Purification and activity enhancement of extracellular tyrosinase from a protease-silenced zygomycete Amylomyces rouxii strain

TL;DR

Researchers purified and enhanced the activity of tyrosinase from a modified zygomycete strain, achieving higher enzyme activity and confirming its active form.

Contribution

A protease-silenced strain of Amylomyces rouxii was developed, leading to enhanced extracellular tyrosinase activity and partial purification of the enzyme.

Findings

The transformant A412-3 showed a 2.14-fold increase in monophenolase and 3.02-fold increase in diphenolase activity compared to the parental strain.

SDS-PAGE analysis revealed three bands of tyrosinase with molecular weights of 40, 53, and 130 kDa.

Purified tyrosinase exhibited higher diphenolase activity than monophenolase activity.

Abstract

The intra- and extra-cellular monophenolase and diphenolase activities of the tyrosinase produced by Amylomyces rouxii were determined in submerged culture using Melin-Norkrans medium supplemented with 12.5 mg/L pentachlorophenol (PCP) and 0.1 g/L tyrosine. Maximal intracellular monophenolase activity was 180 U/mL while maximal extracellular monophenolase activity was 80 U/mL, both using p-cresol as substrate. For diphenolase, the highest intracellular activity was 2233 U/mL using 4-tert-butylcatechol (TBC) as substrate and extracellular diphenolase activity was 975 U/mL with catechol as substrate. The peak tyrosinase activity (mono- and diphenolase) was observed at 48 h of culture. The transformant A412-3 exhibited the highest extracellular activities, with a 2.14-fold increase in monophenolase and a 3.02-fold increase in diphenolase activity compared to the parental strain of A. rouxii. Additionally, it was confirmed that the enzyme secreted was in its active form. Extracellular tyrosinase from the transformant A412-3 was partially purified, achieving a purification factor of 10.6. SDS-PAGE analysis of partially purified tyrosinase revealed three bands of 40, 53, and 130 kDa. These bands were sequenced by LC–MS/MS, revealing eight peptides that showed similarity to tyrosinases from different fungi. It was determined that purified tyrosinase exhibited higher diphenolase activity than monophenolase activity, in line with previous studies on fungal tyrosinases.

Figures

Growth of *A. rouxii* parental and A412-3 in MN medium supplemented with tyrosine (0.1 g/L) and PCP (12.5 mg/L). *A. rouxii* (■), *A. rouxii* A412-3 (□)

Determination of tyrosinase activity over time. Extracellular monophenolease (A) and diphenolase (B) activities produced by *A. rouxii* parental (■) and transformant A412-3 (□) during growth in MN medium supplemented with tyrosine and PCP. Monophenolase was determined using tyrosine as a substrate, while activity was determined diphenolase using 4-tert-butyl catechol (TBC) as substrate. Statistical analysis, one-way ANOVA followed by Tukey’s test (p* ≤ 0.05)

Determination of the isoelectric point of the partially purified extracellular protein of *A. rouxii*. (■) Isoelectric point of the proteins. (△) Diphenolase activity determined with L-Dopa (4.5 mM)

Chromatography of partially purified tyrosinase obtained from the transformant A412-3 at 48 h of culture. A Anionic exchange (DEAE-Sepharose) and B gel filtration column (Sephadex-200). (●) Protein 280 nm, (△) monophenolase activity

SDS-PAGE of tyrosinase produced by the transformant A412-3 of *A*. *rouxii*. Numbers in the gel lanes indicate the fractions obtained in each purification step. M) Molecular weight marker (PageRuler™ prestained Protein Ladder). 1 and 2) Fraction 6 from anionic exchange column; 3) fraction 23 from filtration gel column; 4) fraction 14 from anionic exchange column; 5) commercial tyrosinase from anionic exchange column

Alignment of the amino acid sequence of peptide no. 4 obtained from the extracellular protein of transformant A412-3. A412-3. Identification of the characteristic histidine residue in the CuA copper binding site

Full text

Introduction

Tyrosinase (E.C.1.14.18.1), also known as polyphenoloxidase, is an enzyme that belongs to the largest group of type 3 copper-containing proteins. This enzyme catalyzes two types of reactions that occur consecutively. First, it catalyzes the o-hydroxylation of monophenols (monophenolase activity). In a second step, it oxidizes o-diphenols to produce reactive o-quinones (diphenolase activity), both reactions using molecular oxygen (Min et al. 2019). In general, the initial reaction is less pronounced than the subsequent oxidation. The activity of monophenolase exhibits a lag period, whereas that of diphenolase does not (Ba and Kumar 2017). Tyrosinases are distributed in a wide range of mammals, invertebrates, plants, and both prokaryotic and eukaryotic microorganisms (Nunes and Vogel 2018). These enzymes display a range of characteristics in different organs of the same organism. In plants, tyrosinases are also implicated in the regulation of the oxidation–reduction potential. Furthermore, tyrosinase is primarily responsible for enzymatic browning fruits and vegetables, including but not limited to apples, potatoes, avocados, lettuce, and so forth (Selinheimo et al. 2007; Iqbal et al. 2019; McLarin and Leung 2020). In mammals, the role of tyrosinases is crucial in melanin production (Kanteev et al. 2015). Melanin biosynthesis begins with two enzymatic steps, catalyzed by tyrosinase, and proceeds through a series of non-enzymatic steps to produce dopachrome, the first stable product (Cordero and Casadevall 2020). In filamentous fungi, tyrosinase plays a role in the biosynthesis of spore pigments, typically during secondary metabolism and enzymatic browning of edible mushrooms (Halaouli et al. 2006). Fungal tyrosinases have been identified in Neurospora crassa, Agaricus bisporus, Pycnoporus sanguineus, Aspergillus oryzae, Trichoderma reesei, and Lentinula edodes (Fernandes and Kerkar 2017). While the majority of fungal tyrosinases are intracellular, some fungal species, such as Trichoderma reesei (Selinheimo et al. 2006), Auricularia auricula (Zou et al. 2014), Amylomyces rouxii (Montiel et al. 2004), and Rhizopus oryzae (León-Santiesteban et al. 2008), have the capacity to produce extracellular tyrosinases.

Tyrosinase applications have increased in the last decade; it is used in food, cosmetic, and pharmaceutical industries (Sharma et al. 2023). Fungal tyrosinases have also been used in bioremediation processes of waste and soil contaminated with phenolic compounds (Khan et al. 2023). Kim et al. (2008) reported a tyrosinase-based enzyme electrode improved with gold nanoparticles for pesticide measurements. Immobilized tyrosinase has been used as biosensor to detect phenol in olive oil mill wastewater and penicillamine in pharmaceutical preparation; the detection principle is like biosensors based on substrate competition (Bounegru and Apetrei 2023). Tyrosinase can be used as a food additive due to their crosslinking properties during food processing, and it can also be used to produce cross-linked biopolymers (Nawaz et al. 2017). To the best of our knowledge, commercial tyrosinase has been obtained by extracting it from fungal cells (Min et al. 2019). To obtain intracellular enzymes it is necessary to disrupt the cells, which can be troublesome and sometimes reduce the yield of enzymatic activity (Yoshimoto et al. 1985). Fungal tyrosinases are mainly intracellular; however, many authors have reported extracellular tyrosinase (Halaouli et al. 2006; Min et al. 2019; Li et al. 2021; Zolghadri et al. 2023). The use of extracellular tyrosinase in bioremediation processes offers some advantages particularly in overcoming transport limitations associated with phenolic compounds entering the cell (Inamdar et al. 2014). Gasparetti et al. (2012) reported that extracellular and intracellular tyrosinases from Agaricus bisosporus exhibit different inhibition mechanisms.

Studies on fungal tyrosinase production are very important, so fungal tyrosinases have been described from a structural and functional point of view. Recent insights on the biochemical and molecular characteristics of these enzymes have been gained mainly from ascomycetes, such as Trichoderma and Neurospora, as well as from some basidiomycetes, such as Agaricus, Portabella, Pycnoporus, Pholiota nameko, and Lentinula edodes (Halaouli et al. 2005; Mayer 2006; Ioniţă et al. 2014; Kawamura-Konishi et al. 2007; Sato et al. 2009; Zolghadri et al. 2019; García-Molina et al. 2022). There are two fungal protein purification methods: chromatography and membrane filtration. Chromatography is the most common method, and it involves three steps: the first step is extraction, the second step is precipitation, which can be achieved by adding salt or acetone, and the third step is the chromatographic separation, which can be performed with techniques such as gel permeation, hydrophobic interaction, and affinity.

Membrane filtration consists in using a membrane process as microfiltration and ultrafiltration. The main complication is to select a membrane with the appropriate pore size (Labus et al. 2020). Fungal tyrosinase purification presents several challenges since intra- and extra-cellular tyrosinases are produced at low concentrations. Another difficulty is that fungi produce other enzymes such as laccase, quinone reductase, B-glucosidase, and proteases. All these enzymes can be found as contaminants in the tyrosinase extracts causing problems in the purification processes (Lopez-Tejedor and Palomo 2018).

Tomasini et al. (2001) reported the presence of an extracellular tyrosinase produced by the zygomycte Amylomyces rouxii.

This fungus shows positive results for phenoloxidase activity but lacks peroxidase activity. However, attempts to purify the tyrosinase from A. rouxii were unsuccessful results. A. rouxii has been demonstrated to produce an extracellular aspartic protease that has been shown to exert a deleterious effect on tyrosinase activity. In this work, in order to enhance tyrosinase activity, the gene responsible for encoding the protease was silenced. The A. rouxii strain in which the gene was silenced by RNA interference was designated as A412-3. The mutant strain displayed reduced protease activity and elevated tyrosinase activity in comparison to the parental strain (Marcial-Quino et al. 2023). The objective of this study was to demonstrate that A. rouxii A412-3 produces both intra- and extracellular tyrosinases and to partially purify the extracellular tyrosinase from A. rouxii A412-3.

Material and methods

Microorganisms

Amylomyces rouxii, which was isolated from an effluent from a paper mill (Tomasini et al. 1996), and its transformant strain A. rouxii A412-3, which has a silenced aspartic protease II (aspII) gene (Marcial-Quino et al. 2023), were used.

Media and culture conditions

Spores of A. rouxii and of the transformant A412-3, obtained from Erlenmeyer flasks containing potato dextrose agar, were used to inoculate the submerged fermentation system, performed in flasks containing 50 mL of modified Melin-Norkrans (MN) medium (Tomasini et al. 2001), inoculated with 1 × 10^6^ spores/mL, and incubated at 30 °C in a rotary shaker (125 rpm). Tyrosine (0.1 g/L) and pentachlorophenol (PCP, 12.5 mg/L) were added to the culture medium to increase tyrosinase activity. PCP was added at 24 h, when spores are germinated, since it has been shown that PCP inhibits spore germination (Tomasini et al. 2001). Each experiment was made in triplicate and repeated twice.

Obtaining extracts to determine tyrosinase activity

The extracts of A. rouxii and transformant A412-3 were obtained from 48 h cultures. The mycelia were separated by filtration using Whatman No.1 filter paper. The mycelia-free culture broth was used to determine extracellular activity. The mycelia were suspended in 5 mL of sodium phosphate buffer (0.1 M, pH 6.8) containing sorbitol (0.65 M) and protease inhibitor cocktail (Sigma-Aldrich). The mycelia were then disrupted using a Ten Broeck homogenizing pouring nozzle (#7727). Cell debris were removed by centrifugation at 10 000 × g for 30 min. The resulting supernatant was the cell-free extract and was used immediately for intra-cellular enzymatic assays. All extracts were stored at − 20 °C until use.

Tyrosinase activity assays

Monophenolase activity was determined, from extra- and intra-cellular extracts, by a modified Besthorn’s hydrazone method (Mazzocco and Pefferi 1976). In this case, 0.5 M phenol was used as substrate instead of catechol, and the chromophore used was 3-metyl-2-benzothiazolonone hydrazone (MBTH). Reaction mixture (2.5 mL in volume) contained 0.5 mL of phenol (0.5 M), 1.0 mL of phosphate-citric acid buffer (0.4 M, pH 4.2), 0.5 mL of MBTH (2 mM), and 0.5 mL of enzymatic extract. After 1 min, the reaction was stopped with 0.5 mL H_2_SO_4_ (5%) and 3 mL acetone. The reaction mixture was measured at 495 nm. One unit of enzymatic activity was defined as the amount of enzyme that produces 1 μmol of quinone per minute. The equation, Y = 0.754 X − 0.0099, where Y = absorbance, and X = concentration, was used to calculate enzyme activity. In addition, other substrates were used to determine monophenolase activity, L-tyrosine, 475 nm (Winder and Harris 1991) and p-cresol, 400 nm (Fan and Flurkey 2004), with MBTH dissolved in 50 mM phosphate buffer (pH 6.8) as described by Rodriguez-López et al. (1994) and Espín et al. (2000). The substrate’s concentration was 1 to 5 mM, as mentions in each case, and MBTH concentration was 2 mM. A commercial tyrosinase was used as positive control, and laccase and radish peroxidase were used as negative controls, using tyrosine as substrate. Diphenolase activity was determined also from extra- and intra-cellular extracts, using L-dopa, 475 nm (Halaouli et al. 2005; Selinheimo et al. 2009); catechin, 410 nm; catechol, 510 nm; and 4-tert-butylcatechol (TBC), 400 nm (Fan and Flurkey 2004; Kawamura-Konishi et al. 2007) as substrates in 50 mM sodium phosphate buffer (pH 6.8), as described by Wang et al. (1995) and Zhang et al. (1999). One unit of enzymatic activity was defined as the amount of enzyme that produced 1 µmol MBTH adduct per minute. The experiments were made in triplicate.

One-way ANOVA was used to determine differences in growth and enzyme activity, followed by Tukey’s test using the GraphPad Prism 5 program.

Tyrosinase purification

Crude enzyme extract was obtained from 500 mL of broth culture from the transformant A412-3 at 48 h and was used to purify extracellular tyrosinase. The culture medium was separated from the mycelium by filtration, and a protease inhibitor (1 mM phenylmethylsulphonyl fluoride) was added to the culture. The extract was frozen at − 80 °C and lyophilized. Powder from broth cultures was resuspended in 30 mL of 10 mM phosphate buffer, pH 7.0, and proteins were precipitated with ammonium sulfate to 80% w/v saturation. The extract was then centrifuged at 19 000 × g for 40 min at 4 °C. The proteins were dialyzed against milliQ water for 24 h using a 10 kDa membrane cut-off. Samples containing proteins were poured into an anion-exchange chromatographic column (Bio-Rad, EconoPAC High Q cartridge, 5 mL, USA) equilibrated beforehand with 25 mM Tris HCl buffer, pH 8.1. Proteins were eluted with a linear gradient of 0–0.5 M NaCl in 25 mM Tris–HCl buffer, pH 8.1, at a flow rate of 1 mL/min. Fractions of 2.0 mL were collected, and the protein concentration, and tyrosinase activity were determined in each fraction. The fractions with tyrosinase activities were applied to an anion exchange column (Bio Q5, Bio-Rad) equilibrated with 25 mM Tris–HCl buffer, pH 8.1. Proteins were eluted with a gradient of 0–0.5 M NaCl using the same buffer at a flow rate of 1 mL/min. Fractions of 1.5 mL were collected, and the protein concentration and tyrosinase activity were determined in each fraction. Finally, samples were poured onto a gel filtration column (1 × 14 cm, Econopac Bio-gel, 10 DG desalting) to eliminate excess of salts; 1-mL fractions were collected and concentrated using Amicon tubes (0.5 mL, Millipore, MA, USA).

Electrophoresis analysis

SDS-PAGE was performed on 12% acrylamide/gels according to Laemmli (1970) in order to determine the purity. Proteins were detected by Coomassie Brillant Blue R-250 (Sigma-Aldrich, USA). Isoelectric focusing was performed using a BioRad Rotofor Cell System (USA). The protein sample was diluted into a mixture of water and ampholytes, loaded into the assembled chamber (18 mL) and run for 4 h. Fractions were recovered directly into test tubes using a vacuum manifold. Mass molecular marker used was PageRuler™ Prestained Protein Ladder.

Sequencing of purified tyrosinase

The sample was digested in gel with trypsin, and the resulting peptides were applied to an LC–MS system consisting of an Accela micro-flow liquid chromatograph (Thermo-Fischer Co. San Jose, CA) with splitter (1/20) and an LTQ-Orbitrap XL mass spectrometer (Thermo-Ficher Co.) with nano-electrospray ionization system (ESI). For peptide fragmentation, CID (Collision-Induced Dissociation) methods were used, 2 + and 3 + charged ions were selected for this fragmentation event. Ions with charges higher than 4 + and undefined charges were disregarded. All spectra were acquired in positive detection mode. During the automatic data capture, dynamic ion exclusion was used, with an exclusion time of 60 s. The spectrometric data were searched against the NBCInr database using the Matrix science program (Mascot Search Result). This methodology was made in the Laboratorio Universitario de Proteómica IBT/UNAM.

Results and discussion

Intra- and extra-cellular tyrosinase activity produced by A. rouxii

A. rouxii was grown in submerged culture using MN medium supplemented with 0.1 g/L tyrosine and 12.5 mg/L PCP as mentioned before. Extracellular monophenolase and diphenolase activities were determined from the broth culture free of biomass. The intracellular monophenolase and diphenolase activities were determined from the biomass extract. Tyrosinase activity was measured spectrophotometrically using two monophenol and four o-diphenol substrates. The highest intra- and extra-cellular monophenolase activity was observed when p-cresol was used as a substrate, 180 and 80 U/mL, respectively. The highest extracellular diphenolase activity was obtained with catechol as a substrate, 975 U/mL, and the highest intracellular diphenolase activity was observed with TBC, 2233 U/mL (Table 1). Espín et al. (2000) and Fan and Flurkey (2004), who used a commercial mushroom tyrosinase and Portabella mushroom intracellular tyrosinase, reported similar results. Both studies found the highest diphenolase activity when using catechol as a substrate. The intracellular diphenolase activity from A. rouxii was higher than the extracellular activity, regardless of the substrate used, except when catechol was used as the substrate; in this case, diphenolase activity was similar (Table 1). These results suggest that the tyrosinase is actively secreted by A. rouxii, although its activity is lower compared to the intracellular enzyme. While fungal tyrosinase is generally considered to be an intracellular enzyme, some studies have reported its extracellular presence (Selinheimo et al. 2006; León-Santiesteban et al. 2008; Zou et al. 2014). Table 1. Mono- and diphenolase activities of extra- and intracellular tyrosinase from crude extract produced by A. rouxiiExtracellular (U/mL)Intracellular (U/mL)MonophenolsTyrosine25 ± 2.3238 ± 3.18p-cresol80 ± 5.73180 ± 18.92DiphenolsCatechin72.5 ± 4.3282.5 ± 12.24L-Dopa67 ± 3.11225 ± 33.84Catechol975 ± 88.76875 ± 99.11TBC666 ± 56.332233 ± 204.57

Monophenolase activity is lower than diphenolase activity in A. rouxii, as has also been reported for tyrosinases from plants, mammals, and fungi as Trichoderma reesei and Agaricus bisporus (Selinheimo et al. 2007). The maximal extracellular monophenolase and diphenolase activities in A. rouxii were achieved at 48 h in cultures with and without tyrosine. Montiel et al. (2004) reported that tyrosine and pentachlorophenol increased extracellular monophenolase activity.

Gukasyan (1999) studied the effect of lignin on growth and tyrosinase activity of Aspergillus and reported that tyrosinase from Aspergillus rapidly increased when either lignin or another inducer was added at 48 h. León-Santiesteban et al. (2008) reported that intra- and extra- cellular monophenolase activities produced by Rhizopus oryzae ENHE also increased when tyrosine was added to the culture medium.

Kinetics of growth and production of extracellular tyrosinase

The A. rouxii strain and transformant A412-3 were cultivated in M–N medium to assess the growth of both strains and the extracellular mono- and di-phenolase activity over the time. The biomass obtained was higher in cultures of A. rouxii than in cultures of the transformant 412–3, and the difference was significant (p = 0.001). The biomass produced were 2.7 mg/L and 1.89 mg/L, respectively. This means that the growth presented by the transformant was 1.4 times less than the parental strain (Fig. 1).Fig. 1. Growth of A. rouxii parental and A412-3 in MN medium supplemented with tyrosine (0.1 g/L) and PCP (12.5 mg/L). A. rouxii (■), A. rouxii A412-3 (□)

Furthermore, these cultures were used to measure the extracellular activity of mono- and diphenolase produced by A. rouxii and the transformant A412-3 (Fig. 2). It was observed that maximal tyrosinase activity, both mono- and diphenolase, occurred at 48 h of culture. Monophenolase activity obtained with the transformant A412-3 was 2.14 times higher than with the parental strain (Fig. 2A), while diphenolase activity was 3.02-fold higher with A412-3 than with the parental A. rouxii (Fig. 2B). Statistical analysis performed showed that both mono- and diphenolase activities are significantly different between A. rouxii and A412-3 strains with a p-value of 0.0001.Fig. 2. Determination of tyrosinase activity over time. Extracellular monophenolease (A) and diphenolase (B) activities produced by A. rouxii parental (■) and transformant A412-3 (□) during growth in MN medium supplemented with tyrosine and PCP. Monophenolase was determined using tyrosine as a substrate, while activity was determined diphenolase using 4-tert-butyl catechol (TBC) as substrate. Statistical analysis, one-way ANOVA followed by Tukey’s test (*p ≤ 0.05)

Based on these results, the 48-h cultures were used to purify the extracellular enzyme from the A412-3 transformant because it showed the highest mono- and diphenolase activities (Fig. 2).

Determination of the isoelectric point

The isoelectric point of the extracellular tyrosinase produced by A412-3 transformant strain was determined using a Rotofor system (Bio-Rad). After 4 h of protein separation based on isoelectric points, 20 fractions were collected, and the pH and tyrosinase activity were measured in each one. The highest enzymatic activities were observed in fractions 3 to 7, which had a pI range of 4.5 to 5.5. Fraction 5 with a pI of 5.2 exhibited the highest tyrosinase activity (Fig. 3). These results agree with those reported by Fan and Flurkey (2004), who identified up to four isoforms of tyrosinase during the purification of Portabella tyrosinase, with pI values ranging from 4.5 to 5.2. Similarly, Halaouli et al. (2005) reported the presence of four tyrosinase isoforms in Pycnoporus sanguineus with pI values from 4.5 to 5.0.Fig. 3. Determination of the isoelectric point of the partially purified extracellular protein of A. rouxii. (■) Isoelectric point of the proteins. (△) Diphenolase activity determined with L-Dopa (4.5 mM)

Partial purification of extracellular tyrosinase from A412-3

Extracellular tyrosinase from A412-3 was partially purified according to the procedure summarized in Table 2. The culture broth at 48 h was lyophilized, and then, 100 mL of phosphate buffer (50 mM, pH 7) was added to obtain the crude enzyme extract. Proteins were precipitated by adding ammonium sulfate (35–80%) and then resuspended into 30 mL of phosphate buffer (50 mM, pH 7.0). The protein extract was dialyzed for 24 h. The enzyme was further purified using two columns: the first was an anion exchange (DEAE-Sepharose) an a second one of gel filtration (Sephadex-200). El-Shora and El-Sharkawy (2020) also utilized these columns to purify tyrosinase from Penicillium chrysogenum and reported a purification fold of 90.4. However, they used a higher initial total protein amount (215 mg) compared to the protein used in this study. Moreover, intracellular tyrosinase from Aspergillus terreus and Penicillium copticola was purified using a Sephadex-G200 column, resulting in purification folds of 13.8 and 23.37 for A. terreus and P. copticola, respectively. The molecular weight of the obtained protein was approximately 35 kDa (Salah Maamoun et al. 2021). Table 2. Partial purification of tyrosinase obtained from the transformant A412-3Monophenolaese activityDiphenolase activityPurification stepProtein concentration(mg/mL)Total activity(U/mL)Specific activity(U/mg protein)Purification(-fold)Total activity(U/mL)Specific activity(U/mg protein)Purification(-fold)Crude extract1.66419751186123661422180% (NH_4_)_2_SO_4_1.024180017571.48220021481.5Anion exchange0.448167537383.15193343153.0Gel filtration0.06462597658.2296615,10410.6

Proteins were eluted from an anion exchange chromatography column using a gradient of 0–0.5 mol/L NaCl at a flow rate of 1 mL/min, collecting fractions of 3 mL. A single peak of tyrosinase activity was observed between fractions 13 and 15 (Fig. 4A), showing a threefold increase in specific activity (Table 2). These fractions, containing tyrosinase activity, were mixed and loaded into a gel filtration chromatography column where 70 fractions of 3 mL were collected (Fig. 4B). Tyrosinase activity eluted as a single peak in fraction 23, resulting in a purification of 8.2- and 10.6-fold for monophenolase and diphenolase activities, respectively (Table 2). The purification factor obtained was similar to that of a tyrosinase from Trichoderma reesei as reported by Selinheimo et al. (2006) and for monophenolase and diphenolase purification factors of a tyrosinase from Pycnoporus sanguineus as reported by Halaouli et al. (2005), after an ion exchange column. A final purification step using a hydroxyapatite column is necessary to achieve a high purification factor as suggested by Halaouli et al. (2005) who reported a 37.6-fold increase in purification factor after using a hydroxyapatite column to purify tyrosinase from P. sanguineus. Salah Maamoun et al. (2021) purified tyrosinase from Aspergillus terreus and Penicillium copticola using a Sephadex G-200 column as the final step. They achieved purifications of 13.8-fold and 11.2-fold, respectively, results that are similar to those obtained in our study.Fig. 4. Chromatography of partially purified tyrosinase obtained from the transformant A412-3 at 48 h of culture. A Anionic exchange (DEAE-Sepharose) and B gel filtration column (Sephadex-200). (●) Protein 280 nm, (△) monophenolase activity

Purity of tyrosinase was followed in 12% (w/v) polyacrylamide gel electrophoresis (PAGE), using Coomassie Brillant Blue (R350) for staining the protein bands (Fig. 5). Commercial tyrosinase and the fractions obtained from the anion exchange column (Fig. 4A) were assessed in a polyacrylamide gel electrophoresis. Commercial tyrosinase presented three protein bands with an apparent molecular mass of 128, 75, and 43 kDa (Fig. 5, lane 5). A single band of 70 kDa was observed in fraction 6 (lanes 1 and 2), but it did not show any enzymatic activity. For fraction 14 (lane 4), however, tyrosinase activity was detected, but several bands of varying molecular masses were still visible. Therefore, fractions 13–15 (Fig. 4A) were mixed and were eluted in a gel filtration column (Fig. 4B). Fraction 23 exhibited the highest tyrosinase activity. This fraction presented three bands of 40, 60, and 128 kDa, approximately (Fig. 5, lane 3). The three bands of fraction 23 were sequenced by LC–MS/MS.Fig. 5SDS-PAGE of tyrosinase produced by the transformant A412-3 of A. rouxii. Numbers in the gel lanes indicate the fractions obtained in each purification step. M) Molecular weight marker (PageRuler™ prestained Protein Ladder). 1 and 2) Fraction 6 from anionic exchange column; 3) fraction 23 from filtration gel column; 4) fraction 14 from anionic exchange column; 5) commercial tyrosinase from anionic exchange column

Deduced amino acid sequences of the partially purified tyrosinase protein from A412-3

The amino acid sequences of the 40 kDa band obtained from the acrylamide gel were determined using mass spectrometry. Eight peptide sequences were identified, showing similarities to various fungal species (Fig. 6). The closest match was found between these peptides and the sequence of Agaricus bisporus tyrosinase (100%).Fig. 6. Alignment of the amino acid sequence of peptide no. 4 obtained from the extracellular protein of transformant A412-3. A412-3. Identification of the characteristic histidine residue in the CuA copper binding site

By comparing the peptide sequence number 4 with that of other fungi (Fig. 6), it was possible to identify the amino acid sequence QXXG(I/V)HGXP, which includes the histidine (H) residue, highly conserved in fungi. This histidine residue is part of the CuA binding motif of fungal tyrosinases. Some of the most studied fungi in which this conserved region has been identified include A. bisporus (Wichers et al. 1996), N. crassa (Selinheimo et al. 2006), P. nameko (Kawamura-Konishi et al. 2007), L. edodes (Sato et al. 2009), and Pycnoporus sanguineous (Halaouli et al. 2005). This conserved region is also included in the sequence of the crystallographic structure of Aspergillus oryzae tyrosinase reported by Fujieda et al. (2013).

The results obtained allow us to confirm that the extracellular phenoloxidase produced by A. rouxii 412–3 is a tyrosinase of ~ 45 kDa. The molecular mass of the tyrosinase from A. rouxii reported by Montiel et al. (2004) was between 40 and 43 kDa. These data are very similar to those obtained with the tyrosinase from P. sanguineus, 45 kDa (Halaouli et al. 2005); Pholiota nameko, 45 kDa (Kawamura-Konishi et al. 2007); Trichoderma reesei, 43.2 kDa (Selinheimo et al. 2006); A. bisporus and Portabella, 43–47 kDa (Wichers et al. 1996; Fan and Flurkey 2004); and Lentinula edodes, 45 and 68 kDa (Sato et al. 2009). Espín et al. (2000) and Espín and Wichers (1999) reported a 67 kDa size for a latent tyrosinase from Agaricus bisporus after proteolytic activation with an endogenous fungal protease.

Conclusion

We first isolated from a contaminated effluent a zygomycete that produces tyrosinase (Tomasini et al. 2001). To our knowledge, there are few reports mentioning tyrosinase production from zygomycetes (Montiel et al. 2004; León-Santiesteban et al. 2008). A. rouxii produced lower monophenolase activity than diphenolase activity, as reported also for tyrosinases from plants, mammals, and microorganisms (Selinheimo et al. 2007). The results are highly indicative of the production of an extracellular tyrosinase by A. rouxii, with a molecular mass of 40 kDa. The fungal tyrosinase that has been described is primarily associated with ascomycetes, including species such as Aspergillus, Trichoderma, and Neurospora, as well as some basidiomycetes, such as Agaricus, Pycnoporus, and Lentinula. In this study, a tyrosinase from A. rouxii A412-3, a zygomycete was purified and sequenced to obtain an alignment of amino acids. The sequence was then compared with those of other fungi. To our knowledge, this is the first report of a partial purification and peptide sequencing of a tyrosinase from a zygomycete. Enhancing tyrosinase production in A. rouxii could improve its efficiency in degrading toxic compounds. Additionally, it could enable the extraction of extracellular tyrosinase for use in other industrial and biotechnological processes.