Abstract Extracellular laccase from Panus

tigrinus CBS 577.79 was produced in a bubble-

column reactor using glucose-containing medium

supplemented with 2,5-xylidine under conditions

of nitrogen sufficiency. The main laccase isoen-

zyme was purified to apparent homogeneity by

ultra-filtration, anion-exchange chromatography

and gel filtration that led to a purified enzyme

with a specific activity of 317 IU (mg protein)–1

and a final yield of 66%. Laccase was found to be

a monomeric protein with a molecular mass of

69.1 kDa, pI of 3.15 and 6.9% N-glycosylation of

the high mannose type. Temperature and pH

optima were 55�C and 3.75 (2,6-dimethoxyphenol

as substrate). At 50 and 60�C, the enzyme half-

lives were 281 and 25 min, respectively. The P.

tigrinus laccase oxidized a wide range of both

naturally occurring and synthetic aromatic

compounds: the highest catalytic efficiencies were

for 2,2¢-azinobis-(3-ethylbenzthiazoline-6-sulfonic)

acid and 2,6-dimethoxyphenol (5.99 · 106 and

3.07 · 106 M–1 s–1, respectively). Catalytic rate

constants for typical N–OH redox mediators, such

as 1-hydroxybenzotriazole (2.6 s–1), violuric acid

(8.4 s–1) and 2,2,6,6-tetramethylpiperidin-N-oxide

radical (7.8 s–1), were found to be higher than

those reported for other high redox potential

fungal laccases.

Keywords Bioreactor Fermentation Æ Laccase

Production Æ Panus tigrinus Æ Purification ÆSubstrate specificity

Abbreviations

ABTS 2,2¢-azinobis-(3-

ethylbenzthiazoline-6-sulfonic)

acid

BCR bubble column reactor

DMP 2,6-dimethoxyphenol

Em relative electrophoretic mobility

HBT 1-hydroxybenzotriazole

HPI N-hydroxyphtalimide

IEF-PAGE isoelectric focusing on

polyacrylamide gel

Kcat catalytic rate constant

Km Michaelis Menten constant

LME lignin-modifying enzyme

OMW olive-mill wastewater

PAGE polyacrylamide gel electrophoresis

SDS-PAGE sodium dodecyl sulfate

TEMPO 2,2,6,6-tetramethylpiperidin-N-

oxide radical

VLA violuric acid

XYL 2,5-xylidine

D. Quaratino Æ F. Federici Æ M. Petruccioli ÆM. Fenice Æ A. D’Annibale (&)Dipartimento di Agrobiologia & Agrochimica,Universita degli Studi della Tuscia, Via San Camillode Lellis snc, I-01100 Viterbo, Italye-mail: dannib@unitus.it

Antonie van Leeuwenhoek (2007) 91:57–69

DOI 10.1007/s10482-006-9096-4

123

ORIGINAL PAPER

Production, purification and partial characterisation of anovel laccase from the white-rot fungus Panus tigrinusCBS 577.79

Daniele Quaratino Æ Federico Federici ÆMaurizio Petruccioli Æ Massimiliano Fenice ÆAlessandro D’Annibale

Received: 11 April 2006 / Accepted: 29 May 2006 / Published online: 30 September 2006� Springer Science+Business Media B.V. 2006

Introduction

Lignin is a complex aromatic biopolymer rather

recalcitrant to biological degradation. Fungi

belonging to the ecological group of white-rot

basidiomycetes are considered the most effective

lignin degraders in nature (Field et al. 1993).

Degradation efficiency of these fungi is associated

with the release of extracellular lignin-modifying

oxidases (Tuor et al. 1995). Among these oxi-

dases, laccase (E.C. 1.10.3.2 para-benzenedi-

ol:oxygen oxidoreductase) has been shown to be

unambiguously implicated in the breakdown of

lignin via mechanisms either involving natural

mediators, such as 3-hydroxyanthranilate (Eggert

et al. 1997) or reactive oxygen species (Guillen

et al. 2000). Laccases are extracellular, glycosyl-

ated proteins with two disulphide bridges and

four copper atoms distributed in one mononu-

clear (T1) and one trinuclear (T2/T3) domain

(Thurston 1994). The T1 copper center acts as the

primary electron acceptor from the substrate.

From T1, electrons are then transferred to the

type-3 copper pair center that acts as the final

acceptor. The trinuclear T2/T3 center, that is in-

volved in O2 binding, accepts these electrons with

subsequent reduction of molecular oxygen to two

water molecules. In the last decade, increasing

research effort has been aimed at developing

environmentally sustainable processes in the pa-

per industry based on the use of laccases for

mediator-assisted bleaching of cellulose pulps

(Call and Mucke 1997). Laccases have also found

industrial application in the manufacturing pro-

cess of Denim textiles. In addition, the capability

of laccases to polymerize ubiquitous pollutants,

such as substituted phenols and anilines (Dec and

Bollag 1994) and to oxidize industrial dyes

(Nyanhongo et al. 2002) has suggested possible

usage in wastewater treatment (Duran and

Esposito 2000). In this respect, an efficient use of

laccases and the development of tailor-made

applications require a thorough determination of

their kinetic and stability properties.

The white-rot fungus Panus tigrinus CBS 577.79

was found to be an efficient producer of both Mn-

dependent peroxidase and laccase (Fenice et al.

2003). In addition, it proved to efficiently degrade

and detoxify olive-mill wastewater (OMW), an

agro-industrial phenol-containing effluent, even in

the presence of high organic loads (D’Annibale

et al. 2004; D’Annibale et al. 2006). This promp-

ted us to purify a major laccase isoenzyme and to

characterize its main physicochemical and bio-

chemical properties in order to gain further

information on this promising strain for possible

future applications. These properties are com-

paratively discussed with those reported for the

reference strain P. tigrinus 8/18 (Golovleva et al.

1993; Leontievsky et al. 1994).

Materials and methods

Organism and inoculum preparation

Panus tigrinus (strain 577.79) was obtained from

the CBS culture collection (Baarn, The Nether-

lands). During the study, the strain was main-

tained on potato dextrose agar slants at 4�C and

sub-cultured every month. Inocula were prepared

by growing the fungus for 96 h at 28�C in 1 liter

shaken flasks (150 rpm) filled with 200 ml of a

medium containing 50 g l–1 glucose and 2 g l–1

yeast extract. At the end of incubation, pre-cul-

tures were centrifuged (4,000 g, 10 min) and wa-

shed with deionized water. The mycelium was

homogenized by Ultra-Turrax (IKA Labortech-

nik, Staufen, Germany) (two subsequent steps of

30 s each, at ca. 7,000 rpm) and diluted with

deionized water to yield a biomass concentration

of approximately 10 g l–1.

Enzyme production

Fermentations were carried out in a 3-l jacketed

bubble-column reactor (BCR; diameter 12 cm,

height 27 cm) filled with 2 l of a medium con-

taining 70 mM glucose, 20 mM nitrogen (as

ammonium tartrate), 0.25 mM 2,5-xylidine and

0.01 g l–1 yeast extract (medium GATXY).

Experiments in BCR were performed under the

following conditions: inoculum size 0.35 g l–1;

aeration rate 0.3 vvm; temperature 28� C; incu-

bation time 13 days. Air was injected through a

fritted glass sparger (Diameter 11 cm). The fer-

mentation parameters were monitored in the

bioreactors by an adaptative/PID digital controller,

58 Antonie van Leeuwenhoek (2007) 91:57–69

123

ADI 1030 (Applikon Dependable Instruments,

Schiedam NL). Fungal growth was determined by

measuring biomass dry weight.

Enzyme purification

Ten-day-old BCR cultures were centrifuged

(12,000 · g, 20 min) and culture supernatants

concentrated on a Mini-Sart tangential flow

apparatus (Sartorius, Goettingen Germany) fitted

with a 10 kDa membrane cassette. Further con-

centration and buffer exchange against 10 mM

imidazole–HCl buffer pH 6.0 (buffer A) was per-

formed in stirred cells (Amicon, Lexington, Mass)

fitted with a 10-kDa flat membrane prior to its

application to a 30 ml Q-Sepharose Fast Flow

column pre-equilibrated with buffer A at a flow

rate of 1 ml min–1. After extensive washing with

175 ml equilibration buffer, laccase was eluted by

a linear NaCl gradient from 0 M to 0.25 M

(660 ml) in buffer A. The pooled active fractions

were concentrated as above and applied to a Su-

perdex 75 prep-grade column (100 cm · 1.5 cm)

equilibrated with buffer A added with 0.15 M

NaCl (flow rate 0.54 ml min–1). Active fractions,

exhibiting bright blue color, were pooled, desalted,

filter-sterilized (0.22 lm) and stored at –20�C.

Laccase and analytical assays

Laccase activity was routinely assayed spectro-

photometrically at 35�C using 2,6-dimethoxyphe-

nol (DMP) as a substrate and monitoring the

formation of 3,3¢,5,5¢-tetramethoxy- p-dipheno-

quinone (cerulignone) at 477 nm (e = 14600 M–

1 cm–1) (Slomczynsky et al. 1995). One unit of

enzyme activity (IU) is defined as the amount of

enzyme, which produces 1 lmol of product per

min under the assay conditions. Protein determi-

nation was performed according to the dye-

binding method (Bradford 1976), using bovine

serum albumin as the standard. Total sugars and

ammonium were determined as reported else-

where (Quaratino et al. 2006).

Physico-chemical characterization

The native enzyme molecular mass was deter-

mined by gel filtration chromatography on Super-

dex 75 column as described above. The column was

calibrated with tyroglobulin (670 kDa), c-globulin

(158 kDa), ovalbumine (44 kDa) and myoglobin

(17 kDa), vitamin B12 (1.35 kDa). Polyacrylamide

(12%) sodium dodecyl sulfate gel electrophoresis

(SDS-PAGE) was performed at constant voltage

(200 V) on a Mini-Protean II apparatus (Biorad,

Richmond USA) according to the Laemmli

method (1970). Molecular mass determination

under denaturing conditions was performed by

comparison with low-molecular weight standards

(Roche, Manheim). Analytical isoelectric focusing

on polyacrylamide gel (IEF-PAGE) in the range

2.5–7.0 was performed on a Mini-IEF apparatus

(Biorad, Richmond USA). The pH gradient was

measured by using the following standards: human

carbonic anhydrase (pI = 6.55), bovine carbonic

anhydrase (pI = 5.88), b-glucosidase (pI = 5.20),

soybean trypsin inhibitor (pI = 4.50), glucose oxi-

dase (pI = 4.15), amyloglucosidase (pI = 3.50) and

pepsinogen (pI = 2.80). Proteins were visualized

on gels by the ultra-sensitive colloidal Coomassie

G-250 method (Neuhoff et al. 1988). Native poly-

acrylamide gel electrophoresis (PAGE) was per-

formed using the discontinuous gel system of Davis

(1964). Gels were cast with a 4% stacking gel and

12.5% resolving gel. Proteins were allowed to stack

at 20 mA and separate at 30 mA. Prior to activity

staining, native gels were rinsed three times with

distilled water, equilibrated in 50 mM acetate

buffer pH 4.0 and finally stained with 2 mM 2,2¢-azino-bis-(3-ethylbenzthiazoline)-sulfonic acid

(ABTS) in the presence of 50 IU of catalase. To

quantify the carbohydrate content of the P. tigrinus

laccase, the enzyme (4 lg) was previously degly-

cosylated by incubation with 12 IU of N-glycosi-

dase F (Roche Mannheim) in 0.2 M phosphate

buffer pH 7.2 for 3 h. Quantification was per-

formed by comparing the relative electrophoretic

migrations of native and deglycosylated laccases

on SDS-PAGE gel. UV–vis absorbance spectra of

purified laccase (540 lg ml–1 10 mM imidazole–

HCl buffer at pH 6.0) were recorded in a Lambda

20 spectrophotometer (Perkin Elmer, Sweden).

Lectin assay

Laccase (1 lg) and standard glycoproteins (1 lg

each) supplied with the Dig Glycan Differentiation

Antonie van Leeuwenhoek (2007) 91:57–69 59

123

Kit (Roche, Manheim Germany) were spotted

onto a Protran BA85 nitrocellulose membrane

(Schleicher & Schuell, Dassel Germany) and de-

tected immunologically after binding to lectins

conjugated with digoxigenin according to the

manufacturer’s instructions. The following lectins

were used: Galanthus nivalis agglutinin, specific

for terminal mannose; Sambucus nigra agglutinin

specific for sialic acid a(2–6)galactose; Maackia

amurensis agglutinin, specific for sialic acid a(2–

3)galactose; peanut agglutinin, specific for galac-

tose b(1–3)N-acetylgalactosamine and Datura

stramonium agglutinin specific for galactose b(1–

4)N-acetylglucosamine. UV–vis absorbance

spectra of purified laccase (540 lg ml–1 10 mM

imidazole–HCl buffer at pH 6.0) were recorded

in a Lambda 20 spectrophotometer (Perkin El-

mer, Sweden).

Effect of pH and temperature on enzyme

activity and stability

Laccase activity as a function of pH was measured

at 30�C using DMP as a substrate and 0.1 M cit-

rate–phosphate buffer in the pH range 2.5–7.5.

The temperature–activity profile was determined

in 0.1 M citrate–phosphate buffer pH 3.75 in the

range 10–70�C. To test the pH stability, 10 IU of

laccase were incubated at 25�C for 325 h in 5 ml

0.1 M citrate–phosphate buffer over the pH range

3.0–5.0. Incubations in the same buffer at pH 5.5

and 6.0 were prolonged to 670 h. Aliquots (10–

30 ll) were assayed for residual laccase activity

using DMP as a substrate. Thermal stability

experiments were conducted by incubating 40 IU

of laccase in 4 ml 0.1 M citrate–phosphate buffer

pH 6.0 at 50 and 60 and 70�C for 360, 90 and

12 min, respectively. Aliquots (50 ll) were chilled

on ice and assayed for residual activity as de-

scribed above.

Steady-state kinetic measurements

Kinetic constants of laccase for different phenolic

and non-phenolic substrates were determined at

30�C in air-saturated 0.1 M citrate–phosphate

buffer pH 3.75 using a substrate concentration

range from 0.005 mM to 10 mM. For ABTS,

experiments were performed in the same buffer at

pH 3.5. Table 2 provides wavelengths and

extinction coefficients used for the test substrates.

For kinetic studies on N–OH mediators, enzy-

matic activity was determined by measuring the

oxygen uptake rate with a SA 520 Clark oxygen

electrode (Orion Instruments, Boston MA) con-

nected with a LKB 481 single-channel potentio-

metric recorder. The reaction mixture (10 ml)

containing variable concentrations (generally

from 0.5 mM to 25 mM) of the tested mediator in

0.1 M acetate buffer pH 4.0 was equilibrated at

25�C in the electrode chamber, then the reaction

was initiated by adding appropriate amounts of

laccase. Activity towards N–OH compounds was

calculated from the oxygen uptake rate by

assuming a stoichiometric ratio of 4 moles of

oxidized mediator per mole of oxygen consumed

(Xu et al. 2000). All kinetic constants were cal-

culated by non-linear least squares regression,

fitting experimental data to the Michaelis Menten

equation. Data were then linearised by the

Eadie–Hofstee method.

Results

Laccase production

Figure 1 shows a typical fermentation for

extracellular laccase production by P. tigrinus

CBS 577.79 grown in a bubble column reactor on

a glucose-containing medium supplemented with

2,5-xylidine (XYL), as an inducer. Addition of

2,5-xylidine to the medium led to a ca. twenty-

fold increase in laccase activity (data not shown).

However, although XYL concentration was

rather low (i.e. 0.25 mM), its presence led to

delayed growth with biomass formation increas-

ing very slightly within the first 7 days of fer-

mentation (Fig. 1B). The onset of laccase activity

occurred on day 4 and increased at almost linear

rate up to day 10 when laccase reached its maxi-

mal activity (1.71 IU ml–1). The activity peak was

observed when glucose and ammonium were

depleted by about 85 and 39.5% from the growth

medium, respectively (Fig. 1A). The time course

of extracellular protein production paralleled that

of, laccase activity reaching a concentration of

88 lg ml–1 on day 10 (Fig. 1A): specific laccase

60 Antonie van Leeuwenhoek (2007) 91:57–69

123

activity and volumetric productivity were 19 IU

(mg protein)–1 and 7.12 IU l–1 h–1, respectively.

Native alkaline PAGE analyses followed by

activity staining with ABTS showed the presence

in 10-day-old cultures of a major laccase isoen-

zyme with Em of 0.44 and two minor bands with

Em values of 0.14 and 0.47, respectively (data not

shown). Under these culture conditions, no

manganese-peroxidase activity was detected. Ini-

tial additions of CuSO4 to the growth medium at

concentrations ranging from 0.1 mM to 1.0 mM

did not result in any further stimulation of laccase

production leading, by contrast, to marked inhi-

bition of fungal growth (at concentrations higher

than 0.5 mM, data not shown).

Laccase purification

The purification of P. tigrinus CBS 577.79 laccase

from 10-day-old cultures is summarized in

Table 1. Culture supernatant was 100-fold con-

centrated with 93% recovery of the total activity

with an expected low purification of 1.18 fold. In

the first chromatographic step, that involved an-

ion-exchange chromatography on Q-Sepharose

Fast Flow, laccase eluted at a NaCl concentration

of 0.22 M with a yield of 75% and a purification

fold of 15.97. Laccase was eluted as a single and

symmetric peak (Kav = 0.230) in the second step

that involved gel permeation chromatography on

Superdex 75, resulting in a final purity-fold and

yield of 16.71 and 66%, respectively (Table 1).

The enzyme, which showed a bright blue color and

had a specific activity of 317 IU (mg protein)–1

(DMP as a substrate), turned out to be homoge-

neous by both SDS- (Fig. 2A) and IEF-PAGE

(Fig. 2B), as judged by ultra-sensitive colloidal

Coomassie G-250 staining.

Physico-chemical properties of P. tigrinus laccase

The molecular mass of the native enzyme, as

determined by gel permeation chromatography,

Lac

case

act

ivit

y (IU

ml-1

)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

Ext

race

llula

r p

rote

in (

gm

l-1)

0

10

20

30

40

50

60

70

80

90

Time (days)

0 2 4 6 8 10 12 140,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

Tota

l su

gar

s (

g l-1

)

0

1

2

3

4

5

6

7

8

9

10

11

12

(N

H4)+ (

mM

)

5

8

11

14

17

20B

iom

ass

dry

wei

gh

(tg

l-1

)

A

B

µ

Fig. 1 Time courses oflaccase activity (•),extracellular protein (n),biomass dry weight (,),nutrient nitrogen (s) andtotal sugars (m) in a 3 lbubble-column reactor.Data are themean ± standarddeviation of threefermentations

Antonie van Leeuwenhoek (2007) 91:57–69 61

123

slightly differed from the one determined under

denaturing conditions by SDS-PAGE (69.1 kDa

vs. 68.5 kDa, respectively) (Fig. 2A). The protein

was shown to be rather acidic exhibiting a pI value

of 3.15 (Fig. 2B). The carbohydrate content of the

laccase was found to amount to 6.9% of

the molecular mass. Analysis of the nature of the

protein-bound oligosaccharide showed that the

enzyme was specifically recognized by G. nivalis

agglutinin lectin, which is reported to bind termi-

nal mannose residues. Negative results obtained

with M. amurensis, D. stramonium and S. nigra

and peanut agglutinin lectins suggested absence of

salivated complex type glycans and O-linked

oligosaccharide chains containing b(1–3)-N-acet-

ylgalactosamine at their non-reducing ends. The

UV–vis spectrum showed a maximum of absor-

bance at 610 nm and a shoulder at ca. 330 nm,

which are indicative of the presence of type-1 site

and type-3 binuclear copper, respectively.

Effect of pH and temperature on activity and

stability of laccase

The bell-shaped pH-activity profile showed that

the optimal pH for laccase activity was at 3.75

(DMP as a substrate), while the enzyme exhibited

a relative activity of about 3% at 6.5 (Fig. 3A).

Table 1 Purification scheme of extracellular laccase from P. tigrinus CBS 577.79

Step Total activity(IU)

Total protein(mg)

Specific activityUI (mg protein)–1

Yield(%)

Purity(fold)

Culture supernatant 2000 111.1 19.00 100 1Ultra-filtration (cut-off 10 kDa) 1860 82.75 22.47 93 1.18Q-Sepharose chromatography 1499 4.94 303.5 75 15.97Superdex 75 chromatography 1320 4.16 317.5 66 16.71

Table 2 Kinetic constants (Km and Kcat) and catalytic efficiency (Kcat/Km) of extracellular laccase from P. tigrinus CBS577.79

Substrate Wavelength(nm)

Extinctioncoefficient (M–1 cm–1)

Km (lM) Kcat (s–1) Kcat/Km (M–1 s–1)

Phenol 398 6400 457 ± 12 4.8 ± 0.4 1.06 · 104

Catechol 392 1456 107 ± 8 34.4 ± 2.0 3.21 · 105

4-methyl-catechol 400 1400 132 ± 9 93.9 ± 5.6 7.11 · 105

2-methoxyphenol 465 12100 102 ± 4 22.8 ± 1.3 2.23 · 105

4-methoxyphenol 253 4900 77 ± 8 36.3 ± 2.1 4.71 · 105

2,6-dimethoxyphenol 468 27500 119 ± 5 365.6 ± 32 3.07 · 106

3,4-DHPAa 390 1311 1056 ± 86 49.6 ± 5.0 4.69 · 104

3,4-DOPAb 460 3380 422 ± 34 64.7 ± 7.3 1.53 · 105

Pyrogallol 450 4400 239 ± 12 25.9 ± 0.8 1.08 · 105

o-dianisidine 460 29600 9.1 ± 1.2 24.5 ± 2.2 2.69 · 106

ABTSc 436 29300 31 ± 4 185.7 ± 21 5.99 · 106

1,4-phenylenediamine 487 14685 3340 ± 198 327.4 ± 12 9.80 · 104

o-tolidine 600 6340 609 ± 14 138.5 ± 6.7 2.27 · 105

TEMPOd,e n.d. n.d. 2730 ± 123 7.8 ± 0.2 1.44 · 103

Violuric acide n.d. n.d. 2548 ± 213 8.4 ± 0.3 1.66 · 103

1-hydroxybenzotriazolee n.d. n.d. 8270 ± 345 2.6 ± 0.02 1.52 · 102

N-hydroxyphtalimidee n.d. n.d. 11450 ± 765 0.4 ± 0.05 0.34 · 102

n.d., not determined; a3,4-dihydroxyphenylacetic acidb3,4-dihydroxyphenylalaninec2,2¢-azinobis-(3-ethylbenzthiazoline)-6-sulfonic acidd2,2,6,6-tetramethylpiperidin-N-oxide radicaleActivity determined by the Clark oxygen electrode

Data are the mean ± standard deviation of three experiments

62 Antonie van Leeuwenhoek (2007) 91:57–69

123

The pH optimum for ABTS oxidation was in the

range 2.7–3.2. Figure 3B shows that the optimal

temperature was at 55�C and that a relative

activity of 40% was observed at 15�C.

Laccase stability as a function of pH was

determined at values lower than or equal to 6.0,

due to the negligible activity exhibited by the

enzyme above that pH value. Figure 4A shows

that the enzyme was maximally stable at pH 6.0

and 5.5 with half-lives of 632 h and 607 h. How-

ever, in the pH range 3.0–5.0, laccase proved to be

significantly stable showing half-lives ranging

from 131 h to 372 h. Thermal stability profiles of

P. tigrinus laccase at 50, 60 and 70�C are shown in

Fig. 4B. Half-lives at these temperatures were

281, 25 and 4 min, respectively.

Substrate specificity

Table 2 reports the kinetic constants for the lac-

case-catalyzed oxidation of both naturally occur-

ring and synthetic aromatic compounds. Presence

of an additional methyl or methoxyl group re-

sulted in a notable increase in Kcat. This was

evident by comparing 4-methoxyphenol with

phenol (36.3 s–1 vs. 4.8 s–1, respectively), 2,6-di-

methoxyphenol with 2-methoxyphenol (365.6 s–1

vs. 22.8 s–1, respectively) and 4-methyl catechol

with catechol (93.9 s–1 vs. 34.4 s–1, respectively).

Comparison of the Kcat for 2-methoxyphenol and

4-methoxyphenol oxidation (22.8 s–1 vs. 36.3 s–1,

respectively) showed that the activity was signifi-

cantly affected by the position of methoxyl sub-

stitution on the aromatic ring. In addition, P.

tigrinus laccase also showed higher affinity for the

para- than the ortho-methoxyl-substituted sub-

strate (Km of 77 lM vs. 102 lM, respectively).

The enzyme was unable to oxidize tyrosine and

nitrophenols, whether they be ortho, meta- or

para-substituted (data not shown). Laccase

showed markedly lower affinity for synthetic

substrates than phenolic compounds with the

notable exceptions of o-dianisidine and ABTS,

the Km values of which were 9.1 and 31 lM,

respectively. Table 2 also reports kinetic con-

stants for four distinct redox mediators, namely 1-

hydroxybenzotriazole (HBT), N-hydroxyphthali-

mide (HPI) and violuric acid (VLA), that have

been reported to be very effective in laccase-

mediated reactions (Xu et al. 2000; Fabbrini et al.

2002). Catalytic rate constants for these mediators

were in the following order: VLA ‡TEMPO > HBT > HPI. Lowest affinities were

observed with HBT and HPI (8270 and

11450 lM, respectively). In general, the catalytic

efficiencies for these mediators were lower, by at

least two order of magnitude, than the majority

of substrates under study. For naturally occur-

ring and synthetic substrates, the highest val-

ues of catalytic efficiency were observed with

97.4

MW (kDa)

A

77.0

39.2

26.6

21.5

14.4

Lac

d-Lac

NG -F

1 2 3

B

6.555.85

5.20

4.504.153.50

pI

2.80

1 2

Fig. 2 (A) SDS-PAGE of native (Lane 3, 4 lg, Lac) anddeglycosylated (Lane 2, 4 lg, d-Lac) laccase from P.tigrinus CBS 577.79 and molecular weight standards (Lane1). (B) IEF-PAGE of laccase (Lane 1, 4 lg) and pIstandards (Lane 2). NG-F stands for N-glycosidase F

Antonie van Leeuwenhoek (2007) 91:57–69 63

123

2,6-dimethoxyphenol (3.07 · 106 M–1 s–1) and

ABTS (5.99 · 106 M–1 s–1), respectively.

Discussion

P. tigrinus is a white-rot basidiomycete able to

perform selective degradation of lignin in both

woody and non-woody plants (Costa et al. 2002).

This interesting capability has suggested the use

of this organism in biopulping techniques (Gon-

calves et al. 2002). The relevance of this fungus in

other potential biotechnological applications has

been inferred from its capability to decolorize

agro-industrial effluents (D’Annibale et al. 2006)

and to degrade both textile dyes (Nazareth &

Sampy 2003) and priority pollutants (Leontievsky

et al. 2002). Most data available on the lignin-

degrading system of P. tigrinus refer to strain 8/18

(Maltseva et al. 1991; Leontievsky et al. 1994;

pH2 3 4 5 6 7 8

Rel

ativ

e ac

tivi

ty (

%)

0

20

40

60

80

100

Temperature (˚C)10 20 30 40 50 60 70

Rel

ativ

e ac

tivi

ty (

%)

0

20

40

60

80

100 B

AFig. 3 (A) pH-activityprofiles of P. tigrinus CBS577.79 laccase with 2,6-dimethoxyphenol(DMP, •) and 2,2¢-azinobis-(3-ethylbenzthiazoline)-6-sulfonic acid (s)oxidation at 30�C. (B)Temperature–activityprofile of P. tigrinus CBS577.79 laccase in DMPoxidation (n) in 0.1 Mcitrate phosphate bufferpH 3.75. Data are themean ± standarddeviation of threereplicates

64 Antonie van Leeuwenhoek (2007) 91:57–69

123

Lisov et al. 2003). In particular, extracellular

laccase was purified from both liquid (Leontiev-

sky et al. 1997) and solid-state cultures (Golovl-

eva et al. 1993) of strain 8/18. The enzyme

purified from liquid cultures exhibited a molecu-

lar mass of 64 kDa and typical UV–vis and EPR

spectroscopic features of copper oxidases (Leon-

tievsky et al. 1997). Recently, the enzyme has

been crystallized and a preliminary structure

analysis at 1.4 A resolution has been reported

(Ferraroni et al. 2005). By contrast, laccase ob-

tained from solid-state cultures of the same strain,

albeit containing approx. four copper atoms per

molecule, exhibited atypical spectroscopic fea-

tures and was defined as a yellow laccase (Leon-

tievsky et al. 1997). The authors postulated that

the native enzyme did not maintain its copper

centers in the oxidized state and hypothesized

that this effect was due to the reaction of low

molecular mass lignin degradation intermediates

with ligands of the type-1 copper (Leontievsky

et al. 1997). However, manganese peroxidase was

found to be the main lignin-degrading enzyme in

P. tigrinus 8/18, since it was secreted at signifi-

cantly higher extents than laccase (Maltseva et al.

1991; Leontievsky et al. 1994; Podznyakova et al.

1999). In addition, the total ligninolytic capacity

of this strain was found to be highly correlated

Incubation time (hours)0 50 100 150 200 250 300 350 650 700

Res

idu

al a

ctiv

ity

(%)

0

20

40

60

80

100

120

A

Incubation time (hours)0 50 100 150 200 250 300 350 400

Res

idu

al a

ctiv

ity

(%)

0

20

40

60

80

100

120

B

Fig. 4 (A) Residualactivity of laccase from P.tigrinus CBS 577.79throughout 325 hincubation at 25�C in0.1 M citrate phosphatebuffer at pH 3.0 (•), 3.5(s), 4.0 (.), 4.5 (,), 5.0(n), 5.5 (h) and 6.0 (rÞ.The incubation of theenzyme at pH 5.5 and 6.0was prolonged to 670 h.(B) Residual activity of P.tigrinus CBS 577.79laccase throughout 360, 90and 12 min incubation in0.1 M citrate phosphatebuffer pH 6.0 at 50 (•), 60(s) and 70 (.) �C,respectively. Data are themean ± standarddeviation of threereplicates

Antonie van Leeuwenhoek (2007) 91:57–69 65

123

with manganese peroxidase production (Leon-

tievsky et al. 1994).

By contrast, laccase was the predominant lig-

nin-modifying enzyme (LME) produced by the

strain P. tigrinus CBS 577.79 in both solid (Fenice

et al. 2003) and liquid cultures in OMW-based

media (D’Annibale et al. 2004). The same strain,

grown on an agro-industrial effluent, was able to

both tolerate organic loads as high as 60,000 mg l–1

and to perform significant dephenolization and

decolorization of the waste (D’Annibale et al.

2004). The interesting characteristics of P. tigrinus

CBS 577.79 and the aforementioned differences

with the reference strain 8/18 prompted us to

produce, purify and to characterize the main

laccase isoenzyme produced in liquid cultures. To

this end, a pneumatically agitated reactor, i.e. a

BCR system, was used in the present study; in-

deed, the BCR system proved to be more ade-

quate than mechanically agitated reactors to

support laccase production by this strain (Fenice

et al. 2003) due to lower shear-stressing condi-

tions (D’Annibale et al. 2006). With regard to

nitrogen concentration, a condition of sufficiency

was adopted since it was previously observed that

laccase production levels by P. tigrinus CBS

577.79 were very low or absent under nitrogen

limitation (Quaratino 2005; Quaratino et al.

2006). In this respect, these findings are in

agreement with earlier studies reporting that

culture conditions in which nutrients, including

carbon, sulphur and, above all, nitrogen, were not

limiting resulted in markedly improved laccase

production (D’Souza et al 1999; Medeiros et al.

1999; Galhaup et al. 2002). However, when the

laccase activity peak was reached, the nitrogen

source was found to be only partially depleted

from the medium (present study). The addition of

2,5-xylidine to the liquid growth medium signifi-

cantly stimulated laccase production by P. tigrinus

CBS 577.79 in agreement with an earlier study

conducted with another strain of the same species

(Elisashvili et al. 2002) and with other studies

reporting 20-fold and 9-fold stimulation in Tra-

metes villosa (Yaver et al. 1996) and Pycnoporus

cinnabarinus (Eggert et al. 1996), respectively. By

contrast, both the failure of CuSO4 addition to

enhance laccase production by strain CBS 577.79

and the evident toxic effects of this salt at con-

centrations higher than 0.5 mM were unexpected.

In contrast, a recent study (Chernykh et al. 2005)

showed that laccase production by the reference

strain P. tigrinus 8/18 was 10-fold stimulated by

the combined addition of 2 mM CuSO4 and

1 mM 2,4-dimethylphenol to a glucose/peptone

liquid medium. Although this is a further feature

that differentiates the strain employed in the

present study from P. tigrinus 8/18, the two strains

exhibited some similarities with regard to the

physiological regulation of LMEs production. In

particular, the enhancement of laccase production

was shown to require either nitrogen-sufficient or

nitrogen-rich conditions (Quaratino 2005; Cher-

nykh et al. 2005) while manganese peroxidase

was preferentially produced in nitrogen-limited

cultures in the presence of high concentrations of

Mn2+(Quaratino et al. 2006; Lisov et al. 2003).

The three-step purification scheme employed

in the present study proved to be highly efficient

resulting in a 66% final yield and a purity fold of

16.7. The purified enzyme was found to be N-

glycosylated with a glycosylation pattern of the

high-mannose type and differed slightly from P.

tigrinus 8/18 blue laccase regarding molecular

mass (69 kDa vs. 64 kDa, respectively), pI (3.15

vs. 3.0, respectively), pH and temperature optima

(3.75 vs. 4.8 with DMP as a substrate and 55�C

vs. 60�C, respectively) and apparent Km for

2,6-dimethoxyphenol (119 lM vs. 16.6 lM,

respectively). Albeit being classified as para-

benzenediol:oxygen oxidoreductases, laccases are

rather non-specific as to their reducing substrates

(Thurston 1994; Xu 1996). The P. tigrinus CBS

577.79 laccase did not make any exception since it

was able to oxidize a wide range of aromatic

compounds albeit with different reaction rates

and affinities. The increase in Kcat observed for

compounds bearing an additional methoxyl or

methyl group (2,6-dimethoxyphenol vs. 2-meth-

oxyphenol and 4-methylcatechol vs. catechol,

respectively) might be due to the electron-

donating effect of both substituents leading to

higher density at the phenoxy group. For the

opposite reason, the enzyme was unable to oxi-

dize nitro-substituted phenols. In fact, the nitro

substituent has a very strong electron-withdraw-

ing character thereby impairing the susceptibility

of the substrate to oxidation. With respect to the

66 Antonie van Leeuwenhoek (2007) 91:57–69

123

effect of substituents on laccase-catalyzed reac-

tion rates, Garzillo et al. (1998) emphasized the

polar effect of additional non-bulky substituents

with at least one lone pair electrons on the atom

adjacent to the aromatic ring such as the hydro-

xyl, the methoxyl or the amino-group. The

greater effect of these substituents than other

electron-donating groups such as –CH3 and –

CH=CH–COOH was explained in terms of their

higher impact on the electron density of the pri-

mary oxidizing group (Garzillo et al. 1998). Sev-

eral studies have emphasized the role played by

the difference between the redox potential at site

type 1 of laccase and that of reducing substrates in

governing reaction rates in laccase-catalyzed

oxidations (Xu 1996; Xu et al. 2000). In this re-

spect, the lower redox potentials of both DMP

and 4-methylcatechol than 2-methoxyphenol and

catechol can also account for their higher sus-

ceptibility to laccase-catalyzed oxidation. How-

ever, it has been shown that the catalytic

efficiencies of Trametes trogii, Rigidosporus

lignosus and Pleurotus ostreatus laccases were

only partially due to their specific redox capabil-

ities (Garzillo et al. 2001). In another study, lac2

isoenzyme of basidiomycete C30 was more effi-

cient in phenol oxidation than lac1, although the

redox potential of the T1 copper of the former

was lower by 170 mV than that of lac1 (Klo-

nowska et al. 2002). The same investigators de-

duced that other factors, such as hydrogen

bonding and extent of protonation of ionisable

residues in the vicinity of the T1 copper, might

play considerable effects on the overall activity

(Klonowska et al. 2002). The applicative impli-

cations of laccase-mediated catalysis prompted us

to investigate the kinetic constants of P. tigrinus

laccase for four distinct redox mediators belong-

ing to the N–OH group including HBT, VLA,

TEMPO and HPI. The catalytic rate constants for

HBT and VLA were similar to those of T. villosa

I laccase or better than those reported for other

high redox potential laccases, such as those from

Pycnoporus cinnabarinus or Botrytis cinerea (Li

et al. 1999; Xu et al. 2000). In addition, P. tigrinus

laccase exhibited larger affinity for these com-

pounds than other fungal laccases with the sole

exception of Coriolus cinereus laccase-1, the Km

of which for HBT was 7000 lM (Xu et al. 2000).

In a recent study (Kulys and Vidzuinaite 2005)

aimed at investigating kinetic constants of TEM-

PO oxidation, three out of four recombinant

laccases were found to be scarcely competent on

that mediator. Among them, recombinant T.

villosa laccase exhibited better affinity and cata-

lytic efficiency on this mediator than P. tigrinus

laccase (391 lM and 1.9 · 104 M–1 s–1 vs.

2730 lM and 1.44 · 103 M–1 s–1, respectively).

Although a significant amount of information

is available on the lignin-degrading system of P.

tigrinus, this study reports on a laccase isoenzyme

with properties distinct from those previously

reported for this species and provides further,

important knowledge on the strain CBS 577.79,

the use of which has been shown to be very

promising in OMW treatment (D’Annibale et al.

2004; D’Annibale et al. 2006). The good stability

of the P. tigrinus CBS 577.79 laccase in the pH

range 5.0–5.5, which is generally that encountered

in OMW, and the high values of the specificity

constants (Kcat/Km) for several phenols that are

commonly found in that wastewater (i.e. catechol,

4-methylcatechol, 3,4-dihydroxyphenylacetic acid

and 4-methoxyphenol), clearly indicate the ade-

quacy of this enzyme in OMW treatment.

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