Production and characterization of a secreted,
C-terminally processed tyrosinase from the filamentous
fungus Trichoderma reesei
Emilia Selinheimo
1
, Markku Saloheimo
1
, Elina Ahola
2
, Ann Westerholm-Parvinen
1
, Nisse Kalkkinen
2
,
Johanna Buchert
1
and Kristiina Kruus
1
1 VTT Technical Research Centre of Finland, Espoo, Finland
2 Protein Chemistry Research Group and Core Facility, Institute of Biotechnology, University of Helsinki, Finland
Tyrosinase (monophenol, o-diphenol:oxygen oxidore-
ductase, EC 1.14.18.1) is a copper-containing metallo-
protein that is ubiquitously distributed in nature.
Tyrosinases are found in prokaryotic as well as in
eukaryotic microorganisms, and in mammals, inverte-
brates and plants. Tyrosinase is a mono-oxygenase and
a bifunctional enzyme that catalyzes the o-hydroxyla-
tion of monophenols and subsequent oxidation of
o-diphenols to quinones [1,2]. The activities are also
referred to as cresolase or monophenolase and
catecholase or diphenolase activities, respectively.

putative amino acid sequence (61.151 kDa). According to N-terminal and
C-terminal structural analyses by fragmentation, chromatography, MS and
peptide sequencing, the mature protein is processed from the C-terminus
by a cleavage of a peptide fragment of about 20 kDa. The T. reesei TYR2
polypeptide chain was found to be glycosylated at its only potential N-gly-
cosylation site, with a glycan consisting of two N-acetylglucosamines and
five mannoses. Also, low amounts of shorter glycan forms were detected at
this site. T. reesei TYR2 showed the highest activity and stability within a
neutral and alkaline pH range, having an optimum at pH 9. T. reesei tyros-
inase retained its activity well at 30 °C, whereas at higher temperatures the
enzyme started to lose its activity relatively quickly. T. reesei TYR2 was
active on both l-tyrosine and l-dopa, and it showed broad substrate
specificity.
Abbreviations
TYR2, tyrosinase 2 from Trichoderma reesei; Q-TOF, quadrupole time-of-flight.
4322 FEBS Journal 273 (2006) 4322–4335 ª 2006 The Authors Journal compilation ª 2006 FEBS
In mammals, tyrosinase catalyzes reactions in the
multistep biosynthesis of melanin pigments, being
responsible, for instance, for skin and hair pigmenta-
tion [4]. Tyrosinases play an important role in regula-
tion of the oxidation–reduction potential, and the
wound-healing system in plants [5,6]. They are also
related to browning reactions of fruit and vegetables
[7]. Tyrosinase activity has an essential role in some
plant-derived food products, e.g. tea, coffee, raisins
and cocoa, where it produces distinct organoleptic
properties [8]. Most commonly, tyrosinase-mediated
reactions in plants, however, are related to the brown-
ing reactions that are considered harmful [9]. To date,
the information on the physiologic role of tyrosinases

structure from Streptomyces castaneoglobisporus [17]
became available, and will enable more detailed analy-
sis of the exact reaction mechanisms. The tyrosinase
structure, wherein the active site was located at the
bottom of a large vacant space and one of the six his-
tidine ligands appeared to be highly flexible, was deter-
mined with a help of a caddie protein, ORF378, at
1.2–1.8 A
˚
resolution [17]. Knowledge of fungal tyrosin-
ases is still limited, and the work has been hampered
by relatively low production yields of the enzymes. In
this article, the cloning, production and characteriza-
tion of a novel tyrosinase from the filamentous fungus
T. reesei is reported.
Results
Isolation of a tyrosinase gene from Trichoderma
reesei
A homology search was performed against the genome
sequence of T. reesei ( />trire1.home.html). This revealed two uncharacterized
genes showing clear similarity with known tyrosinase
sequences. Analysis of the deduced protein sequence
encoded by the tyr2 gene with the program signalp
[24] indicated that the protein has a signal sequence,
and should thus be a secreted enzyme. The T. reesei
tyr2 gene and the corresponding cDNA were cloned
by PCR and sequenced in order to verify the sequence
at the genome website, to exclude PCR mutations and
to localize the introns. The gene is interrupted by seven
short introns. The encoded protein consists of 571

supernatants or in cell lysates could be detected.
Therefore, the tyrosinase was overexpressed in T. ree-
sei. An expression construct in which the protein-
coding region of the genomic tyr2 is between the cbh1
promoter and terminator was made by in vivo recombi-
nation with the Gateway recombination system. The
cbh1 promoter is a strong inducible promoter and act-
ive throughout cultivation. The construct (pMS190)
was transformed into T. reesei, and the transformants
were tested with a plate activity assay with tyrosine as
the indicator substrate. A number of transformants
developing a stronger brown color around the streaks
than the parental strain were found (data not shown).
These uninucleate clones were isolated and tested for
tyrosinase production in shake flask cultures. The best
transformant produced 40.1 nkatÆmL
)1
of tyrosinase
activity. The first test cultures were made with 0.1 mm
CuSO
4
in the medium. The effect of copper concentra-
tion on the production level was studied by using
0–6 mm CuSO
4
in the medium in cultures of the best
transformant. The optimal copper concentration was
2mm, but relatively good production was obtained at
1–4 mm. The highest tyrosinase production obtained
in shake flask cultures was 96 nkatÆmL

)1
; Table 1),
the highest activity obtained in fermentation, 300
nkatÆmL
)1
, corresponds to about 1 g of the enzyme
per liter of culture supernatant.
Enzyme purification
Enzyme purification was started with desalting by gel
filtration (Sephadex G25). The following cation
exchange chromatography was performed in 10 mm
Tris ⁄ HCl, pH 7.3. Tyrosinase eluted at an NaCl con-
centration of 120 mm. Because of the high pI of
T. reesei TYR2, most of the Trichoderma cellulases
and hemicellulases could be separated from the tyro-
sinase-containing fractions. The final purification step
was carried out with gel filtration (Sephacryl S-100).
The overall recovery of activity in the three-step purifi-
cation procedure was 15% (Table 1).
Biochemical characterization
IEF of the purified T. reesei TYR2, and subsequent
staining with l-dopa, showed a band in the gel corres-
ponding to a pI around 9.5. The purified T. reesei
TYR2 appeared as a double protein band on
SDS ⁄ PAGE gel (Fig. 2), with an apparent molecular
mass of 43 kDa, which is far below the theoretical
value of 61 151 Da calculated from the encoded amino
acid sequence (including the signal sequence). The
result suggested that T. reesei TYR2 is processed, as
also described for several other fungal tyrosinases [25–

Desalting 43 165 694 62.2 86 1.0
Cation exchange
chromatography
19 120 74 258.4 38 4.2
Gel filtration 7270 24 303.0 15 4.9
MW4321MW
kDa
203.6
116.1
92.3
50.4
37.0
28.9
20.0
6.9
Fig. 2. Purification of T. reesei TYR2 as analyzed by SDS ⁄ PAGE
(12% Tris ⁄ HCl gel). Gel lanes: MW, molecular mass markers; 1,
culture filtrate; 2, desalted culture filtrate, enzyme preparation after
cation exchange; 3, enzyme preparation after gel filtration.
min50
2.5
2.0
1.5
1.0
0.5
0.0
0 10203040
MW 1kDa
97.0
66.0

cing by Edman degradation. No amino acid deriva-
tives comparable to the amount of analyzed protein
(200 pmol) could be obtained, suggesting that the
protein has a blocked N-terminus. For further char-
acterization, the protein was alkylated and fragmen-
ted by trypsin. The tryptic peptides obtained were
first directly analyzed by MALDI-TOF peptide mass
fingerprinting, where most of the obtained peptide
masses could be correlated with theoretical tryptic
peptide masses calculated from the deduced protein
sequence. Notably, no tryptic peptide masses correla-
ting with the C-terminal part after Lys394 (Fig. 1) of
the encoded sequence could be found. The most
N-terminal tryptic peptide found that correlated with
the theoretical tryptic peptide map of the deduced
protein was QNINDLAK (m ¼ 914.482 Da), indica-
ting that the possible signal sequence cleavage site is
located N-terminally to this peptide. Homology
comparisons suggested that the signal sequence clea-
vage site could be at the A(18)–Q(19) bond in the
deduced sequence (shown by an arrow in Fig. 1).
Often, this kind of cleavage is followed by cyclization
of the N-terminal glutamine to form pyroglutamic
acid. The tryptic peptide mass fingerprint of TYR2
contained a peptide mass of 2136.108 Da, which was
suggested to correspond to the N-terminal blocked
tryptic peptide (< QGTTHIPVTGVPVSPGAAVPLR,
m ¼ 2136.196 Da). The identity of this peptide was
then confirmed by MALDI-TOF ⁄ TOF fragment ion
analysis, where partial sequences of this peptide were

mass of the polypeptide chain of TYR2 with the deter-
mined N-terminus and C-terminus is 41 862.7 Da,
whereas the mass determined by MS is about
43 200 Da. Thus, there is a mass difference of about
1300 Da between the determined and calculated mass,
due to post-translational modifications. The purified
polypeptide chain contains one potential N-glycosyla-
tion site in the tryptic peptide SGPQWDLYVQA
MY
NMSK (m ¼ 2016.907 Da). In order to analyze
the possible N-glycosylation, the protein was digested
with trypsin and the potential glycopeptides were
bound to a ConA column. MALDI-TOF MS analysis
of the eluted material revealed a few peptides, of which
the largest had a molecular mass of 3255.309 Da. This
could correspond to the sodium adduct of the above-
mentioned tryptic peptide with a high-mannose-type
glycan, (GlcNAc)
2
(Hex)
5
, attached to it. Further
MALDI-TOF ⁄ TOF MS analysis of this peptide selec-
ted as the precursor ion (Fig. 4) revealed a ladder of
b-ions corresponding to the suggested glycopeptide
with a sequential loss of Na
+
, five hexoses, and two
N-acetylglucosamines, respectively. The resulting prot-
onated mass of 2017.0 Da fits well with the mass of

the enzyme started to lose activity; after 1 h at pH 5,
activity loss was 50%, and after 1 h at pH 4.0, the
enzyme had totally lost its activity. Although l-tyro-
sine was chosen as the substrate to diminish the sub-
strate auto-oxidation effect in the pH optimum and
stability determination, the disturbance of auto-oxida-
tion could not be totally eliminated, because l-tyrosine
is first hydroxylated to diphenolic l-dopa and then
further oxidized to quinones in tyrosinase-catalyzed
reactions. An alkaline environment also changes the
redox potential of the phenolic substrates, making
them more easily oxidized. Therefore, the pH profile
reflects not only the optimal behavior of the enzyme,
but also changes in the substrate. With regard to tem-
perature stability, T. reesei TYR2 was found to be sta-
ble up to 30 °C. However, at higher temperatures it
started to lose its activity relatively quickly; the
enzyme showed half-lives of 18 h, 3 h 45 min and
15 min at 30 °C, 40 °C and 50 °C, respectively.
Among the tested substrates (Table 2), the highest
affinity of T. reesei TYR2 was observed with p-tyrosol
(K
m
¼ 1.3 mm), followed by p-coumaric acid (K
m
¼
1.6 mm) and l-dopa (K
m
¼ 3.0 mm). The highest turn-
over number, k

1.0-
0.5-
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200
m/z
Abs. Int. × 1000
Δ m GlcNAc = 203.20 Da
Δ m Hexose = 162.14 Da
c A N c l G
c
A
N c l G
x e H
x e H
x e H
x e H
x e H
0 . 7 1
0
2
) + a N - ( 0 . 5 3 2 3
0
. 7
5
2 3
3256.309
- L Y V Q A M Y -
670.931
783.800
945.792 1174.001
1045.842 1244.723

better than the d-forms (Table 4).
Various potential inhibitors of T. reesei TYR2 were
tested (Table 5). Kojic acid and b-mercaptoethanol
were the most effective inhibitors, even at low concen-
trations. Sodium chloride and EDTA did not inhibit
the enzyme very efficiently. Glutathione caused only
moderate inhibition, inhibiting the enzyme with 20%
efficiency, as measured with the oxygen consumption
assay. However, as measured with the spectrophoto-
metric assay, inhibition efficiency was 100%, suggest-
ing that glutathione does not inhibit the enzymatic
reaction, but has more effect on the subsequent non-
enzymatic reactions, as also reported in other studies
[31].
T. reesei TYR2 was able to oxidize the tested model
peptides glycine–tyrosine and glycine–glycine–tyrosine
(Table 6). The oxidation rate was dependent on the
Table 2. Determination of K
m
and k
cat
values for T. reesei TYR2 on
L-dopa, p-coumaric acid and p-tyrosol.
Substrate K
m
(mM) k
cat
(s
)1
)

-Dopa 100 100
L-Tyrosine 11 ND
Phenol 8 8
4-Mercaptophenol 0 0
p-Cresol 12 8
4-Aminophenol 1 ND
3-Hydroxyanthranilic acid 0 0
Tyramine 3 3
p-Tyrosol 23 16
p-Coumaric acid 25 16
o-Coumaric acid 0 0
Ferulic acid 0 0
Aniline 1 0
(–)-Epicatechin 96 89
(+)-Catechin hydrate 142 73
Pyrocatechol 87 72
Pyrogallol 66 52
Table 4. Stereospecificity of T. reesei TYR2.
Substrate
(2.5 m
M)
Activity (%) relative to
L-dopa and L-tyrosine
L-Dopa 100
DL-Dopa 46
D-Dopa 18
L-Tyrosine 100
DL-Tyrosine 40
D-Tyrosine 7
Table 5. Degree of inhibition of T. reesei TYR2 as determined in

1 17 100
NaCl 100 15 49
10 1 0
EDTA 10 39 13
111 7
Secreted tyrosinase from Trichoderma reesei E. Selinheimo et al.
4328 FEBS Journal 273 (2006) 4322–4335 ª 2006 The Authors Journal compilation ª 2006 FEBS
length of the peptide, the tripeptide being more readily
oxidized than the dipeptide.
Discussion
Tyrosinase enzymes and their genes have previously
been characterized from bacteria, fungi, plants and
mammals. The most extensively investigated fungal
tyrosinases, from both a structural and a functional
point of view, are from Agaricus bisporus [10] and
N. crassa [1]. Also, a few bacterial tyrosinases have
been reported, of which Streptomyces tyrosinases are
the most thoroughly characterized [32,33]. In addition,
tyrosinases have been reported, for example, from
Pseudomonadacae [34], Bacillus, Myrothecium [35],
Mucor [36], Miriococcum [37], Aspergillus, Chaetoto-
mastia, Ascovaginospora [38], Trametes [39] and Pyc-
noporus [40]. Our aim was to discover novel fungal
tyrosinases, and we used the genome sequence of a
well-known industrial enzyme producer T. reesei for
the search. A homology search in the genome database
of this fungus revealed a new tyrosinase gene tyr2,
which, according to sequence analysis, has a signal
sequence. The gene was overexpressed in the native
host; thus, the gene product was verified to be secre-

addition of copper to the T. reesei medium had a
positive effect on tyrosinase production. Because the
tyrosinase was expressed under the cbh1 promoter,
which is not activated by copper, the improved
production levels were presumably not caused by higher
transcription rates. In addition, no effect of copper
addition on fungal growth was observed, which implies
that the higher enzyme yields may have been due to
improved folding of the active enzyme in the presence
of elevated copper concentrations.
The importance of high copper concentrations has
been reported in laccase production in S. cerevisiae,
where the overexpression of two copper-trafficking
enzymes from Trametes versicolor led to significantly
improved recombinant laccase yields [46]. Added cop-
per can improve correct folding of recombinant lac-
case, as previously detected in Aspergillus nidulans and
Aspergillus niger expressing a laccase from Ceriporiop-
sis subvermispora [47], and in T. reesei producing the
laccase of Melanocarpus albomyces [48].
C-terminal processing of fungal tyrosinases has been
reported previously, and also the molecular mass of
purified TYR2 tyrosinase, 43.2 kDa, suggested exten-
sive processing of the protein. The intracellular tyro-
sinases from N. crassa [26–28] and Agaricus [28] and
Pycnoporus species [25] have an additional C-terminal
domain that is proteolytically released from the cata-
lytic domain. It has been postulated that the function
of the C-terminal domain is to keep the enzyme in-
active until the activity is needed [26]. According to

tidecutter [49], which searches for all known pepti-
dase cleavage sites, did not indicate that the protein
could be cleaved at that site. The C-terminal glycine of
the mature TYR2 is followed in the sequence by the
amino acids Lys–Lys–Arg. This contains a recognition
sequence for the KEX2 ⁄ furin-type protease, which
resides in the Golgi complex and processes a number
of secreted enzymes and other proteins after dibasic
recognition sites [50]. The putatively secreted tyrosin-
ase of G. zeae has Lys–Arg at the same position
(Fig. 1). For these reasons, it is possible that TYR2 is
first cleaved by a T. reesei KEX2-type endopeptidase
during secretion and is further processed by an exo-
peptidase. Further analyses are needed to elucidate the
role of the C-terminal processing.
As for the T. reesei TYR2, the pH optimum in the
alkaline pH range has been reported for Thermomicro-
bium roseum (pH 9.5) [51] and pine needle tyrosinase
(9–9.5) [52]. Many fungal tyrosinases have their pH
optima at neutral and slightly acidic pH, e.g. N. crassa
and Aspergillus flavipes at pH 6.0–7.0 [53,54] and Pyc-
noporus sanguineus at pH 6.5–7 [40]. T. reesei TYR2
was not able to retain substantial activity at tempera-
tures above 30 °C. Longer half-lives have been repor-
ted, e.g. 2 h at 50 °C for P. sanguineus tyrosinase.
However, at 60 °C, P. sanguineus tyrosinase was also
inactivated completely within 20 min [40]. In general,
mammalian and plant-derived tyrosinases are not very
thermostable; even a short incubation at 70–90 °C inac-
tivates the enzymes completely [52,55]. Also, inactiva-

substrate analog for T. reesei TYR2. Also, a thiol
group in the phenolic ring inhibited the enzyme. Tous-
saint and Lerch [62] and Ga˛sowska et al. [63] showed
that N. crassa tyrosinase oxidizes aromatic amines and
o-aminophenols, structural analogs of monophenols
and ortho-diphenols. Similar catalytic reactions, ortho
hydroxylation and oxidation, took place, although the
reaction rates observed for aromatic amines were relat-
ively slow as compared to those for monophenols.
T. reesei TYR2 was also found to oxidize phenylalan-
ine, although extremely slowly.
The tyrosyl residue was oxidized by T. reesei TYR2
in the dipeptide glycine–tyrosine and the tripeptide
glycine–glycine–tyrosine. The relative oxidation rate
increased as the length of the peptide increased. Simi-
larly, protein-bound tyrosyl was oxidized by the
enzyme, and subsequent protein crosslinking was
observed, as analyzed by SDS ⁄ PAGE (data not shown).
Because of difficulties in the production and purifi-
cation of microbial tyrosinases in sufficient amounts,
knowledge of their structure–function relationships
and exact reaction mechanisms is still limited. The
availability of the enzyme has also hampered its testing
and use in applications. We have reported here for the
first time the production, purification and characteriza-
tion of a novel tyrosinase from the well-known protein
producer T. reesei. The high production levels of the
tyrosinase also allow the testing of the enzyme for
applications.
Experimental procedures

reverse primer, GAT CGG TAC CTC ATT ACA GAG
GAG GGA TAT GGG GAA C. The PCR reaction was
done as described above. The amplified PCR product was
inserted into the EcoRI and KpnI sites of the vector pPIC-
Za
´
A (Invitrogen) and the sequence of the product was
verified.
Overexpression of the tyrosinase gene
in Trichoderma reesei
The genomic tyr2 gene fragment was transferred by an LR
recombination reaction from the pDONR221 vector to the
T. reesei expression vector pMS186, giving rise to the plas-
mid pMS190. The pMS186 contains the Gateway reading
frame cassette C (RfC) inserted between the cbh1 (cello-
biohydrolase 1) promoter and terminator, and a hygromy-
cin resistance cassette. The LR recombination reaction was
done with the Gateway Recombination kit (Invitrogen)
according to the manufacturer’s instructions.
The plasmid pMS190 was transformed into the T. reesei
strain VTT-D-00775, essentially as described [19], and
transformants were selected for hygromycin resistance on
plates containing 125 lgÆmL
)1
of hygromycin B. The trans-
formants were streaked on the selective medium for three
successive rounds and tested for tyrosinase activity with a
plate assay. In the assay plates, Trichoderma minimal med-
ium [19] with 2% lactose as a carbon source, 1% potassium
phthalate as a buffering agent (pH 5.5), 0.1 mm CuSO

2
O. The medium pH
was adjusted to 5.5–6 with NH
4
OH and H
3
PO
4
, and the
cultivation temperature was 28 °C. The dissolved oxygen
level was kept above 30% with agitation at 450 r.p.m., aer-
ation at 8 LÆmin
)1
and 0–30% O
2
enrichment of incoming
air. Foaming was controlled by automatic addition of
Struktol J633 polyoleate antifoam agent (Schill & Seilacher,
Hamburg, Germany). After fermentation, cells were har-
vested by centrifugation and the culture supernatant was
concentrated 2.5 times by ultrafiltration.
Protein and enzyme activity assays
Tyrosinase activity was measured according to Robb [2]
with a few modifications, using 15 mml-dopa and 2 mm
l-tyrosine as substrates. Activity assays were carried out in
0.1 m sodium phosphate buffer (pH 7.0) at 25 °C, monitor-
ing dopachrome formation at 475 nm (e
dopachrome
¼
3400 m

Sweden). The sample was applied to a HiPrep
tm
16 ⁄ 10 CM
Sepharose Fast Flow column, in 10 mm Tris ⁄ HCl buffer,
pH 7.3. Bound proteins were eluted with a linear NaCl gra-
dient (0–180 mm in six column volumes) in the equilibra-
tion buffer. Tyrosinase-positive fractions were pooled,
concentrated with a Vivaspin concentrator (20 mL, 10 000
molecular weight cut-off; Vivascience, Hannover, Ger-
many), and subjected to gel filtration in a Sephacryl S-100
E. Selinheimo et al. Secreted tyrosinase from Trichoderma reesei
FEBS Journal 273 (2006) 4322–4335 ª 2006 The Authors Journal compilation ª 2006 FEBS 4331
HR column (1.6 · 90 cm; Pharmacia Biotech, St Albans,
UK) equilibrated with 20 mm Tris ⁄ HCl buffer (pH 7.5),
containing 150 mm NaCl. Active fractions were pooled and
concentrated.
SDS ⁄ PAGE (12% Tris ⁄ HCl Ready Gel; Bio-Rad) was
performed according to Laemmli [20], using prestained
SDS ⁄ PAGE standards [Broad Range, Cat. no. 161-0318
(Bio-Rad); or LMW, Cat. no. 17-0446-01 (GE Healthcare,
Uppsala, Sweden)] and Coomassie Brilliant Blue (R350;
Pharmacia Biotech, St Albans, UK) for staining the proteins.
Determination of isoelectric point
The isoelectric point of the enzyme from culture superna-
tant and purified enzyme was determined by IEF within the
pH range 3.5–9.5 (Ampholine PAGplate 3.5–9.5 for IEF;
Amersham Bioscience, Uppsala, Sweden) and the pH range
8–10.5 (Pharmalyte
TM
carrier ampholyte; Amersham Bio-

2
HPO
4
con-
taining 25 mm citric acid) at a pH range of 3–7, 50 mm
Tris ⁄ HCl buffer at a pH range of 7–8.5, and 50 mm gly-
cine ⁄ NaOH buffer at a pH range of 8.5–10; the activity
was measured by following the oxygen consumption rate.
l-Tyrosine was chosen as the substrate to diminish the
effect of auto-oxidation of diphenols at alkaline pH. The
stability of the enzyme at different pH values was deter-
mined in McIlvaine universal buffer by incubating the
enzyme solution at different pH values at room temperature
for 1 h and for 1, 2 and 3 days. The residual tyrosinase
activity was determined by the spectrophotometric activity
assay using 15 mml-dopa as substrate. Temperature stabil-
ity was determined at 30 °C, 40 °C and 50 °C. The enzyme
solution in 20 mm Tris ⁄ HCl buffer (pH 7.5) was incubated
at different temperatures, and the residual enzyme activity
was determined after certain time periods by the spectro-
photometric activity assay.
Stereospecificity and substrate specificity
The stereospecificity of T. reesei TYR2 was studied by
following the activities on 15 mml-dopa, dl-dopa and
d-dopa and 2.5 mml-dopa, dl-dopa and d-tyrosine, and
the activities were measured by the spectrophotometric
activity assay. The tyrosinase activity was determined on
various compounds: l-tyrosine, phenol, 4-mercaptophenol,
p-cresol, 4-aminophenol, 3-hydroxyanthranilic acid, tyram-
ine, p-tyrosol, p-coumaric acid, o-coumaric acid, ferulic

tides and tripeptides, glycine–tyrosine and glycine–glycine–
tyrosine, were analyzed by following oxidation rate by
oxygen consumption measurements in reaction mixtures.
Reactions were performed with peptide concentration 2.5
mm, in 0.1 m NaCl/P
i
at pH 7.
Inhibition
The inhibition of tyrosinase by benzoic acid, benzaldehyde,
kojic acid, 2-mercaptoethanol, glutathione, EDTA, SDS,
sodium chloride, sodium azide and hydrogen peroxide was
analyzed by determining enzyme activity on 15 mml-dopa
in the presence of the inhibitors. The concentration of the
inhibitor was 0.1, 1, 10 or 100 mm, depending on the in-
hibition efficiency. At least two inhibitor concentrations
Secreted tyrosinase from Trichoderma reesei E. Selinheimo et al.
4332 FEBS Journal 273 (2006) 4322–4335 ª 2006 The Authors Journal compilation ª 2006 FEBS
were tested. Substrate and inhibitor compounds were dis-
solved simultaneously in 0.1 m sodium phosphate buffer
(pH 7.0), and inhibition efficiency was followed with the
oxygen consumption activity assay. Inhibition measure-
ments were also performed with spectrophotometric assay
to detect a possible quinone-binding type of inhibition.
Reversed-phase chromatography, alkylation and
tryptic digestion
For structural characterization, the protein was subjected
to reversed-phase chromatography on a TSKgel TMS-250
(C1, 1.0 · 20 mm or 2.0 · 20 mm, Tosoh Corporation,
Tokyo, Japan) column. Elution was performed with a lin-
ear gradient of acetonitrile (3–100% in 60 min) in 0.1% tri-

PAGE was performed as described by Bamford et al. [22].
MS
MALDI-TOF MS was performed using an Ultraflex TOF ⁄
TOF instrument (Bruker Daltonik, Bremen, Germany). Pro-
teins and cyanogen bromide fragments were analysed in the
linear positive mode using sinapic acid (Fluka Chemie AG,
Buchs, Switzerland) as the matrix. Peptides were analyzed in
the reflector positive mode using a-cyano-4-hydroxycinnamic
acid (Aldrich, Steinheim, Germany) as the matrix. For
MALDI-TOF ⁄ TOF fragment ion analysis of selected pep-
tides, the instrument was operated in the LID-LIFT (Bruker
Daltonik) mode. Electrospray MS was performed using a
Q-TOF instrument (Micromass Ltd, Manchester, UK).
Protein and peptide sequencing
N-terminal protein and peptide sequencing was performed
using a Procise 494A Sequencer (Perkin Elmer, Applied
Biosystems Division, Foster City, CA, USA). Proteins were
analyzed either after reversed-phase chromatography or
after electroblotting from SDS ⁄ PAGE onto a polyvinylid-
ene difluoride membrane followed by Coomassie Brilliant
blue staining [23]. Peptides were analyzed after separation
by reversed-phase chromatography.
Chemical cleavage with cyanogen bromide
The reversed-phase purified protein (about 1 nmol) was dis-
solved in 70% (v ⁄ v) trifluoroacetic acid, and 3.2 lmol of
cyanogen bromide was added. Cleavage was performed at
room temperature in the dark for 16 h. The reaction mix-
ture was then diluted 10-fold with water and dried in a
vacuum centrifuge. For separation by reversed-phase chro-
matography, the fragments were dissolved in 200 lLof

cipates the Commissions’s future policy in this area.
Also, the skillful technical assistance of Sirkka Kanervo,
Riitta Lampinen, Outi Liehunen, Kati Sulin and
Michael Bailey is acknowledged.
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