Tài liệu Báo cáo khoa học: Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei ppt

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.

Tyrosinase thus accepts monophenols and diphenols as
substrates, and the monophenolase activity is the ini-
tial rate-determining reaction [2,3].
Keywords
fungal; secreted; Trichoderma reesei;
tyrosinase
Correspondence
E. Selinheimo, VTT Technical Research
Centre of Finland, PO Box 1000,
Espoo FIN-02044 VTT, Finland
Fax: +358 20 722 7071
Tel: +358 20 722 7135
E-mail: Emilia.Selinheimo@vtt.fi
(Received 16 May 2006, revised 7 July
2006, accepted 27 July 2006)
doi:10.1111/j.1742-4658.2006.05429.x
A homology search of the genome database of the filamentous fungus
Trichoderma reesei identified a new T. reesei tyrosinase gene tyr2, encoding
a protein with a putative signal sequence. The gene was overexpressed
in the native host under the strong cbh1 promoter, and the tyrosinase
enzyme was secreted into the culture supernatant. This is the first report on
a secreted fungal tyrosinase. Expression of TYR2 in T. reesei resulted in
good yields, corresponding to approximately 0.3 and 1 gÆL
)1
tyrosinase in
shake flask cultures and laboratory-scale batch fermentation, respectively.
T. reesei TYR2 was purified with a three-step purification procedure,
consisting of desalting by gel filtration, cation exchange chromatography
and size exclusion chromatography. The purified TYR2 protein had a sig-
nificantly lower molecular mass (43.2 kDa) than that calculated from the

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

in microorganisms is very limited. The most extensively
investigated fungal tyrosinases, from both a structural
and a functional point of view, are from Agaricus
bisporus [10] and Neurospora crassa [1]. Studies with
N. crassa have shown that the enzyme is completely
absent in the vegetative stage. However, under stress
conditions high levels of the enzyme can be induced
[1]. This suggests that tyrosinases are not essential to
the metabolism of the fungi, but improve the survival
and competence of the fungi by producing melanins.
Tyrosinases have been shown to share a similar act-
ive site with catechol oxidase and hemocyanin, a pro-
tein involved in oxygen transport in arthropods and
molluscs [11]. These proteins are type 3 copper pro-
teins with a diamagnetic spin-coupled copper pair in
the active center. Each of the two copper atoms is
coordinated by three conserved histidine residues [12].
Molecular oxygen is used as an electron acceptor and
it is reduced to water in tyrosinase-catalyzed reactions.
On the basis of thorough chemical and spectroscopic
analyses of tyrosinases, the binuclear active site is
known to exist in three states: oxy-tyrosinase, met-
tyrosinase and deoxy-tyrosinase [13–15]; a catalytic
cycle, in which these states are alternated, has been
proposed [16]. The met state is the resting state of the
enzyme, and in the absence of substrate about 85–90%
of the enzyme is in this state [16]. Both the met and
oxy states of tyrosinases can catalyze the diphenoloxi-
dase reaction, whereas the monohydroxylase reaction
requires the oxy state. Just recently, the first tyrosinase

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

amino acids, including a predicted signal sequence of
18 amino acids, and three potential N-glycosylation
sites. The closest homologs of the T. reesei TYR2 pro-
tein are putative tyrosinases from the fungi Gibberella
zeae (46% amino acid identity), N. crassa (35% iden-
tity) and Magnaporthe grisea (34% identity). All these
three proteins are predicted by the signalp program to
have a signal sequence; however, none of them has
been characterized at the protein level. The amino acid
identity of TYR2 to the intracellular tyrosinase from
Pycnoporus sanguineus is 34% [25], whereas the amino
acid identity to other fungal tyrosinases is around
25–33%. Alignment of T. reesei TYR2 with the
G. zeae tyrosinase places the suggested signal sequence
cleavage sites of both enzymes precisely at the same
location (Fig. 1). When these two sequences are
aligned with the intracellular tyrosinase characterized
from N. crassa [26], the suggested N-termini of the
mature secreted enzymes coincide with the N-terminus
of the intracellular enzyme (Fig. 1). The segments in
the N-terminal portion around the copper ligand
amino acids of the active site are well conserved
between the proteins, whereas the C-terminal domains
are less conserved.
Overexpression of the tyrosinase gene in
Trichoderma reesei
Tyrosinase production by an untransformed T. reesei
strain was tested, but no tyrosinase activity in culture
E. Selinheimo et al. Secreted tyrosinase from Trichoderma reesei
FEBS Journal 273 (2006) 4322–4335 ª 2006 The Authors Journal compilation ª 2006 FEBS 4323

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
. The best
tyr2-overexpression transformant was grown in a
laboratory fermenter in a volume of 20 L. The enzyme
production increased continuously during culture, and
the activity level of 300 nkatÆmL
)1
was reached after 6
days of cultivation. Although the activity was still
increasing, the fermentation had to be stopped because
Fig. 1. Alignment of T. reesei TYR2
(TrTYR2) amino acid sequence with the
putatively secreted tyrosinase of Gibberella
zeae (GzTYR) and the intracellular tyrosinase
characterized from Neurospora crassa
(NcTYR1). Identical amino acids are indica-
ted by asterisks, conserved substitutions by
colons, and similar amino acids by dots. The
hisitidines acting as ligands for the Cu
atoms A and B are shaded. The Cys–His
thioester bond in the active site is indicated.
Signal sequence cleavage sites of TrTYR2
and GzTYR are indicated by arrows. The last
amino acids of processed TrTYR2 and
NcTYR1 are marked by triangles. Putative
N-glycosylation sites are in bold.
Secreted tyrosinase from Trichoderma reesei E. Selinheimo et al.
4324 FEBS Journal 273 (2006) 4322–4335 ª 2006 The Authors Journal compilation ª 2006 FEBS
of foaming problems. According to the specific activity
of the purified tyrosinase (300 nkatÆmg

)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–

28]. The purified tyrosinase had an absorption maxi-
mum at around 350 nm, which is an indication of a
T3-type copper pair in its oxidized form with a brid-
ging hydroxyl moiety, assigned as an O
2
2–
fi Cu
2+
charge transfer transition [29].
For molecular characterization, the purified enzyme
was first subjected to reversed-phase chromatography,
where it eluted as one symmetric peak (Fig. 3).
Further analysis of the reversed-phase purified protein
by SDS ⁄ PAGE still gave a double protein band
corresponding to a molecular mass of about 43 kDa.
Table 1. Purification of T. reesei TYR2.
Purification step
Total activity
(nkat)
Total protein
(mg)
Specific activity
(nkatÆmg
)1
)
Activity
yield (%)
Purification
factor
Culture filtrate 50 000 816 61.3

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

45.0
30.0
20.1
14.4
AU 214 nm
Fig. 3. Reversed-phase chromatographic analysis of T. reesei TYR2
from the last gel filtration purification step. Chromatography was
performed on a 1 · 20 mm TSKgel TMS-250 column using a linear
gradient of acetonitrile (3–100% in 60 min) in 0.1% trifluoroacetic
acid and a flow rate of 40 lLÆmin
)1
. The eluted protein peak was
collected in two fractions, which were analyzed by 12%
SDS ⁄ PAGE (insert). Gel lanes: 1 and 2, equal samples from the
first half and second half of the peak, respectively; MW, molecular
mass markers.
E. Selinheimo et al. Secreted tyrosinase from Trichoderma reesei
FEBS Journal 273 (2006) 4322–4335 ª 2006 The Authors Journal compilation ª 2006 FEBS 4325
By MALDI-TOF MS, the reversed-phase purified pro-
tein gave a single peak corresponding to an average
molecular mass of 43.3 kDa (not shown). Further-
more, in an electrospray MS analysis, using a quadru-
pole time-of-flight (Q-TOF) instrument, which has
a considerably better resolution, the same protein
preparation resulted in a set of masses among which
43 124.0 Da and 43 204.0 Da were dominant (not
shown). The results thus indicate that the protein exists
in different post-translationally modified forms. To
further characterize the molecule, the reversed-phase
purified protein was subjected to N-terminal sequen-

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

obtained from the ladders of b-fragment and y-frag-
ment ions. Subsequently, for specifying the C-termi-
nus of the protein, the most C-terminal tryptic
peptide, as compared with the theoretical tryptic pep-
tide map of the deduced protein, was found to be
SQAQIK (m ¼ 673.376 Da). The mass of the follow-
ing theoretical tryptic peptide (SSVTTIINQLYGP-
NSGK, m ¼ 1777.927 Da) could not be found,
indicating that the C-terminus of the protein is within
this sequence. In the peptide mass fingerprint, a mass
corresponding to the peptide SSVTTIINQLYGPNSG
(m ¼ 1649.826 Da) was found. The identity of this
C-terminal peptide was further confirmed by
MALDI-TOF ⁄ TOF fragment ion analysis as well as
by Edman degradation of the corresponding peptide
after purification by reversed-phase chromatography.
During the search for and confirmation of the N-ter-
minus and C-terminus of the protein, many other pep-
tides were also analyzed, and the results confirmed
most of the remaining deduced amino acid sequence.
Edman sequencing of purified tryptic peptides covered
39.1% of the sequence. The molecular masses of the
tryptic peptides, either from the mass fingerprint or
purified peptides, covered 60.3% of the sequence, and
the masses of cyanogen bromide fragments, including
the N-terminal and C-terminal ones, covered 94.9% of
the sequence. Together with the results from the glyco-
peptide analysis (see below), the peptide analyses com-
pletely confirmed the deduced amino acid sequence of
the secreted protein. The calculated average molecular

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 nonglycosylated peptide. Further downstream in
the fragment ion spectrum, a b-ion ladder correspond-
ing to the amino acid sequence LYVQAM was detec-
ted, which confirmed the identity of the peptide. Thus,
the purified protein is N-glycosylated at the asparagine
residue (N62, Fig. 1) having a high-mannose-type
glycan consisting of two N-acetylglucosamines and five
hexoses. From the ConA eluate, other masses corres-
ponding to the same tryptic peptide but with a shorter
glycan (e.g. peptide + 1 · GlcNAc, m ¼ 2220.12 Da)
were also detected, which indicates that the presence of
Secreted tyrosinase from Trichoderma reesei E. Selinheimo et al.
4326 FEBS Journal 273 (2006) 4322–4335 ª 2006 The Authors Journal compilation ª 2006 FEBS
other shorter glycan structures cannot be excluded.
N-Glycans with five mannoses have been found as the
predominant form previously in the Cel7A cellulase of
T. reesei [30]. In the same study, N-glycosylation sites
with a single GlcNAc were also found.
In order to clarify the reason for the existence of
TYR2 as a double band in SDS ⁄ PAGE, the two pro-
tein bands were individually cut out and ‘in-gel’ diges-
ted with trypsin. Mass fingerprint analysis of the
tryptic fragments by MALDI-TOF MS did not show
significant differences in the mass fingerprints, thus
leaving the reason for the double band unclear.
T. reesei TYR2 was shown to be almost fully active
within a pH range of 6–9.5, with an optimum at pH 9.
Considering the stability of T. reesei TYR2 within a
pH range of 2–8, the enzyme showed good stability at
neutral and alkaline pH. When the pH was under 7,

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

cat
, was observed with l-dopa, at
22 s
)1
. Substrate specificity determination for T. reesei
TYR2 showed that the enzyme was able to oxidize
various substituted monophenols, which had the OH
group in the para position (Table 3). The activity of
the enzyme on diphenols was substantially higher than
on monophenols; for example, catechol was oxidized
approximately 10 times faster than phenol. Interest-
ingly, aniline, containing no hydroxyl groups in the
aromatic ring, but an amino group, was also oxidized
by the tyrosinase, although slowly. Any side chain
ortho to the phenolic hydroxyl group prevented
8.5-
8.0-
7.5-
7.0-
6.5-
6.0
5.5-
5.0-
4.5-
4.0-
3.5-
3.0-
2.5-
2.0-
1.5-

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

1375.497
1539.592
Fig. 4. The MALDI-TOF MS ⁄ MS spectrum of a ConA affinity-purified tryptic glycopeptide from T. reesei TYR2. The peptide with a deter-
mined monoisotopic protonated mass of 3256.309 Da (shown in the insert) was selected as the precursor ion and analyzed in the LID-LIFT
mode without collision gas. The resulting fragment ions correspond to a sequential loss of one Na
+
, five hexoses and two N-acetylglucosa-
mines, resulting in a molecule with a protonated mass of 2017.0 Da. The fragmentation ion ladder at the lower molecular mass range corres-
ponds to a sequence LYVQAMY, which confirms the identity of the glycopeptide.
E. Selinheimo et al. Secreted tyrosinase from Trichoderma reesei
FEBS Journal 273 (2006) 4322–4335 ª 2006 The Authors Journal compilation ª 2006 FEBS 4327
oxidation of the substrate, presumably because of ster-
ic hindrance. The presence and the position of an
amine group in the substrate structure appeared to be
critical, considering the oxidation of the substrate by
T. reesei TYR2. The closer to the hydroxyl group of
phenol the amino group was, the slower was the oxida-
tion of the substrate. For most of the substrates stud-
ied, increasing the substrate concentration from
2.5 mm to 10 mm (or to 20 mm, data not shown) did
not substantially affect the activity as calculated
relative (%) to l-dopa (Table 3). However, different
stereo-forms of catechin behaved differently. As the
concentration of (+)-catechin and (–)-catechin was
increased from 2.5 to 10 mm, (–)-catechin was oxidized
faster, suggesting that T. reesei TYR2 has a lower K
m
value for (–)-catechin than for (+)-catechin. Further-
more, T. reesei TYR2 was found to be stereospecific; it
oxidized the l-forms of dopa and tyrosine noticeably

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
)

k
cat
⁄ K
m
(s
)1
ÆmM
)1
)
L-Dopa 3.0 22 7
p-Coumaric acid 1.6 8 5
p-Tyrosol 1.3 7 6
Table 3. Substrate specificity of T. reesei TYR2 as determined
relative to
L-dopa. ND, not determined due to the low solubility.
Relative activity (%) on monophenols and polyphenols from oxygen
consumption (nmolÆL
)1
Æs
)1
) was calculated according to the stoichio-
metry that one monophenol molecule needs one oxygen molecule,
and one polyphenol molecule needs half an oxygen molecule, in
the reaction to form a quinone. c, substrate concentration.
Substrate
Activity (%) relative to
L-dopa
c ¼ 2.5 m
M c ¼ 10 mM
L

-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

the presence of 15 m
ML-dopa, as analyzed by oxygen consumption
and spectrophotometric assay.
Inhibitor
Inhibitor
(m
M)
Degree (%) of
inhibition
as analyzed by
oxygen
consumption
assay
Degree (%) of
inhibition
as analyzed by
spectro-
photometric
assay
Sodium azide 10 50 91
139 75
Kojic acid 10 99 100
195 98
b-Mercaptoethanol 10 100 100
1 100 100
SDS 10 68 73
139 44
Benzaldehyde 10 64 42
130 13
Glutathione 10 18 100

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-

ted. This is exceptional in this class of enzymes, as all
the plant, animal and fungal tyrosinases studied thus
far have been intracellular. The characterized Strep-
tomyces tyrosinases are secreted but do not have signal
sequences; their secretion is assisted by a second pro-
tein that has a signal sequence [41,42]. It appears that
other ascomycetous fungi also have secreted tyrosin-
ases, because the three closest homologs of T. reesei
TYR2 from G. zeae, N. crassa and Magnaporthe grisea
have putative signal sequences. In fact, N. crassa has
both secreted and intracellular tyrosinases; the enzyme
cloned and studied previously is intracellular [26].
From an industrial point of view, a naturally secreted
tyrosinase can be considered beneficial, as such an
enzyme is likely to be compatible with the secretory
system of the host organism in attempts to produce
substantial amounts of enzyme for applications.
Although microbial tyrosinases have been produced
heterologously, e.g. in Eschericia coli [32,43,44] and in
Saccharomyces cerevisiae [45], the expression levels
reported thus far have been relatively low. The avail-
ability of the enzyme has hampered its detailed charac-
terization as well as testing it in various applications.
The T. reesei TYR2 tyrosinase gene was expressed in
T. reesei under the strong cbh1 promoter. In shake
flasks, the highest production level was approximately
320 mgÆL
)1
, whereas production levels were over three
times higher than this in fermenter conditions. The

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

our results, the secreted T. reesei TYR2 is also C-ter-
minally processed (after Gly410) (Fig. 1). However, in
this case the peptidase performing the cleavage must
reside in the secretory pathway or be extracellular. The
precise processing site has previously been determined
only for the N. crassa tyrosinase (after Phe408)
(Fig. 1). According to the alignment of T. reesei TYR2
and the N. crassa tyrosinase, the positions of the pro-
cessing sites in these two enzymes coincide exactly,
even though the sequences are not conserved in that
region. This is compatible with the idea that this site is
at a domain border that would be susceptible to pro-
teases. The processed N. crassa tyrosinase ends with a
phenylalanine, and thus it was assumed that it is
Table 6. Activity of T. reesei TYR2 on 2.5 mM dipeptides and trip-
eptides in relation to
L-tyrosine (%). Y, tyrosine; G, glycine.
Substrate
Activity (%)
relative to Y
Y 100
GY 292
GGY 335
E. Selinheimo et al. Secreted tyrosinase from Trichoderma reesei
FEBS Journal 273 (2006) 4322–4335 ª 2006 The Authors Journal compilation ª 2006 FEBS 4329
cleaved by a chymotrypsin-like enzyme [26]. The
Agaricus bisporus tyrosinase can be processed in vitro
by the serine proteases trypsin and subtilisin [28]. The
processed T. reesei TYR2 ends with a glycine residue.
Analysis of the whole sequence with the program pep-

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-

tion of A. flavipes [54] and N. crassa [56,57] tyrosinases
at relatively low temperatures has been reported.
The enzyme showed relatively high K
m
values for all
tested substrates, l-dopa, p-coumaric acid, and p-tyro-
sol. The values were in accordance with values reported
in the literature. K
m
values for l-dopa were 3.0 mm for
T. reesei TYR2, 0.74–1.09 mm for N. crassa [1,58],
5.0 mm for A. flavipes [54], 5.97 mm for Streptomyces
glaucescens [59] and 8.7–10 mm for pine needle [52].
Trichoderma reesei TYR2 showed surprisingly broad
substrate specificity and higher oxidation activity for
diphenols than for monophenolic substrates. Ferulic
acid, as well as other compounds with a side group
ortho to the phenolic hydroxyl group, was not oxidized
by the enzyme, presumably because of steric hin-
drance. The substituted phenols, such as 2-aminophe-
nol and 4-nitrophenol, or benzene derivatives, such as
benzoic and naphthoic acids, have been reported to be
efficient tyrosinase inhibitors, and the inhibitory mech-
anism is suggested to be competitive docking, due to
the similarity between the structures of these inhibitory
compounds and those of phenol or tyrosine [60,61].
For instance, Piquemal et al. [61] showed in a theoret-
ical study that 2-aminophenol forms a more stable and
energetically favored complex with tyrosinase than
phenol does. The amine group also seemed to act as a

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

Isolation of the tyrosinase gene from
Trichoderma reesei
The tyr2 gene was amplified from genomic T. reesei DNA
with the following primers: forward, GGG GAC AAG
TTT GTA CAA AAA AGC AGG CTA TCA TGC TGT TGT
CAG GTC CCT CTC G; and reverse, GGG GAC CAC
TTT GTA CAA GAA AGC TGG GTC A GT GGT GGT
GGT GGT GGT GCA GAG GAG GGA TAT GGG GAA
CGG CAA A. The PCR reaction was done with the Dyna-
zyme EXT thermostable polymerase (Finnzymes, Helsinki,
Finland) in a reaction mixture recommended by the manu-
facturer. The PCR program comprised an initial denatura-
Secreted tyrosinase from Trichoderma reesei E. Selinheimo et al.
4330 FEBS Journal 273 (2006) 4322–4335 ª 2006 The Authors Journal compilation ª 2006 FEBS
tion step of 3 min at 94 °C, followed by 25 cycles of 30 s at
94 °C, 45 s at 52 °C and 2.5 min at 72 °C. This was
followed by a final elongation step of 5 min at 72 °C. The
tyr1 gene fragment was cloned into the pCR2.1TOPO
vector with the TOPO-TA Cloning Kit (Invitrogen, Carls-
bad, CA, USA) and subsequently transferred into the
pDONR221 vector (Invitrogen) with a BP recombination
reaction carried out with the Gateway Recombination kit
(Invitrogen).
The tyr2 cDNA was isolated by RT-PCR from a cDNA
expression library of T. reesei RutC-30 [18] with primers
that were designed to create an N-terminal His6 tag and
add EcoRI and KpnI restriction endonuclease sites to the 5¢
and 3¢ ends, respectively. The primers used were as follows:
forward primer, GTT GGA ATT CCA TCA TCA TCA
TCA TCA TCA GGG CAC GAC ACA CAT CCC C; and

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

4
and
1% tyrosine as an indicator substrate was used. The trans-
formants were streaked on the plates and grown for 7 days,
and tyrosinase activity was observed on the plates as a
brown color appearing around the streaks. Positive trans-
formants were isolated by single-spore cultures. In order to
quantify tyrosinase production in liquid cultures, the trans-
formants positive in the plate assay were grown in shake
flasks for 8 days in 50 mL of Trichoderma minimal medium
[19] supplemented with 4% lactose, 2% spent grain,
100 mm piperazine-N-N¢-bis(3-propanesulfonic acid) and
0.1–2 mm CuSO
4
, and tyrosinase activity was measured
with 3,4-dihydroxy-l-phenylalanine (l-dopa) substrate as
described below.
Trichoderma reesei was cultivated in a Braun Biostat
C-DCU 3 fermenter (B. Braun Biotech International,
GmbH, Melsungen, Germany) in 20 L of a medium con-
taining (gÆL
)1
): lactose (20), distiller’s spent grain (10), and
KH
2
PO
4
(15), and 2 mm CuSO
4
.5H

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

)1
Æcm
)1
). Tyrosinase activity was also determined by
following the consumption of the cosubstrate oxygen with a
single-channel oxygen meter (Precision Sensing GmbH,
Regensburg, Germany). The activity was determined by
measuring the oxygen consumption during the reaction in a
sealed and a fully filled sample vial (1.8 mL) at 25 ° C. The
reaction was initiated by addition of the enzyme to the sub-
strate solution, and the oxidation rate (nmolÆL
)1
Æs
)1
) was
calculated from the linear part of the oxygen consumption
curve. The protein concentration was determined with the
Bio-Rad DC protein assay kit (Bio-Rad, Richmond, CA,
USA), with BSA as standard. During enzyme purification,
to estimate protein contents for pooling fractions, protein
content determinations were done by monitoring absorb-
ance at 280 nm.
Enzyme purification
The concentrated culture supernatant was first desalted on
a Sephadex G-25 Coarse column (2.6 · 27 cm; Pharmacia
Biotech, Uppsala, Sweden) in 10 mm Tris ⁄ HCl buffer,
pH 7.3. The subsequent purification steps were carried out
with an A
¨
KTApurifier (Amersham Biosciences, Uppsala,

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-

science, Uppsala, Sweden), on an LKB 2117 Multiphor II
Electrophoresis System (LKB Pharmacia, Bromma, Swe-
den) according to the manufacturer’s instructions. Bands
containing tyrosinase activity were visualized by staining
the gel with 15 mml-dopa in 0.1 m sodium phosphate buf-
fer (pH 7.0), and proteins were visualized by Coomassie
Blue staining.
Kinetic parameters, pH optimum and stability
and thermal inactivation
The K
m
and k
cat
values for T. reesei TYR2 using l-dopa,
p-coumaric acid and p-tyrosol as substrates were deter-
mined by following the enzymatic oxygen consumption.
Substrates were dissolved at a concentration of 0.2–15 mm
in 0.1 m sodium phosphate buffer (pH 7.5). Reactions were
carried out at 25 °C; 14.6 lgofT. reesei TYR2 was used
for l-dopa, and 4.9 lg for p-coumaric acid and p-tyrosol.
The Michaelis–Menten curves for determination of K
m
and
k
cat
values were obtained with the graph pad prism 3.02
program (GraphPad Software Inc., San Diego, CA, USA).
Determination of the pH optimum for T. reesei TYR2 was
carried out with 2 mml-tyrosine as substrate, dissolved in
50 mm McIlvaine universal buffer (50 mm Na

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

acid, l-dopa, (–)-epicatechin, (+)-catechin hydrate, pyro-
catechol, pyrogallol and aniline. The activity was measured
relative to the l-dopa activity with substrate concentrations
of 2.5, 10 and 20 mm in 0.1 m sodium phosphate buffer
(pH 7.0) by following the enzymatic reaction with oxygen
consumption measurement. The solubility of some com-
pounds restricted determination at higher concentrations;
for example, l-tyrosine was soluble only up to 2.5 mm, and
l-dopa up to 15 mm. To observe the possible auto-oxida-
tion of diphenols and triphenols, control experiments with-
out enzyme were performed with all of the polyphenolic
substrates. Relative enzymatic activities of T. reesei TYR2
on monophenols and polyphenols was calculated from oxy-
gen consumption (nmolÆ L
)1
Æs
)1
), according to the stoichio-
metry of the supposed tyrosinase reaction pathway [9]: to
form a quinone from the substrate, one monophenolic sub-
strate molecule needs 1.5 O
2
and one polyphenolic substrate
molecule needs 0.5 O
2
in the oxidation reaction. All calcu-
lations were performed in relation to the corresponding
l-dopa activity (%).
Oxidation of model peptides
The activities of T. reesei TYR2 on selected model dipep-

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-

fluoroacetic acid at a flow rate of 0.05 mLÆmin
)1
(1 mm
internal diameter column) or 0.2 mLÆmin
)1
(2 mm internal
diameter column), and absorbance was monitored at
214 nm. Aliquots of the collected protein were directly sub-
jected to MALDI-TOF MS, electrospray MS and N-ter-
minal sequencing. The rest of the protein was dried,
alkylated with 4-vinylpyridine, desalted by reversed-phase
chromatography and subjected to enzymatic digestion with
trypsin (1%, w ⁄ w, Sequencing Grade Modified Trypsin,
V5111; Promega, Madison, WI, USA) overnight at 37 °C,
as described by Kerovuo et al. [21]. Separation of tryptic
peptides was performed by reversed-phase chromatography
on a 0.1 · 15 cm Vydac C8 column (300 A
˚
,5lm; LC-
Packings, Amsterdam, the Netherlands). Elution was per-
formed at a flow rate of 40 lLÆmin
)1
, with a linear gradient
of acetonitrile (0–40% in 120 min) in 0.1% trifluoroacetic
acid. Chromatography was monitored at 214 nm and the
peptides were collected automatically (SMART
tm
System;
Pharmacia Biotech, Uppsala, Sweden). ‘In-gel’ digestion of
Coomassie Brilliant blue-stained protein bands from SDS ⁄

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

50 mm Tris ⁄ Cl (pH 7.5), 150 mm NaCl, and 0.1 m dithio-
threitol.
Purification and analysis of glycopeptides
The reversed-phase purified protein (about 1 nmol) was
cleaved overnight with trypsin (1% w ⁄ w) in 0.1 m ammo-
nium bicarbonate at 37 °C. Glycopeptides were adsorbed
onto a 2 · 20 mm ConA Sepharose (Amersham Pharmacia
Biotech, Uppsala, Sweden) column equilibrated with 20 mm
Tris ⁄ Cl (pH 7.4), 0.5 m NaCl, 1 mm CaCl
2
, and 1 mm
MnCl
2
. Bound glycopeptides were eluted with 0.5 m
methyl-a-d-mannopyranoside in the equilibration buffer
and desalted on a reversed-phase C
18
ZipTip (ZTC 18M
096; Millipore Corporation, MA, USA).
Acknowledgements
This work was carried out with financial support from
the Research Foundation of Raisiogroup (Raisio, Fin-
land), the Finnish Funding Agency for Technology and
Innovation (TEKES) and the Commission of the Euro-
pean Communities, specifically RTD program ‘Quality
of Life and Management of Living Resources’, pro-
posal number QLK1-2002-02208, ‘Novel crosslinking
enzymes and their consumer acceptance for structure
engineering of foods,’ acronym CROSSENZ. It does
not reflect the Commissions’s views and in no way anti-

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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