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Bacterial tyrosinases and their applications
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Greta Faccioa,b, Kristiina Kruusb, Markku Saloheimob, Linda Thöny-Meyera
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a Empa,
Swiss Federal Laboratories for Materials Science and Technology - Laboratory for
Biomaterials, Lerchenfeldstrasse 5, CH-9014 St. Gallen, Switzerland
b VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, 02044 Espoo, Finland
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Greta.Faccio@empa.ch - Kristiina.Kruus@vtt.fi - Markku.Saloheimo@vtt.fi –
Linda.Thoeny@empa.ch
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Corresponding author
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Linda Thöny-Meyer
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Empa, Swiss Federal Laboratories for Material Sciences and Technology
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Laboratory for Biomaterials, Lerchenfeldstrasse 5
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CH-9014 St. Gallen, Switzerland
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Tel. +41 58 765 7792
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Abstract
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Tyrosinases with different physico-chemical properties have been identified from various bacterial phyla
such as Actinobacteria and Proteobacteria and their production is often inducible by environmental stresses.
Tyrosinases are enzymes catalysing the oxidation of mono- and di-phenolic compounds to corresponding
quinones with the concomitant reduction of molecular oxygen to water. Since the quinone produced can
further react non-enzymatically with other nucleophiles, e.g. amino groups, many tyrosinases have a
recorded cross-linking activity on proteins. Various bacterial tyrosinases oxidise tyrosine, catechol, L/DDOPA, caffeic acid and polyphenolic substrates such as catechins. This substrate specificity has been
exploited to engineer biosensors able to detect even minimal amounts of different phenolic compounds. The
physiological role of tyrosinases in the biosynthesis of melanins has been used for the production of
coloured and dyeing agents. Moreover, the cross-linking activity of tyrosinases has found application in food
processing and in the functionalization of materials. Numerous tyrosinases with varying substrate
specificities and stability features have been isolated from bacteria and they can constitute valuable
alternatives to the well-studied tyrosinase from common mushroom.
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Keywords
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Tyrosinase, biosynthesis, biosensors, bioremediation, food, dyeing
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Abbreviations
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ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); COD, chemical oxygen demand; DOPA,
3,4-dihydroxyphenylalanine; SDS PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis;
TYR, tyrosinase.
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1. Introduction
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Tyrosinases are copper-dependent enzymes. They catalyse the ortho-hydroxylation of monophenols such as
tyrosine and the subsequent oxidation to quinones (Figure 1). Tyrosinases comprise the activity of catechol
oxidases (EC. 1.10.3.1), a family of structurally similar enzymes whose activity is limited to diphenolic
substrates. However, the substrate specificity of tyrosinases and catechol oxidases can overlap with the one
of laccases (EC 1.10.3.2) which are structurally different enzymes lacking monophenol hydroxylase activity,
and these enzymes are sometimes grouped under the name ‘polyphenol oxidase’.
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To date, three three-dimensional structures from tyrosinase have been published. In 2006, the first crystal
structure of a tyrosinase from the bacterium Streptomyces castaneoglobisporus was solved [5]. In 2011, the
three-dimensional structure of the bacterial tyrosinase from Bacillus megaterium as well as that of the fungal
enzyme from Agaricus bisporus were published [6, 7]. Additionally, the three-dimensional structure of two
structurally related enyzmes, the catechol oxidases from sweet potato (Ipomoea batata) and the polyphenol
oxidase from grapes (Vitis vinifera) are available [8, 9]. Recent sequence analyses revealed a similarity
between bacterial and fungal polyphenol oxidases (tyrosinases) which carry features that are not present in
the corresponding enzymes from plants [10].
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The application of tyrosinases to different fields, ranging from food to materials, relies on the ability of these
enzymes to oxidise phenolic groups from both small molecules such as tyrosine to polymeric substrates such
as proteins, thus enabling polymer cross-linking. Although many bacterial tyrosinases have been
characterised to some extent, the information about them, e.g. substrate specificity and stability features, is
neither easily accessible nor has it been clearly summarised. Bacterial tyrosinases have been the subject of
two previous review articles dealing with the structural features [11] and the molecular properties [12] of
this class of enzymes. A review focusing on tyrosinases from streptomycetes has been published recently
[13]. The present review provides a more comprehensive overview on the biochemical properties of the
reported bacterial tyrosinases and on their various applications in different fields.
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2. Reaction and structural features of tyrosinase
Tyrosinases oxidise phenolic hydroxyl groups of small molecules or large polymeric substrates such as
proteins. Tyrosinases catalyse first the ortho-hydroxylation of the phenolic substrate and second its
subsequent oxidation to quinone (Figure 1) with the concomitant reduction of oxygen to water. The reaction
is chromogenic as the quinones produced can undergo further non-enzymatic polymerisation to form black
eu-melanins and, when reacting with thiol groups, brownish pheo-melanins [14]. This process can be
inhibited by antioxidants such as ascorbic acid, for example to prevent the browning reaction in food
preparations [15]. Tyrosinase activity is generally measured by either determining the consumption of
oxygen during the reaction or spectrophotometrically by following the increase of absorbance at 475 nm due
The ability of bacteria to produce melanins has long been known. Tyrosinase (EC 1.14.18.1), the key
enzyme initiating the biosynthetic pathway, has been characterised in many species. Genes coding for
proteins that carry the characteristic tyrosinase domain have been identified in many of the bacterial
genomes sequenced to date. Similarly, tyrosinases are present in fungi, plants and animals. The synthesis of
microbial melanin can also involve other enzymes than tyrosinase such as laccases, polyketide synthases, phydroxyphenylpyruvate oxidase and 4-hydroxyphenylacetic acid hydroxylase [1]. Bacterial melanin plays a
protective role in different ways: it protects DNA from the damages of UV radiation and reactive oxygen
species [2], it is able to bind toxic heavy metals [3] and to interact with DNA, possibly slowing down the
metabolism [4]. Tyrosinases are thus important for the survival of the organisms.
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to dopachrome formation. The cross-linking activity of tyrosinase on proteins is usually analysed by SDS
PAGE, size-exclusion chromatography, UV spectroscopy or mass spectrometry [16].
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The active site of tyrosinases interacts with both the phenolic substrates and the co-substrate oxygen and it
alternates among three different oxidation states. When in the oxy state, tyrosinase binds oxygen and is able
to catalyse the hydroxylation of monophenols to diphenols, thus changing into the met form. The met form
of tyrosinase is responsible for the oxidation of diphenols to quinones and the reaction turns the enzyme into
the deoxy form that, upon binding molecular oxygen, returns to the oxy form. The met form is the resting
state of the enzyme and it has been calculated that up to 85% of the enzyme is in this state when in solution
[17, 18]. The inability of most of the enzymes in an enzyme population to act on monophenols explains why
a significant lag phase is detected in the activity when monophenols are the substrate of the reaction.
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Bacterial tyrosinases have been divided in five types according to the organisation of domains and the
possible requirement of a caddie protein for enzyme activity [12]. The necessity of a secondary helper
protein (caddie protein) for secretion, correct folding, assembly of the copper atoms and activity of the
enzyme is common to tyrosinases of type I, e.g. the enzyme from S. castaneoglobisporus and S. antibioticus
[19, 20]. Type II tyrosinases are small, monomeric enzymes containing only the catalytic domain, which do
not require additional helper proteins and are possibly secreted. An example is the tyrosinase from B.
megaterium [6]. Type III tyrosinases are represented by the enzyme from Verrucomicrobium spinosum. Like
the fungal tyrosinases it carries a C-terminal domain whose removal led to about 100-fold higher activity
[21]. This supports the theory that the role of the C-terminal extension in plant and fungal tyrosinases is to
keep the enzyme in an inactive form inside the cell [22-24]. Among the smallest bacterial tyrosinases
reported (Type IV) are the ones produced by Streptomyces nigrifaciens (18 kDa) and Bacillus thuringiensis
(14 kDa) [25, 26]. However, it is debated whether these proteins are true tyrosinases [12]. Type V
tyrosinases include enzymes that do not carry the sequence features of tyrosinases but show features typical
of laccase and have only marginal activity on tyrosine. For example, a membrane-bound tyrosinase active on
the typical laccase substrate ABTS (NCBI ID: AAF75831.2) has been isolated from Marinomonas
mediterranea. A tyrosinase with a classical substrate specificity that is activated by SDS (NCBI ID:
AAV49996.1) has also been reported from the same organism [27].
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Similar to catechol oxidases and the oxygen carrying haemocyanins, tyrosinases are type-3 copper proteins,
containing two copper atoms in the active site. The absorbance spectrum of oxy-tyrosinases has a
characteristic maximum in the UV region (330-345 nm). As reported for the structurally similar catechol
oxidases, a fluorescence intensity maximum at 330 nm upon excitation at 280 nm is also detected [28, 29].
Copper is essential for the catalytic activity of tyrosinases. The crystal structure of these enzymes has
demonstrated the presence of two copper ions in the catalytic core (Table 1). In all tyrosinases of different
origins and in the haemocyanins each of the copper ions is coordinated by three histidine residues that are
found in a characteristic pattern in the primary structure (Figure 2). In the tyrosinase from Streptomyces
glaucescens, for example, the key role of histidines at position 37, 53, 62, 189, 193 and 215 in the
coordination of copper, and thus in catalytic activity, was confirmed by the decrease of activity upon their
substitution with other amino acids [30, 31].
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Various additional residues have been identified to have a function in fungal and bacterial tyrosinases, either
being essential for or modulating tyrosinase activity. Sequence analysis and various mutagenesis studies
have been performed in order to identify the residues necessary for the activity of the enzyme. In tyrosinase
sequences from plants and fungi, the N-terminal signal peptide, when present, is followed by a conserved
arginine residue that marks the beginning of the central catalytic domain and that forms a pi-cation
interaction with a conserved C-terminal Y/FXY tyrosine motif, where X is any amino acid [32]. These
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residues are conserved also in bacterial tyrosinases (Supplementary file 1). Substitution of the N-terminal
conserved arginine (R40) has been reported to abolish the production of tyrosinase from V. spinosum [21].
Two single-amino acid substitutions have been reported to improve the catalytic activity of the tyrosinase
from Rhizobium etli CFN42. The independent replacement of proline at position 334 and of aspartic acid at
position 535 (Supplementary file 1) with a smaller residue such as serine (P334S) or glycine (D535G),
respectively, led to a significant enhancement of the catalytic activity and melanin formation [33-35]. In the
tyrosinase from B. megaterium, a single substitution of arginine by histidine within the copper B binding
region (R209H) has been sufficient for a 1.7-fold improvement of the activity towards tyrosine
(monophenolase) and for a 1.5-fold reduction of activity on L-DOPA (diphenolase), whereby the overall
protein stability was not affected [36]. The crystal structure of the tyrosinase from B. megaterium showed
that this arginine is positioned at the entrance of the active site in a flexible position and plays a role in the
docking of the substrate [6]. However, the conservative substitution of the corresponding residue asparagine
190 to glutamine (N190Q) in S. glaucescens tyrosinase abolished the catalytic activity, indicating that this
residue was possibly involved in hydrogen bonding at the active site [30]. Moreover, the conservative
substitution of the residue aspartic acid 209 (D209E) has been reported to stabilise the oxy-form of the same
enzyme [37]. To our knowledge, no study has investigated the role of the oxygen binding motif PYWDW
[38] with regards to the affinity for oxygen in tyrosinase. The affinity for the co-substrate oxygen has been
evaluated for the tyrosinase from Streptomyces antibioticus that carries the PYWDW motif. It was found
that this enzyme had a three-fold lower dissociation constant (kD) for oxygen than the A. bisporus tyrosinase
[39, 40] that carries a PFWDW motif, i.e. 16.5 μM compared to 46.6 μM. The analysis of the characterised
bacterial tyrosinases evidenced the presence of functionally active variants of this motif (Supplementary file
1 and 2), e.g. PYWNY in the tyrosinase from M. mediterranea, PFWDW in tyrosinase from R. etli,
PYWEW in the tyrosinase from B. megaterium, PYWRF and PYWNW in the tyrosinases from Ralstonia
solanacearum. Mutational studies have also addressed the interaction of tyrosinases from streptomycetes
and their caddie protein. In S. antibioticus, the two histidine residues at positions 102 and 117 of the caddie
protein MelC1 have been found to be crucial for the biosynthesis of active tyrosinase [41].
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The available crystal structures of bacterial tyrosinases and their mutant forms have been obtained from
Gram-positive S. castaneoglobisporus and B. megaterium (Table 1). While the B. megaterium tyrosinase
formed crystals containing only the enzyme, the S. castaneoglobisporus tyrosinase required the presence of
a second protein, referred to as caddie protein, to stabilise its structure [4]. Moreover, the structure of the
Streptomyces tyrosinase has been solved in different states of oxidation. Aiming at understanding the
interaction between tyrosinase and caddie protein, tyrosinase has been crystallised in the presence of mutant
forms of the caddie protein (Table 1). Likewise, the fungal tyrosinase from A. bisporus was crystallised as a
tetramer in a complex with a second protein, a lectin-like protein [7].
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Both intracellular and secreted bacterial tyrosinases have been isolated and characterised. For example, the
tyrosinases from Streptomyces nigrifaciens, Bacillus thuringiensis, M. mediterranea, R. solanacearum and
Thermomicrobium roseum were isolated from cell biomass and the ones from S. antibioticus, S. glaucescens,
S. castaneoglobisporus, Streptomyces albus, B. megaterium, Sinorhizobium meliloti, Aeromonas media, R.
etli and V. spinosum were either isolated from the culture medium or predicted to be secreted [19, 21, 25,
26,42-51]. The twin-arginine signal peptide is often found in cofactor-binding oxidoreductases that undergo
complete folding in the cytoplasm prior to secretion to the periplasmic or extracellular space. Twin-arginine
type signal peptides [52] could be identified in the N-terminal region of tyrosinases from R. solanacearum
(34-amino acid long) and V. spinosum (33-amino acid long). A more detailed analysis of the sequence
retrieved for the tyrosinase from R. etli and the alignment with the other sequences of tyrosinases
(Supplementary File 1) suggests the possibility of incorrect open reading frame prediction. The true N5
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terminal methionine may be M112 (underlined in Supplementary file 1) as it aligns with the initial residue of
the tyrosinase from R. solanacearum (number 15 in Supplementary file 1) and is followed by a predicted
twin-arginine signal peptide of 31 amino acids [51]. Thus, we suggest that these proteins purified from the
cell biomass but carrying a signal peptide for secretion are localised in the periplasm.
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Tyrosinases, also from bacteria, and their caddie proteins generally lack conserved cysteine residues (for
comments see [11, 12]). The paucity of cysteine residues, and thus disulphide bonds, allowed, however, the
isolation of tyrosinases with significant thermal stability, e.g. the enzyme from B. megaterium had an
optimum temperature of 50°C [48]. A single cysteine residue is conserved in proximity of the second
histidine residue of the copper A binding motif in the characterised tyrosinases from M. mediterranea, R.
solanacearum, S. meliloti, R. etli and V. spinosum (Supplementary file 1). A cysteine residue at this position
has been found to be covalently bound to a histidine residue two positions forward in, for example, the
fungal tyrosinase from Neurospora crassa [53], the plant catechol oxidase from I. batata [8] and
haemocyanins from the snail Helix pomatia [54]. The function of this unusual cysteine-histidine bond is not
established, but it could confer structural rigidity to the copper-binding region and affect the redox potential
[8]. Replacement of this cysteine residue (C84) with serine abolished the production of the tyrosinase from
V. spinosum [21]. Type-3 copper proteins carrying six conserved cysteines (forming three in silico predicted
disulphide bonds) and characterised by significant thermal stability have been reported in fungi [26]. No
mutagenesis study has addressed a possible improvement of the thermal stability of bacterial tyrosinases by
introducing disulphide bonds. However, in silico analysis revealed the possible presence of one disulphide
bond in the tyrosinases from R. solanacearum and S. meliloti and two in the enzymes from M. mediterranea
and R. etli (Dianna software, http://clavius.bc.edu/~clotelab/DiANNA). The tyrosinase from S.
castaneoglobisporus and the one from B. megaterium share approximately 30% sequence similarity with a
catechol oxidase from Aspergillus oryzae that showed a melting temperature above 70°C and a half-life of
20 hours when incubated at 50°C [29].
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It should be noted that the tyrosinase from A. media exhibits different sequence features when compared to
the other enzymes. The sequence alignment with bacterial tyrosinases shows that none of the typical
signature motifs (copper A and B regions, oxygen binding motif and tyrosine motif) are present (see
Supplementary file 1). Moreover, this enzyme has a predicted 23-amino acid long signal peptide [50] and
shows strong sequence similarity to bacterial periplasmic proteins that are responsible for the uptake of
peptides and involved in nutrition and sensing of the environment [55].
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3. Biochemical properties and distribution of bacterial tyrosinases
Although the ability of bacteria to synthesise melanin has been reported for various species (the latest
example being the proteobacterium Brevundimonas sp. SGJ [56]), the information concerning the
characterisation of purified bacterial tyrosinase enzymes is limited and not easily available. Tyrosinases with
different biochemical properties and cellular localisation have been identified from organisms belonging to
different bacterial phyla (Figure 3), particularly from streptomycetes.
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Many bacterial genomes carry more than one operon containing tyrosinase-coding genes. The genome
analysis of the Gram-positive streptomycetes, for example, revealed two operons melC and melD,
responsible for the production of two tyrosinases, MelC2 and MelD2, respectively, with quite different
properties [57]. The melD operon was identified in all the genomes analysed, while the melC operon was
only present in the melanin-producing Streptomyces strains. Tyrosinase MelC2 is secreted and has activity
on a wide range of substrates, whereas MelD2 is intracellular (membrane-associated) and has a narrower
substrate specificity, i.e. it is not active on ortho-aminophenol and caffeic acid [57]. Melanin production is
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associated with the presence of the operon containing the tyrosinase MelC2, and albino mutants have been
found to harbour only the melD operon. MelC2 has been shown to promote catechol uptake by the cells, by
oxidising them to more hydrophobic quinones. MelD2 has been suggested to play a protective role against
the toxic oxygen-reactive species that can be spontaneously produced from phenolic compounds in the cell
[57]. Two genes coding for tyrosinase have also been identified in the genome of the Gram-negative plant
pathogen R. solanacearum (NCBI ID: NP_518458 and NP_519622). Although both corresponding
intracellular proteins carried the sequence features typical of tyrosinases, their biochemical characterisation
revealed that the former had a significant preference for monophenols and the latter for diphenols, like a
catechol oxidase [43]. In cases when the tyrosinase requires the co-expression of a caddie protein for its
secretion and assembly such as the tyrosinase from S. castaneoglobisporus [20] and from M. mediterranea
[58], both genes are located within the same operon.
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The available information suggests that in general bacterial tyrosinases have a pH optimum around 7.5 and
an optimal working temperature around 40°C (Figure 3). Bacterial tyrosinases are generally monomeric
enzymes with a molecular mass between 20 and 60 kDa (Figure 3). A dimeric bacterial tyrosinase has been
isolated from the highly thermophilic bacterium T. roseum (Figure 3, number 21) [44]. Robust bacterial
tyrosinases with physico-chemical characteristics ideal for industrial applications have been identified from
various strains. For example, a 31 kDa tyrosinase with a maximum activity at 50°C was described from B.
megaterium and tyrosinases with enhanced activity in the presence of organic solvents have been isolated
from B. megaterium and Streptomyces REN-21 [48, 59] and a tyrosinase with optimal working temperature
of 70°C and pH 9.5 from T. roseum [44]. Typical substrates for tyrosinases are monophenols such as
tyrosine and modified tyrosine, phenol and coumaric acid and diphenols such as the model substrate LDOPA or caffeic acid; polyphenols of different sizes including pyrogallol and catechins can also be
substrates for tyrosinases (Table 2). The substrate specificity of tyrosinase has been altered not only by
mutagenesis [37] but also by changing the reaction conditions. For example, the B. megaterium tyrosinases
showed a 5-fold higher monophenolase/diphenolase activity in the presence of ionic liquids [60]. The
oxidation of protein-bound tyrosines to quinone-like structures by tyrosinase promotes their reaction with
other tyrosine, cysteine or histidine residues of the same or a different polypeptide chain thus forming intraand inter-molecular cross-links [61]. Recently, the cross-linking activity of the V. spinosum tyrosinase has
been demonstrated on tyrosine-containing model proteins of different sizes, e.g. cytochrome c (11.7 kDa) or
lipase B from Candida antarctica (33.4 kDa) [62].
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4. Production of bacterial tyrosinases
The production of tyrosinase and the associated synthesis of melanin are reported to be naturally induced by
exogenous stresses, such as heat and hyperosmotic stress, and by specific compounds such as tyrosine as
well as in the presence of copper, the essential metal cofactor (Table 3). In S. antibioticus, the induction by
L-methionine promotes fast secretion of the enzyme without intracellular accumulation, and cultivation in
the absence of copper resulted in the production of the enzyme in the apo-form [19]. Studies in Streptomyces
species revealed that induction is regulated at both the transcriptional and translational level [19, 63].
Similarly, wounding and methyl jasmonate have been identified as inducers of tyrosinase production in
plants [64]. The expression of fungal tyrosinases is known to be triggered by bacterial infection [65],
exposure to light [66], culture medium composition [67] and the presence of copper [68]. By contrast,
specific compounds have a repressive effect on the production of tyrosinase. In bacteria, ammonium has
been identified as a repressor for the production of tyrosinase from Streptomyces michiganensis [69, 70] and
in fungi, amino acids and analogues such as D-tyrosine act as repressors in the production of a tyrosinase
from Neurospora [71].
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Currently, the tyrosinase from the fungus A. bisporus is still the only commercialised tyrosinase [72]. The
production of bacterial tyrosinases has not only taken advantage of the natural production, but it has also
been carried out heterologously in host strains. Scarce data are available about the production of bacterial
tyrosinases that is often identified only by the production of melanin. S. albus is a natural tyrosinase
producer and 0.2 mg/l of purified tyrosinase could be isolated from the culture medium [47]. A production
level of 20 mg of purified protein per liter of culture was achieved when the operon containing the gene
coding for the tyrosinase from S. antibioticus was overexpressed in the native host [73]. Upon
overexpression in E. coli, production levels of 86 mg/l of purified tyrosinase from B. megaterium [48] and
approximately 20 mg/l of tyrosinase from V. spinosum [21] were achieved. The tyrosinase from
Streptomyces REN-21 showed activity on tyrosine-containing peptides and was recombinantly produced in
E. coli with a production level of 54 mg/l [74]. Among others, the tyrosinases from Pseudomonas
maltophila and R. etli have also been recombinantly produced in E. coli, but there is no information on the
production levels obtained [32, 51, 75, 76].
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5. Applications of tyrosinases
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Most suggested applications of tyrosinases have been tested with the commercially available mushroom
enzyme. However, considering the similar reactivities, these applications are conceivable also for bacterial
tyrosinases. The ability of tyrosinase to act on catechol-like substrates was the subject of patenting in 1970
[77] and the cross-linking activity of tyrosinases on peptides was patented in 2003 [78, 79]. In some cases,
the enzyme was not used in an isolated form, and natural tyrosinase-producing strains were employed in the
process, e.g. in bioremediation [80].
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Applications of tyrosinase rely on the ability of tyrosinases to oxidise both small phenolic molecules and
protein-associated phenolic groups, i.e. the side chain of the amino acid tyrosine [16, 77, 78]. Due to the
various potential applications of tyrosinases (Figure 4) not only have fungal enzymes been subjects of patent
applications but also bacterial enzymes such as the tyrosinases from V. spinosum, R. etli, S. antibioticus and
Pseudomonas sp. DSM13540 [46, 81, 82, 83]. An overview of representative applications of tyrosinases in
different fields is given in Table 4.
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Various aspects of tyrosinase activity are desirable. In molecular biology the chromogenic reaction catalysed
by the tyrosinase from S. glaucescens has been used as a reporter for gene expression [84], and the enzyme
from R. etli has served as a tool to detect bacterial strains producing L-tyrosine [34, 35]. The ability of
tyrosinases to oxidise small phenolic molecules can be exploited for the removal of these substrate
compounds from environmentally polluted samples (bioremediation), for the synthesis of secondary
compounds and for initiating the chromogenic melanin-synthesis process (biocatalysis and dyes production).
The ability of tyrosinases to act on larger molecules such as peptides and proteins containing tyrosine (crosslinking activity) has been exploited, for example, to prepare adhesive solutions and to modify the protein
structure of food [85, 86].
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Tyrosinase has been used not only in free form but also in an immobilised [87] and cross-linked aggregated
form [88]. Immobilization increased its stability and facilitated reusability. A review focusing on the
applications of immobilised tyrosinase was published in 2012 [89]. A similar effect has been reported for
tyrosinase immobilised on solid supports such as silica [90], magnetic beads [91] and embedded in selfadhesive layers made of plant-derived agarose and guar gum [92]. In addition, the immobilisation of
tyrosinase on clay coated with hydroxyl-aluminium not only increased the specific activity of the tyrosinase
but also its temperature stability [93]. Aiming at L-DOPA production, mushroom tyrosinase has also been
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immobilised using glutarhaldehyde on chemically modified nylon [94], on sodium aluminosilicate and on
calcium aluminosilicate, and two modified forms of zeolite [95].
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5.1 Tyrosinase and the production of dyes
Considering the physiological role of tyrosinase in the synthesis of melanin from tyrosine, the enzyme has
been applied to the synthesis of dyeing and colouring solutions. For example, tyrosinase from R. etli has also
been used for melanin production in E. coli, and production levels of melanin reached 6 g/l [76]. As early as
1947, tyrosinase was proposed for the dyeing of animal fibres [96]. More recently, a colouring composition
containing the enzyme from A. bisporus and L-DOPA has been proposed for colouring hair [97]. However,
tyrosinase from mushroom (possibly A. bisporus) has also been tested for colouring goat hair, but little
effect was noted [98].
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5.2 Tyrosinase and biosensors
Various biosensors have been developed using mushroom and bacterial tyrosinases to monitor even subpicomolar amounts of phenolic compounds in a sample [99-101]. Concerning application to food products, a
tyrosinase/beta-galactosidase-coupled reaction has been exploited to develop a disposable biosensor able to
quantify the amount of toxic cyanogenic glycosides from foods such as the kernels of apricot, peach and
cherry [102]. Mushroom tyrosinase has also been applied for the determination of the phenolic content of
fruit juices, tea, infusions and jams [103, 104]. The development of biosensors for phenol detection has been
reviewed recently [105].
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5.3 Tyrosinase for biosynthesis and medical applications
The substrate specificity of most tyrosinases is generally wide and substrates include various mono-, di- and
poly-phenolic compounds. This qualified tyrosinase for the production of ortho-diphenols, also-called
substituted catechols that are essential intermediates in the synthesis of pharmaceuticals, plastics,
antioxidants and agrochemicals. For example, the use of tyrosinase in combination with toluene-4monooxygenase has improved the production of 4-fluorcatechol [106].
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The first product of the tyrosinase reaction with tyrosine, L-DOPA, has a high economical value because it
is the main drug for the treatment of Parkinson’s disease and a forecast of predicted sales for 699 MUS$ in
2019 [107]. L-DOPA is currently produced at industrial scale by chemical synthesis, and many studies have
been targeted at providing an alternative enzyme-based process using free or immobilized tyrosinase [91, 94,
95, 108]; however, the approach using the enzyme tyrosine-phenol lyase that does not catalyse the further
oxidation of L-DOPA might be more promising [109-110]. The di-phenolic product of the reaction with
tyrosinase is able to react with cysteine, and tyrosinase can thus be used to produce catecholamines such as
cysteinyl-DOPAs [111].
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Mushroom tyrosinase has been applied to the synthesis of natural compounds with estrogenic activity such
as coumestan and derivatives that are generally isolated from plant material [112]. The same enzyme has
also been used for the production of the antioxidant hydroxytyrosol in the presence of ascorbic acid (vitamin
C), and the final reaction mixture could be used directly as food additive [113, 114]. In principle, bacterial
tyrosinases should also be able to catalyse all these reactions and might allow whole cell biotransformations.
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Medical applications of tyrosinase include also the production of melanins as natural antibacterial
compounds for the treatment of wounds, i.e. the local application of melanin precursor and tyrosinase in the
form of a cream or ointment [115]. The involvement of tyrosinases in the nervous system is not clear. In an
old US patent it was claimed that melanin plays a protective role in the nervous system [116]. Tyrosinase
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has also been tested for the treatment of neuronal diseases; it has been reported to enhance dopamine
toxicity, but a genetic association with Parkinson’s disease has not been described [117]. Furthermore,
tyrosinase has been suggested as a reporter enzyme for the measurement of cholesterol levels in a skin test
[118].
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5.4 Tyrosinase in bioremediation of wastewaters
Bacterial tyrosinases have been tested in the detoxification of wastewaters by removal of phenolic
compounds and decolourisation. The tyrosinase from S. antibioticus, for example, had activity on industrial
pollutants such as 3- and 4-chlorophenols [119] and 3- and 4-fluorophenols [120]. The application of
bacterial tyrosinase to the treatment of contaminated wastewaters has recently been reviewed [121, 122] and
can be done either with tyrosinase-producing strains [80] or with the enzyme in an immobilized form as
protagonist [123]. For example, tyrosinase (mushroom) has been reported to proceed in a precise order of
efficiency in the oxidation of phenolic compounds from wastewaters, e.g. favouring catechol, to p-cresol, pchlorophenol, phenol and p-methoxyphenol [124]. Similar effects have been observed after immobilization
of the mushroom tyrosinase on chitosan beads, which allowed the removal of chlorophenols and alkylsubstituted phenols from artificial wastewaters [125]. Bacterial tyrosinase may also have a potential
application in decolourization of effluents, as it has been reported for two different species of
Basidiomycetes, e.g. Trichosporon akiyoshidainum and Trichosporon beigelii NCIM-3326 [126, 127] to be
involved in the degradation of different coloured dye molecules commonly used by the textile industry.
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5.5 Tyrosinase and materials
The cross-linking activity of tyrosinase has been used to functionalize materials such as chitosan by crosslinking particular enzymes of interest to this biomaterial. Target enzymes can be substrates for the crosslinking reaction in their native form if they have surface exposed tyrosyl groups. For example,
organophosphorus hydrolase, chloramphenicol acetyltransferase and cytochrome c have been shown to
retain activity upon coupling to chitosan [128].
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The reactivity of the oxidised tyrosyl group, a quinone produced by tyrosinase (A. bisporus), with free
amino groups of a polymer has been exploited to functionalise chitosan with the tyrosine-containing peptide
YGG(KVSALKE)5GGC (Kcoil) that is able to recruit proteins carrying the partner peptide (EVSALEK)5
(Ecoil) via coiled-coil interactions [129]. In a similar manner, mushroom tyrosinase has been used to form
covalent protein-polysaccharide bioconjugates by oxidising the tyrosine residues of silk proteins sericin and
sericin-derived peptides such that they subsequently react with the free amino groups of chitosan [130, 131].
The tyrosinase-catalysed binding of silk proteins to chitosan reduced the particle size of the material, made it
more compact, increased its thermal stability and reduced its adhesiveness, making it suitable for medical
applications [130, 131]. Mushroom tyrosinase has also been suggested for the site-directed attachment of
tyrosine-containing proteins characterised by specific affinity properties to substrates carrying amino groups,
e.g. antigens or antibodies to polyallylamin surfaces [132, 133].
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Tyrosinase has shown activity on tyrosines of large polymers, such as wool fibres and silk fibroin that could
be functionalized with different proteins, e.g. collagen, elastin and gelatine for acquiring bactericidal and
fungicidal properties [134-136]. As a result of its activity on tyrosine-containing proteins and peptides,
mushroom tyrosinase has also been applied to production of adhesives, starting from a polyphenolic protein
with an enzyme to protein ratio of 5-50 units of enzyme per microgram of protein [85]. Moreover, the
activity of mushroom tyrosinase (A. oryzae) in the presence of dopamine conferred adhesive properties to a
diluted solution containing chitosan [137]. The viscosity of the solution increased with the progression of the
reaction, probably due to the interaction of the quinone-like products of the enzymatic reaction with the
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amino groups of chitosan. The adhesive strength of the enzyme-based preparation was higher than an
analogous one prepared with the chemical cross-linker glutaraldehyde [137].
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5.6 Tyrosinase in food and feed applications
Tyrosinase has been proposed for application in food processing not only for the melanogenic reaction it
catalyses, e.g. in tea production [138], but also for its cross-linking activity that can modify the structure of
food. Tyrosinases of different origins have been tested as cross-linking agents on a wide variety of proteins
from milk, meat and cereals. In contrast to traditional cross-linking agents, tyrosinases are characterised by
high specificity in the reaction they catalyse and, furthermore, they utilize food matrix components like
proteins in their reaction and do not require the addition of any chemical or food additive. The improvement
of the textural properties of food can be achieved by using carbohydrate-based gelling agents. Recently, a
comparative study assessed the ability of different tyrosinases from fungi and bacteria, e.g. common
mushroom A. bisporus, the plant pathogen Botryosphaeria obtusa, and the Gram-negative V. spinosum, to
cross-link the commonly used gelling agent gelatine and revealed that the addition of phenolic compounds
to the reaction mixture significantly accelerated the reaction [139]. Tyrosinase could be used for crosslinking the proteins of food matrices. For instance, in dairy and meat applications it can be applied in the
production of low-calorie and low-fat food [140]. Caseins are generally good substrates for tyrosinase
because their structure is flexible [141] and more accessible for the action of tyrosinase, e.g. the enzyme
from V. spinosum [62]. The addition of tyrosinase from Trichoderma has been reported to improve the
firmness of gels from raw milk and sodium caseinate [86]. This tyrosinase has also been reported to be able
to form protein-oligosaccharide conjugates, where the protein was alpha-casein [142], but the reaction was
not very efficient. Furthermore, this fungal tyrosinase has also been effective in improving the firmness of
gels containing with low-meat chicken breast and created a network between the collagen molecules [143].
In baking, tyrosinase (Trichoderma) has also been able to enhance the hardness and reduce the extensibility
of dough [144] and to modify the structure of baked bread by cross-linking the cereal proteins [145].
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The suggested application of tyrosinase to animal feed is quite recent. Aiming at the improvement of the
nutritional value of animal feed, mushroom tyrosinase has recently been able to increase the bioavailability
of iron from phytase treated fava bean-based preparations [146].
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6. Conclusion
Many intracellular and secreted tyrosinases have been reported from bacteria, and four of them have already
been subjects of patent applications. The production of novel bacterial tyrosinases has been eased by the
increasing number of bacterial genomes sequenced. However, the identification of novel tyrosinases through
genome mining studies is hindered by their strong sequence similarity with catechol oxidases. This review
shows that a certain degree of sequence variation in residues and length is present even among reported and
biochemically characterised tyrosinases. On the other hand, the classification of an enzyme according to its
activity on typical tyrosinase substrates, i.e. an activity as mono- and diphenol oxidase could be misleading
as some enzymes such as laccases have been reported to have tyrosinase activity. One of the advantages of
bacterial tyrosinases is the ease of their production in recombinant form in a model host such as E. coli. This
makes production in good quantities and protein engineering studies straight-forward and time-efficient and
bears new potential for future applications.
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Author’s contribution
GF conducted the literature search and drafted the manuscript. KK, MS and LTM have contributed to the
discussion and provided a critical evaluation of the information collected. All authors have read and
approved the final article.
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Acknowledgements
GF was funded by TYROMAT, a project within the Empa Postdocs programme that is co-funded by the
FP7: People Marie-Curie action COFUND. GF was also financially supported by the Finnish Cultural
Foundation and Zerazyme, a project funded by the Finnish Agency for Technology and Innovation (funding
decision 40161/10). We are grateful to Sarah Tighe-Jordan for language revision.
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Figure legends
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Figure 1 Oxidation of L-tyrosine to L-dopaquinone by tyrosinase.
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Figure 2 Three-dimensional structure of tyrosinase from B. megaterium and characteristic sequence motifs
of tyrosinases (PDB ID: 3MN8). The conserved sequence motifs identifying the copper A and B binding
sites of tyrosinases are reported. Protein-specific variation in the distance between the conserved histidine
residues is reported and X is any amino acid.
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Figure 3 Biochemical properties of bacterial tyrosinases.
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Molecular weight (MW, empty symbols), optimum pH (filled symbols) and temperature (grey symbols) of
known bacterial tyrosinases belonging to the phyla of Actinobacteria, Firmicutes, Proteobacteria,
Verrucomicrobia and Chloroflexi are shown. Whenever the optimum pH and temperature values were not
available, the assay conditions for activity are reported (square symbol). Tyrosinases considered were from 1
Streptomyces antibioticus, 2 Streptomyces glaucescens, 3 Streptomyces nigrifaciens, 4 Streptomyces
castaneoglobisporus, 5 Streptomyces lavendulae, 6 Streptomyces michiganensis, 7 Streptomyces sp. KY453, 8 Streptomyces albus, 9 Streptomyces sp. REN-21, 10 Bacillus megaterium, 11 Bacillus thuringiensis,
12 Marinomonas mediterranea, 13 Pseudomonas putida F6, 14-15 Ralstonia solanacearum, 16
Sinorhizobium meliloti, 17 Aeromonas media, 18 Pseudomonas sp. DSM13540, 19 Rhizobium etli CFN42,
20 Verrucomicrobium spinosum, 21 Thermomicrobium roseum. For details and references see
Supplementary file 2.
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Figure 4 Fields of application of tyrosinase based on the activity on phenolic compounds and the crosslinking activity.
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Supplementary file 1
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Sequence alignment of the characterised bacterial tyrosinases that are available at NCBI database
(http://www.ncbi.nlm.nih.gov/). When significant, the consensus sequence (Jalview,
http://www.jalview.org) is reported at the bottom of the alignment. Possible N-terminal arginine residues
and the conserved cysteine residues in the copper A binding region are in grey. The C-terminal tyrosine
motif and conserved sequence motifs involved in copper or oxygen binding are indicated and the key
residues are indicated by a plus. Residues subject to mutagenesis and mentioned in the text are boxed. For
details and references see Supplementary file 2. Tyrosinases considered were from 2 Streptomyces
glaucescens, 4 Streptomyces castaneoglobisporus, 5 Streptomyces lavendulae, 10 Bacillus megaterium, 12
Marinomonas mediterranea, 14-15 Ralstonia solanacearum, 16 Sinorhizobium meliloti, 17 Aeromonas
media, 19 Rhizobium etli CFN42, 20 Verrucomicrobium spinosum.
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Supplementary file 2
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Summary of the bacterial tyrosinases considered in this review, the reference to their sequence and to
literature.
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875
876
[186] Pinero S, Rivera J, Romero D, Cevallos MA, Martinez A, Bolivar F, Gosset G. Tyrosinase from
Rhizobium etli is involved in nodulation efficiency and symbiosis-associated stress resistance. J Mol
Microbiol Biotechnol 2007;13:35-44.
877
26
●
Figure
1
878
Figure 1
879
27
T optimum
Temperature optimum ( C)
Molecular weight (kDa)
120
Actinobacteria
pH optimum
12
Chloroflexi
Verrucomicrobia
10
Firmicutes Proteobacteria
100
8
80
6
60
4
pH optimum
MW
140
40
2
20
0
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Identifier
Figure 3
880
881
882
883
Supplementary file 1
884
885
886
887
888
889
890
891
892
2
4
5
10
16
20
15
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
28
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
19
14
12
17
MPWLVGKPSLERSWNAILSFPESGFQLECRNTIGSSVFSSHFTLHFRVARRLLHFSCRRF
------------------------------MRIDFTINNGGDAAARYLTWAPSPLRLRLL
-----------------------------------------------------------------------------------------------------------------------
1
2
4
5
10
16
20
15
19
14
12
17
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------MSPPTTSRRQFLVTAGA
----------------------------------------------MVVRRTVLKAIAGT
TETQKEPTQALWWCELPTAPAPRRRGTGLKAALILAKDNSNPRESKMSITRRHVIVQGGV
DATPGPDVVATLSEDRQPNGGSIRFCATPDGNFTPTLKVPLPASGASVTVYVRGKFGTPS
------------------------------------------------MIRVRKNVNELT
-------------------------------------------------------MKKGL
1
2
4
5
10
16
20
15
19
14
12
17
Cons
-------------------MT----VRKNQASLTAEEKRRFVAALLELKR--TGRYDAFV
-------------------MT----VRKNQATLTADEKRRFVAAVLELKR--SGRYDEFV
-------------------MT----VRKNQATLTADEKRRFVAAVLELKR--SGRYDEFV
-------------------MT----VRKSVAALTPDEKRAFVNAVLELKR--TGVYDRYV
-------------------MSNKYRVRKNVLHLTDTEKRDFVRTVLILKE--KGIYDRYI
-------------------------MTSADGQKDLQSYMDAVTAMLKLPP--SDRRNWYR
AAASAGWSFGQEPAQAATAKYHRLNLQNPAAAPFLESYKKAITVMLQLPP--SDARNWYR
SVATVFAGKLTGLSAVAADAAPLRVRRNLHGMKMDDPDLSAYREFVGIMK--GKDQTQAL
IAAGLLASGLPGTKAFAQIPS-IPWRRSLQGLAWNDPIIETYRDAVRLLN--ALPASDKF
QADGDVSIVVGGPASELGRLPVMVRVRKNANQLTPAERDRFISAMAQINNRGTGRFTDFR
DDTLLWYSKAVESMKQKDITDPSSWWYQGAIHGYGLDKRPNLANNESWSE--SSVWEQAE
SKIAMALFAAGLAFNVSAADIKVAVASDATSLDPQEQLSGQTLEMSHLVFDPLMRYTQDL
SAAGL-ASK+GGPAAVAA+MTP--+VRKNQA+LTADEKRRFVAA+LELKR--SGRYDQ+V
N-terminal Arginine
29
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
1
2
4
5
10
16
20
15
19
14
12
17
Cons
TTHNAFILGDTDNGE--RTGHRSPSFLPWHRRFLLEFER----ALQSVD--ASVALPYWD
TTHNAFIIGDTDAGE--RTGHRSPSFLPWHRRYLLEFER----ALQSVD--ASVALPYWD
RTHNEFIMSDTDSGE--RTGHRSPSFLPWHRRFLLDFEQ----ALQSVD--SSVTLPYWD
NAHNYYLMSDSDFGP--RIGHRTPSFLPWHRRFLLDFEA----SLQRVD--RNVALPYWD
AWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFER----DLQSIN--PEVTLPYWE
----------NGFIHLMDCPHGDWWFTSWHRGYLGYFEE----TCRELSGNPDFALPYWD
----------NGFIHTLDCPHGNWWFVVWHRGYTGWFER----TVRELSGDPNFAFPYWD
SWLGFANQHGTLNGGYKYCPHGDWYFLPWHRGFVLMYER----AVAALTGYKTFAMPYWN
NWVNLSKIHGSGD-VVKYCPHGNWYFLPWHRAYTAMYER----IVRHVTKNNDFAMPFWD
NMHVAGRA--------DQQAHGGPGFLPWHRAYLLDLER----ELQAID--PAVTIPYWR
GFPPSEGLVNSQFWQ--QCQHGTWFFLPWHRMYLQFFEAIVAKTVVELGGPKDWTLPYWN
QFEPRLAEKYERIDDKTVRFHLRKG VKFHSGNDFTADDVVWTVNRLKASPDFKAIFDPI
NTHNAFIIGDTDFGE-KRCGHGSPSFLPWHRRYLLDFER----ALQSVDGNPDVALPYWD
+ Copper A region
+
+
Oxygen-binding motif + + +
1
2
4
5
10
16
20
15
19
14
12
17
Cons
WSADRSTR-------------------------------SSLWAPDFLGGTGRSRDGQVM
WSADRTAR-------------------------------ASLWAPDFLGGTGRSLDGRVM
WSADRTVR-------------------------------ASLWAPDFLGGTGRSTDGRVM
WTVDRAAN-------------------------------SPLWASDFMGGSRRGRDGQVL
WETDAQMQDPSQ---------------------------SQIWSADFMGGNGNPIKDFIV
WTANPEVLPPLFGTILDPVNSSAYIPDHNRFQDIMQEPIKAYWDSLSPAQLQQQNLRGYP
WTALPQVPDSFFNGVLDPNNP-AFIASYNEFYSQLSNPMSALWNSFSTAQLQQMRNRGFQ
WTEDRLLP------------------------------------EAFT-AKTYNGKTNPL
WTDNPYLP------------------------------------EVFTMQKTPDGKDNPL
FDRPAPNL----------------------------------FTTDFIGVPDALGTVSFS
YCDANNPA---------------------------------------LNPTEQLQALKLP
AEAKKVDDFTVDLVTAKPFPLVLQTVT-----------YIFPMDSKFYSGKDEAGKDKAA
WTADRQVPDP-F---LDP-N--A-I---N-F------P-SSLWASDFLGGTGRSGDGGVL
+
1
2
4
5
10
16
20
15
19
14
12
17
Cons
D-GPFAASAGNWP----INVRVDGRTFLRRALGAG--VSELPTRAEVDSVLAMATYDMAP
D-GPFAASAGNWP----INVRVDGRAYLRRSLGTA--VRELPTRAEVESVLGMATYDTAP
D-GPFAAFTGNWP----INVRVDSRTYLRRSLGGS--VAELPTRAEVESVLAISAYDLPP
D-GPFAAGGGKWP----VTVGVDRRDYLRRVLGSG--VPQLPTRAEVDAVLAMPVYDTAP
DTGPFAA--GRWTT---IDEQGNPSGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPP
DFDALWSDAMAS-----FANQPNARFLTAQNPKLNPATQTAVDIDTIKASLAPTTFANDA
SVNDVWQAVRDSPM---FFPRGRARTLTRQNPGFDATTRRAVSIGTIRNALAPTDFIT-YVPNRNELTGPYAL---TDAIVGQKEVMDKIYAETNFEVFGTSRSVDRSVRPPLVQNSLD
YVSSR-TWPITQPM---PDNIVG-PQVLNTILTAKPYEVFGTTR--------PEGQNSLD
PANPLQFWATDG-----------VQGILRRQLGASPGAQAAPNILTEAQTLALGSAYR-SEFGTNTPNPDFPG---LWMKERAQYQLSSQADASCSVAMKLQNFTASSPATSFGGVQTG
IVKNGDSYASTHVSGTGPFSVKFREQGVKLEYARNANYWDKASKGNVQNLTVVPIKEDAT
DVGPFAASAGDWPM---IDVRVDARTYLRRNLGASP-VAELPTRAEVDSVLAPTTYDTAP
30
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
1019
1020
1021
1022
1023
1024
1025
1
2
4
5
10
16
20
15
19
14
12
17
Cons
-------WNSGS-DGFRNHLEGWR-GVNLHNRVHVWVGG-----QMATG-VSPNDPVFWL
-------WNSAS-DGFRNHLEGWR-GVNLHNRVHVWVGG-----QMATG-MSPNDPVFWL
-------YNSAS-EGFRNHLEGWR-GVNLHNRVHVWVGG-----QMATG-VSPNDPVFWL
-------WNSSS-SGFRNHLEGWR-GTNLHNRVHVWVGG-----HMATA-ASPNDPVFWL
-------WDMTSQNSFRNQLEGFINGPQLHNRVHRWVGG-----QMGVVPTAPNDPVFFL
GAPGLAFNSPVSSSHQVAPVGFSILEGQPHNRVHMSVGGQSAPYGLMSQNLSPLDPIFFL
------FGSGKTANHSESAT-QGILESQPHNNVHNNIGG------FMQDLLSPTDPVFFA
-----------PKWVPMGGGNQGILERTPHNTVHNNIGA------FMPTAASPRDPVFMM
-----------PSWVTTSSGTQGALEYTPHNQVHNNIGG------WMPEMSSPRDPIFFM
-------------NFRG-------MQGNPHGSAHVSYFSGS----ISSIPTAAKDPLFFL
--------------FSHDSGTFGAVENNPHNLVHVDIGG-----AMGDPNTAALDPIFWL
-----------RVAALLGGDVDMIYPVAPNDLERVKNGKD----SQLVTLSGTRAIIIEL
------FWNSASSNGFRNHLEGGILEVNPHNRVHVWVGG-S---QMATGLTSPNDPVFFL
+
+
Copper B region
1
2
4
5
10
16
20
15
19
14
12
17
HHAYIDKLWAEWQRRHPSSPYLPGGGTPNVVDLN----------ETMKPWNDTTPAALLD
HNAYVDKLWAEWQRRHPGSGYLPAAGTPDVVDLN----------DRMKPWNDTSPADLLD
HHAYVDKLWAEWQRRHPDSAYVPTGGTPDVVDLN----------ETMKPWNTVRPADLLD
HHAFIDKLWADWQARNPKAGYLPSGRTQNVIDLR----------GVLPPWNNVTPADMLD
HHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFR----------DPMYPWN-TTPEDVMN
HHCNIDRLWDVWTRKQQAMGLPVGPTADQQTQYDPEPYLFYVNADGSPVSDKTRAADYLE
HHSNIDRLWDVWTRKQQRLGLPTLPTGANLPLWANEPFLFFIGPDGKPVA-KNKAGDYAT
HHGNIDRVWATWNALGRKNSTDPLWLGMKFPNNY---------IDPQGRYYTQGVSDLLS
HHCNIDRIWATWN-LRNANSTDRLWADMPFTDNF---------YDVDGNFWSPKVSDLYV
LHCNVDRLWAKWQSQVGRYDANVAAAYDAGPTPTSLLAG-HNLHDTLWPWNGIVTPPRPS
HHANIDRLWQCWIDQGRENTNDITWLNQVFDFHN-----------ADSLPDTLSVKDVLS
NQNTNPALKDKRVRQAINYAINQVGIVDKINKGFG---------TPAGQLSPKGYAGYNE
+
1
2
4
5
10
16
20
15
19
14
12
17
Cons
HTRH-YTFDV-------------------------------------------------HTAH-YTFDTD------------------------------------------------HTAY-YTFDA-------------------------------------------------HRRF-YTFDKP------------------------------------------------HRKLGYVYDIELRKSKRSS----------------------------------------IGDFDYDYDPGSGEEVIPVATAGRSAPIPALEAAVSASAAVAINKPATAKL--------IGDFDYNYEPGSGEAVIPAASRPGEMNNKVWLGTLGA-AVPNFSASARADV--------TEALGYRY------DVMPRADNKVVNNARAEHLLALFKTGDSVKLADHIRLRSVLKGEHP
PEELGYNYGFRTYFKVAAASAKTLALNDKLTSVIAATATDAAIAGVTTTSTDNSKAATEN
TAPGGAMAGSSCVSAPGNAPRVSDMLDFQGVVSSSAKLGFAYDDVPLP-----------TEALGFTYSDSYSSSAAPTDSKVFALASAGGSGMFDTIAATTKPFLLGSQSTSAQLEFLP
ALKPQYDLAKAKELTKEAGYEKGFKMTFISPAARYVNDVKIAQAVSAMLSKINIKVDLKT
HEALGYTYDKGSGEAVAPAASKGFALNFKA-SA-AAA-AAAAIAVPATASL-N-K----P
+ +Tyrosine motif
31
1026
1027
1028
1029
1030
1031
1032
1033
1034
1035
1036
1037
1038
1039
1040
1041
1042
1043
1044
1045
1046
1047
1048
1049
1050
1051
1052
1053
1054
1055
1056
1057
1058
1059
1060
1061
1062
1063
1064
1065
1066
1067
1068
1069
1
2
4
5
10
16
20
15
19
14
12
17
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TVSQELVDVAAKPSEQSRQFAKVSIAPPMDVGGL
--------------------------MVPEAVPEAAMK-ADGPAVFAKITIAPPMDVAGV
V---------ATAVEPLNSAVQFEAGTVTGALG-ADVGTGSTTEVVALIKNIRIP-YNVI
VPLSLPIKIPAGALQEIVRQPPLPSGMDTMDFGAAQEQAASAPRVLAFLRDVEITSASTT
-----------------------------------------------------------E------------------------KQRAAQVPVLGASNSQTPNQVIIVLDNVTGSGVVA
MP--------------------------VAQYWPEFDKCASDMQLIGWHSDTEDSANFFE
1
2
4
5
10
16
20
15
19
14
12
17
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------NFLVFISPEGTTPDLNPDGPDFAGSFEFFG--VRHHHTDTVSFTIPIDKALDRLIDDGRL
EFHVLVNPPENVSHVDFDSPSFAGTFSVFGKQLGGHKNQPLSFLMPLTEAVKKLQETNEL
SIRVFVNLPNANLDVPETDPHFVTSLSFLTHAAGHDHHALPSTMVNLTDTLKALN---IR
SVRVFLGKNDLKADTPVTDPHYVGSFAVLGHDG--DHHRKPSFVLDLTDAIQRVYGGRGQ
-----------------------------------------------------------PVSVYVKASANSERVLVGKIGLFGLTQSSTPSSTSCLEQGISIELDVTDALQQLRSQTNW
FLTFTKDAKTGMGQYNCGGYANAEADKMVMEANTETDPAKRAAILQKVEAMLIDDAAYVP
1
2
4
5
10
16
20
15
19
14
12
17
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------KAGEPIDFAVVVAQEGKRVEGSMPAKAQLTDIQVGSF
KPGQPLRVQVVAERKGVNLT---PLQAKVSEISVGTF
DDNFSINLVAVPQPGVAVESSGGVTPESIEVAVI--TDGEAIDLQLIP-VGSGAGKPGAVEPAKLEIAIVSA------------------------------------NLENLQIELEPGRELGNASVTVGRVSIKAEVV----LHWEDLAYGAKKNVDIKPVVNVMNFPYLGDLVVSK--
Identifier Organism
Enzyme Sequence
Reference
1
Streptomyces antibioticus
TYR
NCBI Protein ID: AAA88571.1
[181]
2
Streptomyces glaucescens
TYR
NCBI Protein ID: AAA26834.1
[30, 45, 149]
3
Streptomyces nigrifaciens
TYR
na
[25]
4
Streptomyces
castaneoglobisporus
TYR
NCBI Protein ID:AAP33665.1
[46]
5
Streptomyces lavendulae
TYR
NCBI Protein ID:ABQ41256.1
[183]
6
Streptomyces michiganensis
TYR
na
[70 150]
32
1070
7
Streptomyces sp. KY-453
TYR
na
[183]
8
Streptomyces albus
TYR
na
[47]
9
Streptomyes sp. REN-21
TYR
na
[59, 74]
10
Bacillus megaterium
TYR
NCBI Protein ID:ACC86108.1
[6]
11
Bacillus thuringiensis
TYR
na
[26]
12
Marinomonas mediterranea
TYR
NCBI Protein ID:AAV49996.1
[42, 185]
13
Pseudomonas putida F6
TYR
na
[186]
14
Ralstonia solanacearum
TYR
NCBI Protein ID:NP_518458
[43]
15
Ralstonia solanacearum
TYR
NCBI Protein ID:NP_519622
[43]
16
Sinorhizobium meliloti
TYR
na
[49]
17
Aeromonas media
TYR
NCBI Protein ID:ACD40043.1
[50]
18
Pseudomonas sp.
DSM13540
TYR
na
[81]
19
Rhizobium etli CFN42
TYR
NCBI Protein ID: AAM54973.1
[51, 184]
20
Verrucomicrobium
spinosum
TYR
NCBI Protein ID:ZP_02925214.1
[21]
21
Thermomicrobium roseum
TYR
na
[44]
Abbreviations: na, not available
1071
1072 Table 1 Three-dimensional structures of bacterial tyrosinases and features.
Organism
Conditions
Streptomyces
castaneoglobisporus
1. Copper-free
Caddie
protein
Yes
2. Copper-bound
3. oxy-form
Yes
Yes
4. met-form
Yes
5. deoxy-form
Yes
6. Copper-bound form Yes
(deoxy-form, crystal
soaked in O2saturated solution)
7. Different copper
Yes
occupancy
33
PDB Identifier
PDB ID:1WX5
PDB ID: 1WXC
PDB ID:3AWU
PDB ID: 1WX2
PDB ID: 1WX4
PDB ID:2AHK
PDB ID:2ZMY
PDB ID:2ZMX
PDB ID:2AHL
PDB ID: 2ZMZ
PDB ID:2ZWD
PDB ID: 2ZWE
PDB ID: 2ZWF
PDB ID: 2ZWG
PDB ID: 3AWS
PDB ID: 3AWV
(low)
Resolution Reference
(Å)
2.02
[5, 147]
1.20
1.16
1.80
1.50
1.71
1.45
1.33
1.60
1.37
1.35
1.32
1.40
1.32
1.24
1.40
1.35
8.
Bacillus megaterium
1.
2.
3.
4.
5.
PDB ID: 3AWT
PDB ID: 3AWW
(high)
With mutant caddie Yes (H82Q) PDB ID: 3AWX
Yes (M84L) PDB ID: 3AWY
Yes (H97Q) PDB ID: 3AWZ
Yes (Y98F) PDB ID: 3AX0
Copper-bound
No
PDB ID: 3NM8
PDB ID: 3NPY
Copper-bound
No
PDB ID: 3NQ0
(absence of zinc)
(only CuA
occupied)
PDB ID: 3NTM
(CuB partially
occupied)
Mutant form R209H No
PDB ID: 3NQ5
With the inhibitor
No
PDB ID: 3NQ1
kojic acid
In presence of SDS No
PDB ID: 4D87
1073
34
1.35
1.25
1.58
1.43
1.40
2.00
2.19
2.20
2.30
2.30
2.30
3.50
[6,60]
1074
1075
Table 2 Substrate specificity of some bacterial tyrosinases.
Organism
Monophenols
Diphenols
Polyphenols
Reference
Bacillus
megaterium
L/D-tyrosine,
tyramine
L/D-DOPA, caffeic
acid, catechins,
catechol, chlorogenic
acid
pyrogallol,
phloroglucinol
[36, 48]
Bacillus
thuringiensis
L-tyrosine
catechol, L-DOPA,
4-methyl catechol,
dopamine, 3,4diihydroxymandelic
acid, hydroquinone,
3,4-dihydroxyphenylacetic acid,
resorcinol
nr
[26]
Rhizobium etli
L-tyrosine, nacetyl-Ltyrosine
L-DOPA, catechol,
caffeic acid
nr
[51]
Streptomyces
antibioticus
L-tyrosine
p-aminophenol,
2/3-Cl-phenol,
tert-butylcathechol,
dopamine, L-DOPA,
hydro-quinone,
p-cresol, p-nitrophenol, dopamine,
adrenalin,
noradrenalin, 4methyl-catechol, 4nitrocatechol
nr
[19, 40,
101, 148]
Verrucomicrobium
spinosum
L-tyrosine
L-DOPA
Sodium
caseinate,
proteinsa
[21, 62]
1076
1077
1078
35
1079
Table 3 Inducible bacterial tyrosinases.
1080
Organism
Bacillus megaterium
Bacillus thuringiensis
Pseudomonas sp. DSM13540
Streptomyces antibioticus
Streptomyces castaneoglobisporus
Streptomyces glaucescens
Streptomyces michiganensis
Vibrio cholera
Abbreviations: nr, not reported.
Enzyme
TYR
TYR
TYR
TYR
TYR
TYR
TYR
nr
Inducing conditions
Tyrosine and copper
Heat (42°C)
L-tyrosine, wool fibres
L-methionine
Methionine, copper
L-tyrosine, L-methionine
Copper
Osmotic stress, heat (>30°C)
1081
1082
36
Reference
[48]
[26]
[81]
[148]
[63]
[149]
[150]
[151]
1083
Table 4 Representative applications of tyrosinase in different fields.
Field
Production of
dyes
Cosmetic
applications
Biosensors
Biosynthesis
and
medical
applications
Bioremediatio
n of
wastewaters
Materials
Food
applications
Others
Mode of action
Reference
1. Oxidation of an aromatic
phenolic compound by
tyrosinase and production of
coloured compounds
1. Self-tanning agent
[97,152, 153]
1. As single enzymes
2. In a coupled assay to detect
catechol-producing compounds,
e.g. hormones, salicylate
1. Production of L-DOPA
2. Production of substituted
catechols
3. Production of food additives
4. Production of estrogenic
compounds
5. Production of melanins for
therapeutic uses
6. Treatment of neurological
diseases
7. Production of mosquito
repellants
8. Assay of cholesterol on the skin
[155]
[156-160]
1. Removal of phenolic and
substituted phenolic
contaminants
2. Reduction of COD
3. Decolourisation (degradation of
dyes)
1. Production of polyphenolic
polymers
2. Modifications of materials
3. Production of adhesives
4. Film production
5. Treatment of textiles
[119-122, 163-167]
1. Production of tea
[178, 179]
2. Production of dairy products
[86, 180]
3. Cross-linking of meat proteins
1. As a reporter protein for
counting microorganisms
2. Identification of bacteria
producing L-tyrosine
[143]
[181]
[154]
[55, 87, 88, 91, 94, 95]
[106, 111]
[113, 114]
[112]
[115, 161]
[116]
[162]
[118]
[168]
[126, 127, 169-172]
[173]
[125, 134-137, 174]
[85]
[175]
[134-137, 176, 177]
[34, 35]
1084
1085
37