reesei tyrosinase gene tyr2, encoding a protein with a putative signal sequence.. The purified TYR2 protein had a sig-nificantly lower molecular mass 43.2 kDa than that calculated from the
Trang 1C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei
Emilia Selinheimo1, Markku Saloheimo1, Elina Ahola2, Ann Westerholm-Parvinen1, Nisse Kalkkinen2, Johanna Buchert1and Kristiina Kruus1
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 oxidao-hydroxyla-tion 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 reeseiidentified 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 30C, 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.
Trang 2In 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 oxidaregula-tion–reducregula-tion 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-met-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 (http://gsphere.lanl.gov/trire1/ 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% ideniden-tity) 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 zeaetyrosinase 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
Trang 3supernatants 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 recombirecombi-nation 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 CuSO4 in the medium The effect of copper concentra-tion on the producconcentra-tion level was studied by using 0–6 mm CuSO4 in the medium in cultures of the best transformant The optimal copper concentration was
2 mm, 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)1was 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.
Trang 4of 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 O22–fi Cu2+ 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
Cation exchange
chromatography
MW 4
3 2 1
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.
min 50
2.5 2.0 1.5 1.0 0.5 0.0
MW 1 kDa 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.
Trang 5By 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 (m¼ 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,
m¼ 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 (m¼ 673.376 Da) The mass of the
follow-ing theoretical tryptic peptide
(SSVTTIINQLYGP-NSGK, m¼ 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
(m¼ 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-terN-ter-minus 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 glyco-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 MYNMSK (m¼ 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
Trang 6other 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 30C 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 30C, 40 C and 50 C, respectively Among the tested substrates (Table 2), the highest affinity of T reesei TYR2 was observed with p-tyrosol (Km¼ 1.3 mm), followed by p-coumaric acid (Km¼ 1.6 mm) and l-dopa (Km¼ 3.0 mm) The highest turn-over number, kcat, 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
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.
Trang 7oxidation 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 Km
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 reeseiTYR2 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.
k cat ⁄ K m (s)1ÆmM)1)
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)1Æs)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
Table 4 Stereospecificity of T reesei TYR2.
Substrate (2.5 mM)
Activity (%) relative to L-dopa and L-tyrosine
Table 5 Degree of inhibition of T reesei TYR2 as determined in the presence of 15 mM L-dopa, as analyzed by oxygen consumption and spectrophotometric assay.
Inhibitor
Inhibitor (mM)
Degree (%) of inhibition
as analyzed by oxygen consumption assay
Degree (%) of inhibition
as analyzed by spectro-photometric assay
Trang 8length 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-tomycestyrosinases 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
Trang 9cleaved 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 30C Longer half-lives have been
repor-ted, e.g 2 h at 50C for P sanguineus tyrosinase
However, at 60C, 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 70–90C
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 Kmvalues for all
tested substrates, l-dopa, p-coumaric acid, and
p-tyro-sol The values were in accordance with values reported
in the literature Kmvalues 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 reeseiTYR2 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
orthoto 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-nitrophe2-aminophe-nol, 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 AGT 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 (FinnDyna-zymes, Helsinki, Finland) in a reaction mixture recommended by the manu-facturer The PCR program comprised an initial
Trang 10denatura-tion step of 3 min at 94C, followed by 25 cycles of 30 s at
94C, 45 s at 52 C and 2.5 min at 72 C This was
followed by a final elongation step of 5 min at 72C 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 reeseiexpression 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)1of 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% potassmed-ium
phthalate as a buffering agent (pH 5.5), 0.1 mm CuSO4and
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 CuSO4, 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
KH2PO4 (15), and 2 mm CuSO4.5H2O The medium pH was adjusted to 5.5–6 with NH4OH and H3PO4, and the cultivation temperature was 28C The dissolved oxygen level was kept above 30% with agitation at 450 r.p.m., aer-ation at 8 LÆmin)1 and 0–30% O2 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 mm l-dopa and 2 mm
l-tyrosine as substrates Activity assays were carried out in 0.1 m sodium phosphate buffer (pH 7.0) at 25C, monitor-ing dopachrome formation at 475 nm (edopachrome¼
3400 m)1Æcm)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 25C The reaction was initiated by addition of the enzyme to the sub-strate solution, and the oxidation rate (nmolÆL)1Æs)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 HiPreptm
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 fracequilibra-tions 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