Journal of Biotechnology 130 (2007) 471–480
Comparison of the characteristics of fungal and plant tyrosinases
Emilia Selinheimo a , Deirdre NiEidhin b , Charlotte Steffensen c , Jacob Nielsen c , Anne Lomascolo d ,
Sonia Halaouli d , Eric Record d , David O’Beirne b , Johanna Buchert a , Kristiina Kruus a,∗
a VTT Technical Research Centre of Finland, P.O. Box 1000, Espoo FIN-02044 VTT, Finland
Food Science Research Centre, Department of Life Sciences, University of Limerick, Limerick, Ireland
c Danish Institute of Agricultural Sciences, Department of Food Science, Research Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmark
d UMR 1163 INRA-Universités de Provence et de la Méditerranée, Faculté des Sciences de Luminy, Case 925, 13288 Marseille Cedex 09, France
b
Received 29 January 2007; received in revised form 16 April 2007; accepted 8 May 2007
Abstract
Enzymatic crosslinking provides valuable means for modifying functionality and structural properties of different polymers. Tyrosinases catalyze
the hydroxylation of various monophenols to the corresponding o-diphenols, and the subsequent oxidation of o-diphenols to the corresponding
quinones, which are highly reactive and can further undergo non-enzymatic reactions to produce mixed melanins and heterogeneous polymers.
Tyrosinases are also capable of oxidizing protein- and peptide-bound tyrosyl residues, resulting in the formation of inter- and intra-molecular
crosslinks. Tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT), the filamentous fungus Trichoderma
reesei (TrT) and the edible mushroom Agaricus bisporus (AbT) were compared for their biochemical characteristics. The enzymes showed different
features in terms of substrate specificity, stereo-specificity, inhibition, and ability to crosslink the model protein, ␣-casein. All enzymes were found
to produce identical semiquinone radicals from the substrates as analyzed by electron spin resonance spectroscopy. The result suggests similar
reaction mechanism between the tyrosinases. PsT enzyme had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic
l-tyrosine and diphenolic l-dopa, although the tyrosinases generally had noticeably lower activity on monophenols than on di- or triphenols. The
activity of AT and PT on tyrosine was particularly low, which largely explains the poor crosslinking ability of the model protein ␣-casein by these
enzymes. AbT oxidized peptide-bound tyrosine, but was not able to crosslink ␣-casein. Conversely, the activity of PsT on model peptides was
relatively low, although the enzyme could crosslink ␣-casein. In the reaction conditions studied, TrT showed the best ability to crosslink ␣-casein.
TrT also had the highest activity on most of the tested monophenols, and showed noticeable short lag periods prior to the oxidation.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Tyrosinase; Plant; Fungal; Specificity; Inhibition; Crosslinking
Tyrosinases (monophenol, o-diphenol:oxygen oxidoreductase, EC 1.14.18.1), often also called polyphenol oxidases,
are copper containing metalloproteins and essential enzymes
in melanin biosynthesis. Tyrosinases are widely distributed in
nature; they are found both in prokaryotic as well as in eukaryotic
microbes, in mammals and plants. These enzymes are known
as type 3 copper proteins having a diamagnetic spin-coupled
copper pair in the active centre (Lerch et al., 1986). Both cop-
Abbreviations: PT, tyrosinase from potato; AT, tyrosinase from apple; TrT,
tyrosinase from Trichoderma reesei; PsT, tyrosinase from Pycnoporus sanguineus; AbT, tyrosinase from Agaricus bisporus; Y, tyrosine; G, glycine
∗ Corresponding author at: P.O. Box 1500, Espoo FIN-02044 VTT, Finland.
Tel.: +358 50 520 2471; fax: +358 20 722 7071.
E-mail address: [email protected] (K. Kruus).
0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2007.05.018
per atoms (CuA and CuB) are coordinated by three conserved
histidine residues (Klabunde et al., 1998).
Tyrosinases are bifunctional enzymes, which catalyze the
o-hydroxylation of monophenols and subsequent oxidation of
o-diphenols to quinones (Lerch, 1983; Robb, 1984). Thus,
tyrosinases accept both mono- and diphenols as substrates. The
hydroxylation ability of the enzyme is also referred to cresolase
or monophenolase activity (EC 1.14.18.1), and the oxidation
ability to catecholase or diphenolase activity (EC 1.10.3.1).
Monophenolase activity of tyrosinases is known to be the initial
rate-determining reaction (Robb, 1984; Rodriguez-Lopez et al.,
1992). In tyrosinase-catalyzed reactions, molecular oxygen is
used as an electron acceptor and it is reduced to water.
Tyrosinases and the corresponding genes have been characterized from various sources, including bacteria, fungi, plants
and mammals (for reviews see: van Gelder et al., 1997; Halaouli
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E. Selinheimo et al. / Journal of Biotechnology 130 (2007) 471–480
et al., 2006a; Marusek et al., 2006; Mayer, 2006). The most
thoroughly characterized fungal tyrosinases both from structural and functional point of view are from Neurospora crassa
(Lerch, 1983) and Agaricus bisporus (Wichers et al., 1996).
Microbial tyrosinases have been also disclosed from Pseudomonas (McMahon et al., 2007), Bacillus and Myrothecium
(Echigo and Ohno, 1997), Mucor (Yamada et al., 1983), Miriococcum (Yamada et al., 2002), Aspergillus, Chaetotomastia,
Ascovaginospora (Abdel-Raheem and Shearer, 2002), Trametes (Tomsovsky and Homolka, 2004), Pycnoporus (Halaouli
et al., 2006b), Trichoderma (Selinheimo et al., 2006) and Streptomyces (Della-Cioppa et al., 1998a, 1998b). Tyrosinase-related
proteins from higher animals comprise human- (Kwon et al.,
1987) mouse- (Kwon et al., 1988) and frog-derived (Takase et
al., 1992) enzymes. From fruits and vegetables, at least tomato(Newman et al., 1993), potato- (Hunt et al., 1993) bean- (Gary
et al., 1992), spinach- (Hind et al., 1995), apple- (Espı́n et al.,
1995b; Ni Eidhin et al., 2006), artichoke- (Espı́n et al., 1997d),
avocado- (Espı́n et al., 1997c), pear- (Espı́n et al., 1997a), and
strawberry-derived (Espı́n et al., 1997b) tyrosinases have been
reported.
The sequence comparison of the recently published tyrosinases reveals high heterogeneity concerning the length and
overall identity. However, highly conserved regions among all
tyrosinases can be found in the active site area (van Gelder et
al., 1997; Halaouli et al., 2006a; Marusek et al., 2006). Although
the structural data on microbial tyrosinases have been limited,
the first high resolution three-dimensional structure from the
actimomycete S. castaneoglobisporus recently became available (Matoba et al., 2006). The data confirms that despite of low
sequence identity, the tyrosinase shares similar overall structure
with plant catechol oxidase from Ipomoea batatas (Klabunde et
al., 1998) and with hemocyanin, an oxygen carrier protein, from
Octopus dofleini (Cuff et al., 1998). It is assumed that all plant
and microbial tyrosinases or polyphenol oxidases have similar
architecture, represented by the structure of Ipomoea batatas
(Klabunde et al., 1998) and S. castaneoglobisporus (Matoba et
al., 2006; Decker et al., 2006).
Tyrosinases are involved in several biological functions.
Their role in melanogenesis, i.e. the biosynthesis of melanin
pigments, which are heterogeneous polyphenolic polymers distributed in all living organisms, is well accepted. In mammals,
tyrosinase-related melanogenesis is responsible for skin, eye
and hair pigmentation. Pigmentation has a fundamental role in
the protection of the skin via absorbing UV radiation (Hearing
and Tsukamoto, 1991; del Marmol and Beermann, 1996).
Tyrosinases are also suggested to be potential tools in treating
melanoma (Morrison et al., 1985; Jordan et al., 1999, 2001). Furthermore, the role of tyrosinase in neuromelanin production and
damage of neurons related to Parkinson’s disease has been extensively studied (Greggio et al., 2005). In invertebrates, tyrosinase
interfaces with defence reactions and sclerotization (Sugumaran,
2002). To date, the information on the physiological role of the
tyrosinases in microbes has been limited. However, it has been
proposed that melanin has a role in the formation of reproductive organs, spore formation, the virulence of pathogenic, and the
tissue protection after damage (Lerch, 1983). Tyrosinases also
play an important role in regulation of the oxidation–reduction
potential, and the wound healing system in plants (Mayer, 1987;
Walker and Ferrar, 1998).
Presently there is an increasing interest in using tyrosinases
in industrial applications. Traditionally tyrosinases have been
exploited in plant-derived food products, e.g. tea, coffee, raisins
and cocoa, where they produce distinct organoleptic properties
(Seo et al., 2003). However, in fruits and vegetables, tyrosinases are also related to undesired browning reactions (Martı́nez
and Whitaker, 1995; Ramirez et al., 2003), whereupon, methods for controlling tyrosinase activity are constantly searched in
the food industry. Tyrosinases can have many interesting applications in food and non-food processes, especially due to their
crosslinking abilities. It has been shown that tyrosinases can catalyze formation of covalent bonds between peptides, proteins
and carbohydrates (Åberg et al., 2004; Halaouli et al., 2005;
Freddi et al., 2006). Tyrosinase has proved to be applicable
enzyme in structure engineering of meat-derived food products
(Lantto et al., 2007). Tailoring polymers in material science, for
instance, grafting of silk proteins onto chitosan via tyrosinase
reactions has also been reported (Freddi et al., 2006; Anhgileri
et al., 2007).
The aim in this study was to compare different plant and
fungal tyrosinases from apple (AT), potato (PT), P. sanguineus
(PsT), T. reesei (TrT) and the commercially available A. bisporus
(AbT), in terms of substrate and stereo-specificity, semiquinone
production, activity on peptides, ability to crosslink a model
protein, and inhibition of the enzymatic activity.
1. Materials and methods
1.1. Enzymes
Purified and characterized fungal tyrosinase enzymes were
from P. sanguineus (PsT) (Halaouli et al., 2005) and T. reesei
(TrT) (Selinheimo et al., 2006). Plant-derived tyrosinases were
from apple (AT) (Ni Eidhin et al., 2006) and potato (PT) provided by the University of Limerick. Fungal tyrosinase from A.
bisporus (AbT) (Fluka) was also included in the experiment as
a reference enzyme. Some essential biochemical characteristics
of the tyrosinases are shown in Table 1. Protein concentration
of the enzyme preparations was determined by the Bio-Rad DC
protein assay kit (Bio-Rad, Richmond, CA, USA) with bovine
serum albumin as standard.
1.2. Enzyme activity assays
Tyrosinase activity was measured according to Robb
(1984), with 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 25 ◦ C monitoring dopachrome formation at 475 nm
(εdopachrome = 3400 M−1 cm−1 ). Tyrosinase activity was also
determined by following the consumption of the co-substrate
oxygen, with a single channel oxygen meter (Precision sensing GmbH, Regensburg, Germany). Oxygen consumption assay
was performed as described by Selinheimo et al. (2006).
E. Selinheimo et al. / Journal of Biotechnology 130 (2007) 471–480
Table 1
Molecular weight, isoelectric point (pI), pH optimum and temperature stability
(T1/2 ) of plant tyrosinases from apple (AT) (Ni Eidhin et al., 2006) and potato
(PT) (from University of Limerick), and fungal tyrosinases from P. sanguineus
(PsT) (Halaouli et al., 2005), T. reesei (TrT) (Selinheimo et al., 2006) and A.
bisporus (AbT) (from Fluka)
Enzyme source
AT
45a
PT
45a
PsT
45a
Mw (kDa)
pI
–
–
4.5–5
pH-optimum
6.0–6.5
6.0–6.5
6.5–7
Active at a pH range 5.5-8
5.5–8.5
5–8
T1/2 at 30 ◦ C (h)
>72
>72
>2d
∼12
∼5
2
T1/2 at 50 ◦ C (h)
a
b
c
d
TrT
43.5b
9
8–9.5
6–10
18
0.25
AbT
13.4 + 43c
4.7–5
6–7
–
–
–
Determined by SDS-PAGE.
Determined by MS.
Two subunits of each, total Mw 112.8 kDa.
Not defined longer than 2 h.
SDS-PAGE (12% Tris–HCl Ready Gel, Bio-Rad Laboratories, Hercules, CA, USA) was performed according to Laemmli
(1970), using pre-stained SDS-PAGE Standards (Broad Range
Cat. no. 161-0318, Bio-Rad) and Coomassie Brilliant Blue
(R350; Pharmacia Biotech, St. Albans, United Kingdom) for
staining the proteins.
1.3. Substrate and stereo-specificity
The activity of the enzymes was determined on various
compounds: l-tyrosine, phenol, p-cresol, tyramine, p-tyrosol,
p-coumaric acid, ferulic acid, 3,4-dihydroxy-l-phenylalanine
(l-dopa), (−)-epicatechin, (+)-catechin hydrate, pyrocatechol,
caffeic acid, pyrogallol and aniline. The substrates were from
Sigma, except ferulic acid and pyrocatechol were from Fluka.
The activity was measured at the substrate concentration of
2.5 mM in 0.1 M sodium phosphate buffer, pH 7.0, by following
the enzymatic reaction with the oxygen consumption assay. To
observe the possible auto-oxidation of di- and triphenols, control experiments without enzyme were performed with all of the
polyphenolic substrates. The calculations for the activities of the
enzymes on mono- and polyphenols were performed in relation
to the corresponding l-dopa activity (%).
Stereo-specificity of PT, AT, TrT, PsT and AbT was studied
by following the activities on 15 mM l-, dl- and d-dopa and
on 2.5 mM l-, dl- and d-tyrosine (from Sigma). Activities were
measured by the spectrophotometric activity assay.
1.4. Oxidation of model peptides
Activity of the enzymes was measured on selected model
dipeptides (from Fluka) and tripeptides (from Bachem),
containing tyrosine in different positions in the peptide chain. Enzymatic activity on glycine–tyrosine (GY),
tyrosine–glycine (YG), glycine–glycine–tyrosine (GGY),
glycine–tyrosine–glycine (GYG) and tyrosine–glycine–glycine
(YGG) was analyzed by following oxidation rate by the oxygen
consumption assay. Peptides (2.5 mM) were dissolved in 0.1 M
sodium phosphate buffer, pH 7.
473
1.5. Electron spin resonance (ESR) experiment
ESR experiments for semiquinone detection were performed
on a Bruker EMX X-band ESR spectrometer equipped with
an ER4119HS cavity (Bruker Analytische Messtechnik, Rheinstetten, Germany). The operating conditions were as follows:
microwave power, 20 mW; modulation frequency, 100 kHz; field
modulation amplitude, 0.2 gauss; receiver gain, 2 × 103 ; time
constant, 2.56 ms; and conversion time, 2.56 ms. After initiation
of the enzymatic reaction, reaction mixture was immediately
transferred to an ESR flat cell, mounted within the ESR cavity, after which the measurement was started instantly. ESR
signal appearance and disappearance was followed as a function of time: one measurement, a sum of 34 successive scans,
took 3.5 min, after which the ESR was restarted without a
delay. Substrates in the ESR tests were l-tyrosine (2.5 mM),
phenol (15 mM), l-dopa (15 mM) and pyrocatechol (15 mM),
prepared in 0.2 M acetate buffer (pH 6.5) containing 0.05 M
Zn2+ . Zinc ions were included in the reaction mixtures to stabilize the possible formation of ortho-semiquinones (Yamasaki
and Grace, 1998). Reactions were made at ambient temperature and in a volume of 0.5 ml. For each individual substrate
studied, enzyme dosing between the tyrosinases in ESR tests
was set according to reaction rates calculated from the substrate
specificity determination to correspond equal activity on the
substrate.
1.6. Crosslinking of model proteins
Ability of PT, AT, TrT, PsT and AbT to crosslink a model protein ␣-casein (Bovine Milk, Calbiochem, CN Biosciences Inc.)
was studied. Casein was dissolved in 50 mM sodium phosphate
buffer pH 7.0 at concentration of 3 mg/ml. To study the effect
of a small phenolic molecule on a crosslinking process, l-dopa
(2 mM) was also added to the reaction mixture. Enzyme dosages
were 100 and 1000 nkat per gram casein. Incubations were made
at 30 ◦ C for 2 and 24 h. Enzymes were inactivated at 95 ◦ C for
10 min, after which crosslinking of casein was monitored by
SDS-PAGE analysis.
1.7. Inhibition studies
Inhibition of tyrosinases by benzaldehyde, kojic acid, 2mercaptoethanol, glutathione, ethylenediaminetetraacetic acid
(EDTA), sodium dodecyl sulphate (SDS), sodium chloride and
sodium azide, was analyzed by determining the enzyme activity
on 15 mM l-dopa in the presence of the inhibitors (1–100 mM).
Substrate and inhibitor compounds were dissolved simultaneously in 0.1 M sodium phosphate buffer, pH 7.0, and inhibition
efficiency was followed with the spectrophotometric activity
assay.
2. Results
Amino acid sequence alignments of PsT (AAX44240;
Halaouli et al., 2005), AbT (CAA59432, AbPPO1 and
CAA61562, AbPPO2; Wichers et al., 2003) and TrT
474
E. Selinheimo et al. / Journal of Biotechnology 130 (2007) 471–480
Fig. 1. Amino acid sequence alignments of the studied fungal tyrosinase enzymes at the conserved CuA and CuB sites. T. reesei (TrT) (CAL90884, Selinheimo et
al., 2006); P. sanguineus (PsT) (AAX44240, Halaouli et al., 2005); and A. bisporus (AbPPO1 and AbPPO2) (CAA59432 and CAA61562, Wichers et al., 2003). The
sequences of polyphenoloxidases from potato Solanum tuberosum (PotPPO, AAA85122) (Thygesen et al., 1995); and apple Malus domestica (AppPPO, AAA69902)
(Boss et al., 1995) are also presented. Copper site histidines are shown as bold. The amino acids of AbT and TrT identical to the PsT sequence are shown under grey.
(CAL90884; Selinheimo et al., 2006) in the conserved CuA
and CuB binding area are shown in Fig. 1. In comparison, also
the sequences from polyphenol oxidases from potato Solanum
tuberosum (PotPPO, AAA85122) (Thygesen et al., 1995); and
apple Malus domestica (AppPPO, AAA69902) (Boss et al.,
1995) are presented (Fig. 1). It should be pointed out that the corresponding sequences of PT and AT studied in this work are not
known. Amino acid identities between the fungal tyrosinases,
PsT to AbT (AbPPO1), PsT to TrT, and TrT to AbT (AbPPO1),
determined by NCBI Blast2 (http://www.ebi.ac.uk/blastall/),
are 46, 34, and 27%, respectively. Sequence homologies of
PotPPO and AppPPO between the fungal tyrosinases were
around 10–20%.
2.1. Substrate specificity
Comparison of the substrate specificity of PT, AT, TrT,
PsT and AbT on various mono- and diphenols indicated that
PT and especially AT tyrosinase had clearly lower activity
on monophenols than on diphenols (Table 2). In fact, AT
could not oxidize phenol and p-coumaric acid. Interestingly,
the monophenols p-cresol and p-tyrosol were relatively well
oxidized by AT and especially by PT. The activity on the
tested phenolic acids varied depending on the enzyme and the
acid. Ferulic acid was not a substrate to any of the tyrosinases, and p-coumaric acid was rapidly oxidized only by TrT.
On the other hand, diphenolic caffeic acid was oxidized relatively fast by all tyrosinases, except only moderately by
PsT. Stereo-specificity of AT, PsT and TrT was found to be
rather similar (Table 3); l-forms of dopa and tyrosinase were
much better substrates than the corresponding d-forms. Interestingly, PT and AbT oxidized l- and d-forms with similar rate.
Because the activity of the AT on tyrosine was practically nondetectable, no significant differences between the oxidation rates
on the d-, dl- and d-forms of tyrosine could be measured for
AT.
2.2. ESR measurements with mono- and diphenols
Structurally similar compounds, l-tyrosine versus l-dopa
and phenol versus pyrocatechol, were used to examine possible
differences in the semiquinone formation from mono- and
diphenolic compounds by different enzymes. Using l-tyrosine
as substrate, ESR spectra with two overlapping low intensity
multiline ESR signals were detected (Fig. 2A). TrT produced a
short-term ESR signal in the very beginning of the measurement, whereas with AbT and PsT a lag phase was observed
prior to the signal (data on lag periods and signal intensities not
shown). Furthermore, the signal was present clearly longer with
AbT and PsT. PT and AT did not give any ESR signals with
Table 2
Activity (%) of the tyrosinases from apple (AT), potato (PT), P. sanguineus
(PsT), T. reesei (TrT) and A. bisporus (AbT) on mono and polyphenolic compounds as calculated in relation to l-dopa
Substrate (2.5 mM)
l-Dopa
l-Tyrosine
Phenol
p-Cresol
Tyramine
p-Tyrosol
p-Coumaric acid
Ferulic acid
(−)-Epicatechin
(+)-Catechin hydrate
Pyrocatechol
Caffeic acid
Pyrogallol
Enzyme source
AT
PT
PsT
TrT
Activity in relation to l-dopa (%)
AbT
100
1
0
14
2
9
0
0
330
347
158
153
133
100
20
17
21
17
32
0
0
116
114
132
194
100
100
2
3
31
9
29
3
0
215
140
131
180
90
100
51
11
16
35
33
6
0
330
347
191
26
51
100
17
20
29
7
57
65
0
106
220
88
211
55
Oxygen consumption is based on the linear part of the O2 consumption curve.
Relative activity (%) on mono- and polyphenols from oxygen consumption
(nmol l−1 s−1 ) was calculated according to the stoichiometry that one monophenol molecule needs one oxygen molecule and one polyphenol molecule needs
0.5 oxygen molecule in the reaction to form a quinone.
E. Selinheimo et al. / Journal of Biotechnology 130 (2007) 471–480
Table 3
Stereo-specificity of the tyrosinases from apple (AT), potato (PT), P. sanguineus
(PsT), T. reesei (TrT) and A. bisporus (AbT)
Substrate (2.5 mM)
l-Dopaa
dl-Dopaa
d-Dopaa
l-Tyrosineb
dl-Tyrosineb
d-Tyrosineb
a
b
Enzyme source
AT
PT
Relative activity (%)
PsT
TrT
AbT
100
86
60
100
110
107
100
59
39
100
76
46
100
118
106
0
0
0
100
67
100
100
45
25
100
36
7
100
102
61
Activity in relation to 15 mM l-dopa (%).
Activity in relation to 2 mM l-tyrosine (%).
l-tyrosine, which is consistent with the substrate specificity
determination, where the oxidation of tyrosine was found
to be almost negligible. On the other hand, with phenol a
9-line ESR signal was detected with all enzymes (Fig. 2C),
although the signal intensity with PT and especially with
AT was clearly lower than with TrT, AbT and PsT. The lag
phase prior to the ESR signal from phenol was most clearly
observed with AT, PT, and also with AbT and PsT enzymes.
However, with TrT the ESR signal from phenol was detected
immediately, similarly as from l-tyrosine. Simulations of the
9-line ESR signal from the phenol-derived radical revealed
hyperfine coupling to two times two equivalent aromatic protons
(2aH = 3.71G, 2aH = 0.47G), consistent with the enzymatic
hydroxylation and oxidation of phenol to an o-semiquinone
475
radical. The symmetry of the phenol-derived o-semiquinone
is not present in l-tyrosine-derived semiquinones, as the
(s)-2-amino-propionic acid group is substituted to the aromatic
ring. As a consequence, the l-tyrosine-derived semiquinone
radical has no equivalent hydrogen atoms. Simulations of the
overlapping multi-line ESR signals from l-tyrosine-derived
radicals reveal two radicals, one characterized by hyperfine
coupling to four inequivalent protons (aH = 5.46G, aH = 3.66G,
aH = 0.6G, aH = 0.25G) and the other by the hyperfine coupling
to 3 inequivalent protons (aH = 3.36G, aH = 0.91G, aH = 0.74G),
which are consistent with the formation of o-semiquinones
from both protonated and unprotonated l-tyros ine
(Kalyanaraman, 1990).
Due to fast auto-oxidation of the diphenolic substrates l-dopa
and pyrocatechol, a semiquinone radical was present without any
enzyme addition. l-dopa and pyrocatechol gave semiquinonederived signals similar to l-tyrosine and phenol, respectively,
which is explicable by their structural similarity. Pyrocatechol
was the only substrate, from which an ESR signal, although a
very weak and short-term one, was detected also without an addition of Zn2+ . In the presence of zinc ions, which were included
in the reaction mixtures to stabilize the possible formation of
ortho-semiquinones (Yamasaki and Grace, 1998), pyrocatechol
gave clearly the most intense signal when compared to the
other substrates. Thereby, probably the ESR measurement was
not sensitive enough to detect any signals from l-tyrosine, ldopa and phenol without zinc addition. Nevertheless, a signal
from pyrocatechol could be caught also without zinc, which
proved that the ESR signal was not only related to the effect of
zinc.
Fig. 2. ESR signals derived from the semiquinone radicals formed from l-tyrosine (A), l-dopa (B), phenol (C) and pyrocatechol (D) in the reactions catalyzed by
apple, potato, P. sanguineus, T. reesei and A. bisporus tyrosinases. The semiquinone-derived signals shown are the signals detected with the highest intensity, i.e.
signals for l-tyrosine, phenol, l-dopa and catechol are from the reaction with T. reesei, P. sanguineus, A. bisporus and A. bisporus tyrosinases, respectively (Y-axis,
arbitrary units).
476
E. Selinheimo et al. / Journal of Biotechnology 130 (2007) 471–480
Table 4
Activity of the tyrosinases from apple (AT), potato (PT), P. sanguineus (PsT), T.
reesei (TrT) and A. bisporus (AbT) on di- and tripeptides, calculated in relation
to l-tyrosine (%)
Peptides (2.5 mM)
Ya
YGb
GY
YGG
GGY
GYG
a
b
Table 5
Degree of inhibition (%) of the tyrosinases from apple (AT), potato (PT), P.
sanguineus (PsT), T. reesei (TrT) and A. bisporus (AbT) by various inhibitors
Inhibitor (mM)
Enzyme source
Enzyme source
AT
PT
PsT
TrT
Activity in relation to l-tyrosine(%)
AbT
0
0
0
0
0
0
100
140
115
140
110
110
100
450
520
350
430
480
100
30
30
40
60
60
100
130
290
110
340
230
Y equals tyrosine.
G equals glycine.
2.3. Model peptides
Tyrosinase activities were compared on the different model
peptides containing tyrosine residue in different positions in
the peptide chain (Table 4). PT, TrT, PsT and AbT were able
to oxidize all tested model peptides, whereas the activity of
AT on tyrosine and tyrosine-containing peptides was scarcely
detectable. Oxidation rate of the peptides was found to depend
on the length and the position of a tyrosyl residue. The di- and
tripeptides, especially with PT, but also by TrT, were oxidized
faster than tyrosine. Oxidation of the peptides YG and YGG,
in which the tyrosyl residue is in the N-terminus, was relatively
slower, especially by TrT, but to some extent also with PT. On
the other hand, with PsT and AbT, the location of the tyrosine
in the peptide chain did not affect substantially on the oxidation rate. Interestingly, with PsT the oxidation rate, decreased
with increasing the length of the peptide chain, whereas PT oxidized four to five times better the peptide-bound tyrosine when
compared to tyrosine.
2.4. Inhibition
Kojic acid, ␤-mercaptoethanol and glutathione were the
strongest inhibitors among the tested for all tyrosinases
(Table 5). It should be pointed out that thiol compounds, such as
glutathione, do not always inhibit the enzymatic catalysis, but
affect on the subsequent non-enzymatic reactions as quinone
binders (Moridani et al., 2001; Garcia-Molina et al., 2005; Land
et al., 2004). Sodium chloride and EDTA did not cause severe
inhibition to any of the tyrosinases. SDS behaved interestingly
in the inhibition assay: SDS inhibited TrT moderately, while
the activity of AT and PT increased in the presence of SDS. It
has been reported that SDS participates in activation of some
tyrosinases. The mechanism is likely related to conformational
changes, which open the active site and permit substrate’s access
(Moore and Flurkey, 1990; Jiménez and Garcı́a-Carmona, 1996;
Espı́n and Wichers, 1999).
2.5. Crosslinking of α-casein
The crosslinking ability of AT, PT, TrT, PsT and AbT was
studied using ␣-casein as a model protein. In the incubation for
AT
PT
PsT
TrT
AbT
Activity in relation to 15 mM l-dopa (%)
Sodium azide
10
1
50
16
78
54
69
17
91
75
100
92
Kojic acid
10
1
100
89
100
82
99
77
100
98
73
47
␤-Mercaptoethanol
10
1
100
100
100
100
100
100
100
100
100
100
SDS
10
1
−4
−1
−77
−64
0
5
73
44
9
2
Benzaldehyde
10
1
40
28
82
36
34
11
42
13
93
89
Glutathione
10
1
100
88
100
94
100
100
100
100
100
100
NaCl
100
10
24
7
25
8
12
4
49
0
53
18
EDTA
10
1
−1
1
1
4
−1
3
13
7
−2
10
24 h, PsT and TrT were found to crosslink ␣-casein directly, as
visualized by the decrease in the intensity of the casein subunits
and by the formation of the higher molecular weight proteins in
the SDS-PAGE analysis (Fig. 3, gel A: lane 5 and gel B: lanes 4
and 5). Notable crosslinking of ␣-casein was also observed with
a lower TrT dosage (100 nkat/g casein), whereas PsT induced
crosslinking only with a dosage of 1000 nkat/g casein. The
crosslinking of ␣-casein by TrT was already detectable within a
2 h reaction time, whereas no noticeable changes were observed
with the other tyrosinases (data on 2 h incubations not shown).
AT, PT and AbT did crosslink ␣-casein. According to the
literature, small phenolic compounds could enhance tyrosinasecatalyzed crosslinking of proteins (Thalmann and Lötzbeyer,
2002); therefore, an addition of l-dopa was also tested in the
crosslinking experiment. When l-dopa was added to the reaction mixture, crosslinking of ␣-casein was observed also by AT,
PT and AbT. Crosslinking was enhanced in PsT catalyzed reactions in presence of l-dopa as well (Fig. 3, lanes 6 and 7). In
contrast to the other enzymes, crosslinking efficiency of TrT
decreased when l-dopa was added to the reaction mixture.
3. Discussion
Tyrosinases generally show much lower specific activity
for hydroxylation of monophenols than for oxidation of odiphenols. Especially plant tyrosinases are typically found to
have low or no monophenolase activity (Mayer and Harel,
1979; Robb, 1984; Mayer, 1987; Espı́n et al., 1995a, 1998;
Martı́nez and Whitaker, 1995). However, a tyrosinase from Rastonia solanacearum was recently found to have a clearly higher
monophenolase/diphenolase activity ratio when compared to the
other reported tyrosinases (Hernández-Romero et al., 2006). Of
E. Selinheimo et al. / Journal of Biotechnology 130 (2007) 471–480
477
Fig. 3. Crosslinking of ␣-casein proteins by P. sanguineus (gel A) and T. reesei (gel B), apple (gel C), potato (gel D), and A. bisporus (gel E), tyrosinases (TYR) after
24 h incubation time. Lanes: (1) molecular weight marker, (2) ␣-casein, (3) ␣-casein + l-dopa, (4) ␣-casein + TYR 100 nkat/g protein, (5) ␣-casein + TYR 1000 nkat/g
protein, (6) ␣-casein + l-dopa + TYR 100 nkat/g protein, (7) ␣-casein + l-dopa + TYR 1000 nkat/g protein, (8) molecular weight marker.
the tyrosinases examined in this study, AT, PT, TrT and AbT
showed higher activity on diphenols than on monophenols. All
enzymes oxidized monophenolic and diphenolic compounds,
showing, thus, characteristic of tyrosinase activity. The highest
monophenolase/diphenolase activity ratio, as determined on ltyrosine and l-dopa following dopachrome formation at 475 nm,
was detected for the PsT enzyme (data not shown). The activity ratio for PsT was 0.5, whereas for AT, PT, TrT and AbT
the ratio was 0, 0.008, 0.053 and 0.058, respectively. Thereby,
of the enzymes studied, PsT showed atypically high activity on
l-tyrosine in relation to the l-dopa activity.
None of the tyrosinases was able to oxidize ferulic acid, most
presumably because of steric hindrance caused by the methoxy
group next to the phenolic hydroxyl group. The presence and the
position of an amine group in the substrate molecule appeared to
decrease the substrate oxidation rate, especially by TrT, and also
to some extent by AT and PT. Substituted phenols, for example aminophenols, have been reported to be strong tyrosinase
inhibitors, and the inhibitory mechanism is suggested to be competitive due to the structural similarity between these inhibitory
compounds and substrates (Conrad et al., 1994; Piquemal et al.,
2003). The negative effect of the amino group on the activity
of TrT was also observed in the oxidation of the tripeptides,
where the oxidation rate of YG and YGG, i.e. tyrosine locating
in the amino terminus of the peptide, was clearly reduced when
compared to oxidation rate of GY, GGY and GYG. On the con-
trary, the catalytic activity of PsT and AbT decreased when a
carboxyl group was present in the substrate structure, as in the
p-coumaric acid molecule. Kubo et al. (2004), Lim et al. (1999)
and Winkler et al. (1981) have reported that p-coumaric acid
can preferentially bind to the active site of some tyrosinases also
with the carboxylic group and, thus, compete with the hydroxyl
group present in the substrate. TrT showed clearly higher relative activity on the carboxylated substrates when compared to
PsT and AbT enzymes. The result might indicate differences
in the substrate binding area or the active site of these tyrosinase enzymes. Interestingly, also the stereo-specificity varied
between the tyrosinases. Whereas the l-forms were much better
substrates than the d-forms for AT, PsT and TrT, PT and AbT
oxidized l- and d-forms with a similar rate.
All enzymes were found to produce semiquinones
(Kalyanaraman, 1990) as analyzed by ESR. The semiquinonederived ESR signals from diphenolic l-dopa and pyrocatechol
and from monophenolic phenol and l-tyrosine substrates were
similar in all tested tyrosinase-catalyzed reactions. However, AT
and PT did not produce any detectable semiquinones from ltyrosine, most likely due to the low activity on the substrate. The
identical semiquinone radicals produced by the studied tyrosinase suggest that similar reaction mechanisms are involved in
the reactions catalyzed by the tyrosinases.
Enzymes capable of introducing crosslinks in protein or
carbohydrate matrix have recently been used to improve the
478
E. Selinheimo et al. / Journal of Biotechnology 130 (2007) 471–480
functionality of food and non-food proteins (Chen et al., 2003;
Åberg et al., 2004; Freddi et al., 2006; Lantto et al., 2007).
Transglutaminases have been used to improve the technological or nutritional properties of protein-based food ingredients,
by incorporating enzymatically lysine and lysine dipeptides to
casein proteins (Nonaka et al., 1996). Furthermore, production of ␤-lactoglobulin emulsion gels by transglutaminase has
been studied by Dickinson (1997), and the enzyme-crosslinked
ovomucin-␣s1 -casein conjugate has been suggested to have better emulsifying properties than pure ␣s1 -casein (Kato et al.,
1991). In the present study, the suitability of five tyrosinase
enzymes from plant and fungal sources to crosslink ␣-casein
model proteins was analyzed. The ability of PT, AT, TrT, PsT
and AbT to crosslink ␣-casein proteins was different. From the
tested enzymes only TrT and PsT were able to directly crosslink
␣-casein, whereas AT, PT, and AbT were able to from crosslinks
in the presence of l-dopa. Halaouli et al. (2005) have reported
PsT to crosslink casein proteins and Thalmann and Lötzbeyer
(2002) have reported that AbT could crosslink ␣-lactalbumin,
but crosslinking of lysozyme and ␤-lactoglobulin by AbT was
possible only in the presence of low molecular weight phenolic
compound, caffeic acid. The authors suggested that the phenolic
compounds act as bridging agents between the protein subunits.
Although the TrT enzyme could efficiently crosslink ␣casein, interestingly the crosslinking efficiency by TrT was
substantially reduced when l-dopa was added to the reaction
mixture. The reason behind the phenomenon is not known. It
could be that the highly reactive dopaquinones resulting from
dopa oxidation could have reacted non-enzymatically further to
the side groups of amino acid residues participating in crosslinking, thus, blocking crosslinking of ␣-casein. Quinones have been
reported to condense with phenolic coupling or to react with
amino acid side groups, such as sulfhydryl, amine, amide, indole
and imidazole groups (Bittner, 2006). TrT and PsT showed differences in oxidizing l-tyrosine and l-dopa: activity of TrT on
l-dopa was found to be nineteen times higher than on l-tyrosine,
whereas PsT oxidized tyrosine and dopa with a rather similar
rate. Because TrT favored l-dopa over l-tyrosine, the oxidation
could primarily lead to melanogenesis.
The observed differences in the direct crosslinking ability
of the enzymes might be related to the different readiness of
the enzymes to oxidize monophenolic tyrosine in proteins. In
the tyrosinase-catalyzed reactions, a lag period for the oxidation
reaction of monophenols is usually present. The lag phase is
known to be related to the state of the active site of tyrosinase
enzymes. The resting state of the enzyme usually consists of
85–90% of a met-form that can perform only the catalysis with
diphenols, whilst 10–15% of enzyme is in an oxy-form that is
capable for the monophenol oxidation (Solomon et al., 1996).
During the lag period, the oxy-form of tyrosinase is generated
from the met-form and the rate of oxidation accelerates to reach
the maximum (Cooksey et al., 1997; Land et al., 2004). When
comparing the duration of a lag period for the oxidation of ltyrosine by the tyrosinases, the TrT enzyme showed the shortest
lag period with the monophenols studied, suggesting the fastest
transformation to the oxy-form. According to the substrate specificity determination and the ESR experiment, TrT had only a
very short lag phase prior to l-tyrosine oxidation, whereas all
the other tyrosinases showed clearly longer lag periods. Therefore, the efficient crosslinking of ␣-casein by TrT might relate to
the high natural readiness of TrT to oxidize monophenols, such
as l-tyrosine.
The differences in the protein crosslinking ability of the
tyrosinases might also be related to the differences in the accessibility of the tyrosine residues of ␣-casein to active site of the
enzymes. As PsT showed some crosslinking ability, but displayed a lag period like AbT, a lag period alone cannot explain
the protein crosslinking efficiency of the tyrosinases. The inability of AbT to crosslink ␣-casein appears even more interesting
when compared to PsT, as AbT was observed to be able to oxidize peptide-bound tyrosine similarly as free tyrosine, which is
contrary to PsT, which showed reduced oxidation rate for the
peptides. Besides, PT oxidized much better the peptide-bound
tyrosine, when compared to l-tyrosine, but neither PT could act
on protein-bound tyrosine. Thereby, the inability of AbT and PT
to oxidize casein-bound tyrosine might relate to poor accessibility of protein-bound tyrosine to the active site of AbT. It has been
postulated that differences in the so-called gate residue, locating above the active site of tyrosinase, result in the differences in
monophenolase activity of plant, fungal and bacterial tyrosinases
(Matoba et al., 2006; Marusek et al., 2006). Bulky phenylalanine
as a gate residue in plant tyrosinases is suggested to cause blocking of substrate’s access and binding to CuA site (Klabunde et
al., 1998). As the corresponding residue in fungal and bacterial
tyrosinases is usually either leucine or proline, the entrance to
the active site cavity is suggested to be more open (Matoba et
al., 2006; Marusek et al., 2006). Furthermore, in TrT the amino
acid sequence in the gate residue area is interestingly different
from AbT and PsT (Fig. 1), and for instance, the methionine
conserved in both plant and fungal tyrosinases (Halaouli et al.,
2006a; Marusek et al., 2006) is missing in TrT. Nevertheless, to
understand the structure function of these tyrosinases in detail,
the structural data of the proteins or their close homologies is
needed.
We have shown that the different tyrosinases from plant and
fungal origin show interesting differences in their ability to oxidize and crosslink the model substrates. All the tested tyrosinases
crosslinked the model protein either directly or in presence of
small molecular weight phenolic compound. The enzymes are,
thus, potential for crosslinking applications.
Acknowledgements
This paper has been carried out with financial support
from the Research Foundation of Raisiogroup (Raisio, Finland)
and the Commission of the European Communities, specific
RTD programme “Quality of Life and management of Living Resources,” proposal number QLK1-2002-02208 “Novel
crosslinking enzymes and their consumer acceptance for structure engineering of foods,” acronym CROSSENZ. It does not
reflect Commissions’s views and in no way anticipates the Commissions’s future policy in this area.
Markku Saloheimo is acknowledged for the expression construct for TrT production and Michael Bailey for the TrT
E. Selinheimo et al. / Journal of Biotechnology 130 (2007) 471–480
production. The skilful technical assistance of Riitta Isoniemi
is also acknowledged.
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