-dimethoxy-

Various process parameters were tested to enhance laccase production. The medium described above showed
much higher yields than malt extract medium, no laccase
activity was detected in CzapekDox medium after 14 days
of incubation. Ferulic acid and ligninsulfonic acid both
increased laccase activity in the medium 3-fold, but with
ligninsulfonic acid a higher specific activity was obtained.
Cu2 addition only slightly stimulated laccase production.
Higher concentrations of copper or ligninsulfonic acid decreased laccase activity in the culture fluid. A 21-fold increase in enzyme production was obtained when incubating
P. ostreatus in indented shake flasks compared to regular

Table 1
Purification of extracellular laccase of Pleurotus ostreatus
Purification step

Volume
ml

Activity
U

Total protein
mg

Specific activity
U mg1

Yield
%

Purification
fold

Culture fluid
5080% (NH4)2SO4
Q-Sepharose
Sephadex G 75
Hydroxyapatite

500
30
280
1200
4800

1735
1504
1211
990
806

156
97.8
10.1
5.4
2.3

11.1
15.4
120
183
351

100
86.7
69.8
57.1
46.5

1
1.4
10.8
16.5
31.7

G. Hublik, F. Schinner / Enzyme and Microbial Technology 27 (2000) 330 336

Fig. 1. The pH-dependent adsorption profile of laccase to anionic and

cationic ionic exchangers. Laccase activity in the supernatant of
Q-Sepharose; ----- laccase activity in the supernatant of S-Sepharose.

flasks. The mycelia were sheared on the notches of the flask

and formed small pellets with increased surface. The much
higher surface to volume ratio resulted in a better nutrient
and oxygen supplementation of the cells and though to an
increased enzyme production.
3.2. Purification and molecular properties
Laccase was the only phenoloxidase secreted by P. ostreatus RK 36. The protein was purified to homogeneity
according the procedure summarized in Table 1. The resultant pool with laccase activity was found to contain a single
polypeptide with a molecular mass of 66 800 600 Da as
determined by SDS-PAGE, respectively, 67 000 2000 Da
estimated by calibrated gel filtration chromatography. Isoelectric point estimation was performed by a pH-dependent
binding analysis to ionic exchangers that showed a pI of 3.6
(Fig. 1). Similar methods for estimating the isoelectric point

Fig. 2. Absorbance spectrum of laccase from P. ostreatus (0.74 mg ml1

in 10 mM sodium phosphate buffer, pH 7.2). Native laccase (oxidized
form); ----- reduced laccase with 10 mM ascorbic acid.

333

Fig. 3. Effect of the pH on the reaction rate of laccase using the assay with
syringaldazine.

of proteins were used by Lampson and Tytell [15] and Yang

and Langer [16].
The nature of the catalytic center of P. ostreatus laccase
was determined spectrophotometrically in native (oxidized)
and reduced form (Fig. 2). The UV/Vis spectrum of the
purified native laccase showed an absorption peak at 604
nm, typical for the type I Cu2 center, that is responsible for
the blue color of the enzyme. The shoulder at around 324
nm suggests the presence of the type III binuclear Cu2
center [9].
3.3. Activity and stability characteristics
P. ostreatus laccase was active in a range of pH 59,
showing a peak at pH 5.8 with syringaldazine as substrate
(Fig. 3). When the activity was studied as a function of
temperature the reaction rate increased by about 50% when
raising the temperature by 10C. The maximum activity was
reached at 50C (Fig. 4). Laccase was tested with variable
ionic strengths from 10 mM to 1 M citrate-NaOH buffer.
The enzyme was not sensitive to ionic strength showing
only a 9% difference between its maximum activity in 40

Fig. 4. Effect of the temperature on the activity of laccase using the assay
with syringaldazine.

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G. Hublik, F. Schinner / Enzyme and Microbial Technology 27 (2000) 330 336

Table 2
Activity of immobilized laccase
Immobilization procedure

Laccase activity
(U g1 Eupergit)

Eupergit C
Eupergit C
Eupergit C
Eupergit C
Eupergit C
Eupergit C
Activity of

9.1
18.3
23.4
14.6
16.1
20.2
6.75

at 10C
at 20C
at 20C and 10 mM benzoate
250L at 10C
250L at 20C
250L at 20C and 10 mM benzoate
free laccase in 5 ml culture fluid

The concentration of laccase in 5 ml of culture fluid corresponds to the

amount of enzyme bounded to 1 g of Eupergit C.
Fig. 5. Stability of laccase in buffers with variable pH-values. The syringaldazine assay was used to measure the residual activity after different
times of preincubation. Citrate buffer was used for pH 2 to 6, Tris-HCl for
pH 7 and 8 and glycine-NaOH for pH 9 to 13. No residual activity after
100 h was determined when laccase was stored at pH 2.

mM buffer and its minimal activity in 1 M buffer. A similar

activity was achieved in tap water. When comparing different buffers laccase activity decreased in the order phosphate-buffer sodium-acetate-buffer citrate-buffer. The
stability of purified laccase increased with the ionic strength
of the buffer, tested from 0.01 to 1 M. The activity was
preserved in alkaline buffers reaching a maximum at pH 10
(Fig. 5), whereas the activity decreased rapidly in acid
buffers. Laccase was stabilized with increasing concentration of ethylene glycol up to 50%, at concentrations of 75
and 100% the stability decreased dramatically. Storage of
laccase in 0.1 M Tris-buffer, pH 8.0, proved to be the best
condition to achieve high activity as well as stability (Fig.
6). Addition of 10 mM benzoate resulted in a 3-fold higher
residual activity after 500 h of preincubation.

Fig. 6. Stability of laccase in 0.1 M Tris-HCl, pH 8.0 at various temperatures. The syringaldazine assay was used to measure the residual activity
after different times of preincubation.

3.4. Immobilization of laccase

Laccase from P. ostreatus was covalentely immobilized
to EupergitC after the standard procedure of the manufacturer [17]. Best activity yield was achieved when binding
laccase to EupergitC at 20C in the presence of 10 mM
benzoic acid (Table 2). All immobilization variations showed
an increase in laccase activity compared to free laccase. The
operational stability was examined for 10 days at a storage and
working temperature of 25C. Within this period the activity of
the immobilized laccase decreased for 2% on average whereas
the free enzyme lost all activity (Fig. 8).
3.5. Continuous elimination of 2,6-dimethoxyphenol
Culture fluid from P. ostreatus was concentrated by
lyophilization and immobilized to EupergitC in the presence of 10 mM benzoic acid. Fig. 7 shows the process for
the continuous elimination of phenolic pollutants from an
aqueous solution. 200 ml of 1 mM 2,6-dimethoxyphenol
dissolved in 10 mM phosphate buffer, pH 7.0, passed the

Fig. 7. Process scheme for the continuous elimination of 2,6-dimethoxyphenol by immobilized laccase.

G. Hublik, F. Schinner / Enzyme and Microbial Technology 27 (2000) 330 336

packed bed reactor at a flow rate of 1 ml min1 and 25C.

The brownredish reaction product was collected in the
reaction vessel where oxidative coupling and the resulting
precipitation took place. After 3, 5, and 20 h the fluid with
the oxidative coupling products were filtered through a
paper filter. Gas chromatographical analysis showed that
there was no 2,6-dimethoxyphenol dissolved in the filtrate.
The resolubility of the oxidative coupling products of
syringaldazine, 2,6-dimethoxyphenol and o-dianisidine was
examined with respect to temperature, pH and organic solvents. The UV/Vis scans showed that the reaction product of
2,6-dimethoxyphenol was not soluble under the applied conditions (see Section 2). The polymerisate of o-dianisidine was
soluble in acetone and chloroform, the polymerisate of syringaldazine was soluble in chloroform and in water at a temperature above 80C as well at a pH of 13 and 14.

4. Discussion
The laccase activities of P. ostreatus strain RK 36 and a
locally isolated strain were compared. Strain RK 36 showed
a 13 times higher relative and specific activity and was
therefore used as laccase production strain. The culture fluid
of P. ostreatus RK 36 was screened for laccase, tyrosinase,
peroxidase, lignin-peroxidase, manganese-peroxidase, and
veratrylalcohol-oxidase activities, but only laccase activity
was detected. Laccase activity in the culture fluid increased
after the addition of ferulic acid or ligninsulfonate according
to previous reported results that laccase is inducible [18,19].
During the incubation of P. ostreatus the pH of the culture
fluid reached up to 8.5 correlating with the laccase activity
in the medium. The reason for the alkalization of the growth
medium is probably similar to the investigation of Akamatsu et al. [20] with Phanerochaete chrysosporium. They
state that the fungus secrete oxalic acid to improve the
environmental conditions for the wood degrading enzymes
that have maximum activity at pH-values from 3 to 5. The
substrate is first oxidized by the ligninolytic enzymes and
then reduced by oxalic acid that results in the cleavage of
oxalic acid into two molecules CO2 and subsequently to an
alkalization of the culture fluid.
The catalytic center of the laccase of P. ostreatus RK 36
has the characteristics of a typical laccase, the blue color
due to its A604 and the binuclear Cu2 in the type III
configuration that show A324 [9]. Molecular mass determination under denaturating and nondenaturating conditions
both showed 67 000 Da that proves the monomeric structure
of the protein. Although the molecular mass and the isoelectric point at pH 3.6 are generally in keeping with those
for laccases obtained from a wide variety of fungal genera
[3,10], they are at variance in particular with the molecular
mass (59 000) and pI (2.9) of P. ostreatus strain Florida
described by Sannia et al. [21]
Laccase activity reached a maximum at pH 5.8 with
syringaldazine as substrate, whereas the maximum stability

335

Fig. 8. Activity loss of free and immobilized laccase in tap water at 25C
during 10 days.

was attained at pH 10. The preservation of activity in extreme alkaline environment is very unusual and has not yet
been described for other laccases. The most surprising result
was the stability at pH 13 where all amino acids are negatively charged and should cause electrostatic repulsion resulting in the unfolding of the protein. Nevertheless the
residual activity of laccase at pH 13 was as high as at the
pH-value with the enzymes maximum reaction rate. The
phenoloxidase showed the typical activity curve as a function of temperature for an enzyme. Investigation of temperature stability led to expected results; cooler storage of the
enzyme prolonged its activity. The best stability of laccase
in unpurified culture fluid was obtained at 20C, the
lowest temperature investigated. The reaction rate was not
dramatically influenced by the buffers ionic strength
whereas laccase was stabilized in solutions with high ionic
strength. The addition of ethylene glycol resulted in a stabilization of the enzyme due to water bond formation and a
resulting decrease in water activity [22]. Good stabilization
was obtained by adding benzoate in a final concentration of
10 mM to the enzyme solution. The structure of the molecule and the high residual activity of laccase leads to the
assumption that this effect is due to substrate stabilization.
Various immobilization procedures of laccase have been
reported, including adsorption to porous glass [23,24], entrapment in alginate beads [25,26] and gelatin gel [27] and
covalent methods [28,29]. The advantage of the immobilization of laccase on EupergitC is the extremely easy technical procedure, comprising mixing of the components, incubation and washing of the product. The oxirane groups of
EupergitC function as active components for the covalent
binding of ligands containing amino, mercapto, or hydroxyl
groups [17]. Table 2 shows that each immobilization procedure increased the activity yield compared to free laccase.
Preincubation of free laccase with benzoate further increased the activity of the immobilized enzyme due to a
saturation and resulting protection of the reactive thiol and
aminogroups in the catalytic center against the oxirane
groups of EupergitC. The stability of free and immobilized

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G. Hublik, F. Schinner / Enzyme and Microbial Technology 27 (2000) 330 336

laccase at 25C (Fig. 8) and the increased activity after

immobilization shows that this covalent binding method is
very effective. Furthermore the matrix remains electroneutral, the reactors dont get clogged at higher flow rates and
the strong covalent binding prevent the release of the catalysts. The precipitates resulted from oxidative coupling of
laccase substrates are found to be insoluble at conditions
predominating in industrial waste waters.
Studies on the substrate specifity and the potential to
detoxify a variety of phenolic pollutants that are environmentally relevant are undertaken. So far this method of
continuous elimination of phenolic pollutants by immobilized laccase followed by filtration of the precipitate should
demonstrate a potential perspective in environmental technology for the future.
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