A novel laccase from fresh fruiting bodies of the wild medicinal mushroom Tricholoma matsutake

The knowledge about biological activities of constituents from medicinal mushrooms belonging to the genus Tricholoma is limited. A 59-kDa laccase has now been purified from fresh fruiting bodies of the mushroom Tricholoma matsutake. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, affinity chromatography on ConA-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. Of the various affinity and ion exchange chromatographic media employed, the laccase bound only on Con A-Sepharose. The activity of the laccase did not undergo major changes over the temperature range 20–80°C. However, all activity vanished following exposure to 100°C for 10 minutes. The enzyme activity varied only slightly over the pH range 3-5, with the optimal pH of 5, but exhibited a precipitous decline when the pH was increased to 6, and was undetectable at pH 8 and 9. The laccase showed activity in the decolorization of azo dyes without a mediator. Its N-terminal sequence demonstrated only slight resemblance to those of other mushroom laccases. The newly described laccase is distinctive from the previously isolated Tricholoma mushroom laccases in a number of aspects.

Fungi are the main producers of laccases, especially basidiomycetes (Garzillo et al., 2001;Coll et al., 1993;Shin & Lee, 2000;Cambria et al., 2000;Wang & Ng, 2004a;Wang & Ng, 2004b;Zhang et al., 2010;Li et al., 2010).Fungal laccases are particularly abundant in many white-rot fungi, however, very few laccases have been reported from ectomycorrhizal fungi.Tricholoma matsutake, a well-known wild edible mushroom, is an ectomycorrhizal fungus belonging to Tricholomataceae family (Tong et al., 2013).Due to its unique flavor and taste, it is a highly prized mushroom compared with the much less expensive T. mongolicum and T. giganteum.The knowledge about the constituents of mushrooms belonging to the genus Tricholoma is limited.Polysaccharopeptides with immunomodulatory and antitumor activities have been prepared from cultured mycelia of T. mongolicum and T. lobayense (Wang et al., 1996;Wang et al., 1995).A laccase has been purified from T. mongolicum (Li et al., 2010).A nuclease (Hatakeyama et al., 2010) and polysaccharide (Tong et al., 2013) have been isolated from the fruiting body of T. matsutake.Lectins with antiproliferative, antitumor and immunoenhancing activities have been isolated from fruiting bodies of T. mongolicum (Wang et al., 1995).One of these lectins produces hypotensive effects in rats (Wang et al., 1996).Hence it would be a worthy undertaking to examine additional Tricholoma species for the presence of bioactive constituents.The present investigation disclosed that fresh fruiting bodies of T. matsutake produce a laccase with some unique characteristics and its potential application in the decolorization of various dyes has been examined.

Isolation of laccase.
Fresh fruiting bodies (900 g) of the mushroom Tricholoma matsutake were homogenized in distilled water (3 mL/g) using a Waring blender.Distilled water was used instead of physiological saline to obviate the need for subsequent dialysis.The homogenate was centrifuged at 18 500 × g for 30 minutes.Tris-HCl buffer (1 M, pH 7.4) was added to the resulting supernatant to 10 mM.The supernatant was then loaded on a 5 × 20 cm column of DEAE-cellulose (Sigma) in 10 mM Tris-HCl buffer (pH 7.4).Unbound proteins were collected in the flowthrough fraction D1.Bound proteins were eluted with 0.8 M NaCl in the starting buffer and collected in fraction D2.Fraction D1 was then subjected to affinity chromatography on a 2.5 × 20 cm column of Affi-gel blue gel (Bio-Rad) in 10 mM Tris-HCl buffer (pH 7.4).Unbound proteins were eluted with the same buffer and collected as fraction B1.Adsorbed proteins were eluted with 1.5 M NaCl in 10 mM Tris-HCl buffer.Fraction B1 was fractionated on a 2.5 × 20 cm column of CM-cellulose (Sigma) in 10 mM NH 4 OAc buffer (pH 4.5).This step was in conformity with the frequently reported use of ion exchange chromatography at an acidic pH for laccase purification.The unbound fraction CM1 was eluted with the same buffer.The bound fraction CM2 was eluted with 1 M NaCl in starting buffer.Fraction CM1 was further purified on a 2.5 × 20 cm column of Con A-Sepharose (GE Healthcare).Following elution of unbound proteins (fraction Con A1) with 50 mM Tris-HCl buffer (pH 7.4) containing 0.5 M NaCl, 10 mM CaCl 2 and 10 mM MgCl 2 , the bound proteins (fraction Con A2) were eluted with 0.4 M a-methyl-D-glucopyranoside (Sigma) in the 50 mM Tris-HCl buffer.
Assay of laccase activity.Laccase activity was assayed by measuring the oxidation of 2,7'-azinobis [3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS).A modification of the method of Shin and Lee (2000) was used.An aliquot of enzyme solution was incubated in 1.3 mL of 67 mM sodium acetate buffer (pH 4.5) containing 1.54 mM ABTS at 30°C.One unit of enzyme activity was defined as the amount of enzyme required to produce an absorbance increase at 405 nm of one per min per mL of reaction mixture under the aforementioned conditions.All determinations were performed in triplicate.
Molecular mass determination by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by FPLC gel filtration.SDS-PAGE was carried out in accordance with the procedure of Laemmli and Favre (1973) using a 12% resolving gel and a 5% stacking gel.At the end of electrophoresis, the gel was stained with Coomassie brilliant blue.FPLC gel filtration in 0.2 M NH 4 HCO 3 buffer (pH 8.5) at a flow rate of 24 mL/h and with a fraction size of 0.8 mL was carried out using a Superdex 75 column which had been calibrated with molecular mass standards (GE Healthcare).
Analysis of N-terminal amino acid sequence.Amino acid sequence analysis was carried out using an HP G1000A Edman degradation unit and an HP1000 HPLC system (Lam & Ng, 2001).
Assay of pH and temperature optima.In the assay for optimal pH value, a series of solution of ABTS in buffers with different pH values was used.The assay buffers were prepared in 0.1 M NaOAc buffer (pH 3.0, pH 4.0 and pH 5.0), 0.1 M Mes buffer (pH 5.0, pH 6.0 and pH 7.0), and 0.1 M Hepes buffer (pH 7.0, pH 8.0 and pH 9.0).The assay temperature was 37°C.To determine the optimal temperature, the reaction mixture was incubated at 20°C to 90°C in 0.1M NaOAc buffer (pH 5.0).
Assay of substrate specificity.In order to determine the substrate specificity of the purified laccase, several aromatic substrates were added to the assay media in place of ABTS.The substrates used (5.0 mM) were ABTS, N,N-dimethyl-1,4-phenylenediamine, hydroquinone, catechol, 2-methylcatechol, pyrogallol, and tyrosine.The enzyme assay was performed as described above in 50 mM Na 2 HPO 4 -citric acid buffer (pH 3.0).The substrate oxidation rate was followed by measuring the absorbance change with the molar extinction coefficients (ε) obtained from the literature (EggertTemp & Eriksson, 1996;Galhaup et al., 2002;Younes & Sayadi, 2011).
Assay for HIV-1 reverse transcriptase inhibitory activity.The assay for HIV-1 reverse transcriptase inhibitory activity was carried out according to instructions supplied with the assay kit from Boehringer Mannheim (Germany).The assay takes advantage of the ability of reverse transcriptase to synthesize DNA, starting from the template/primer hybrid poly (A) oligo (dT) 15 .Digoxigenin-and biotin-labeled nucleotides in an optimized ratio are incorporated into the DNA molecule synthesized by the reverse transcriptase (RT).The detection and quantification of synthesized DNA as a parameter for RT activity follows a sandwich ELISA protocol.Biotin-labeled DNA binds to the surface of microtiter plate precoated with strepatavidin.In the next step, an antibody to digoxigenin, conjugated to peroxidase, binds to the digoxigenin-labeled DNA.In the final step, the peroxidase substrate is added.The peroxidase catalyzes the cleavage of the substrate, producing a colored product.The absorbance of the samples at 405 nm is determined by a microtiter plate (ELISA) reader and is directly proportional to the level of RT activity.A fixed amount (46 ng) of recombinant HIV-1 reverse transcriptase was used.The inhibitory activity of the isolated laccase was calculated as percent inhibition as compared to a control without the laccase.
Decolorization of textile dyes using purified laccase.Bromothymol blue, Congo red, and Eriochrome black T at a final concentration of 100 mg/L , and malachite green and Evans Blue with a final concentration of 10 mg/L were incubated with 10 μL (0.2 U) enzyme in 10 mM NaAc-HAc buffer (pH 5.0) at 40°C for 3 h.Enzyme preparation boiled for 20 min was used as a Laccase activity was determined at 30°C, pH 4.5.A laccase from Tricholoma matsutake negative control.The decolorization was monitored at λmax of the respective dye using a UV-visible spectrophotometer.

Isolation of laccase
The protocol utilized in the present investigation was efficacious for purifying T. matsutake laccase, as summarized in Table 1.Proteins with little or no activity were removed from the laccase-enriched fraction in each of the chromatographic steps on DEAE-cellulose, Affigel blue gel, and CM-cellulose.Affinity chromatography of CM-cellulose flow-through on Con A-Sepharose yielded a large unbound peak (fraction Con A1) and a much smaller bound peak (fraction Con A2) (Fig. 1).Laccase activity resided in fraction Con A2 only (Table 1).Con A2 was resolved on Superdex 75 into two peaks of similar size (Fig. 2).Laccase activity was enriched in the first peak SU1.The second fraction SU2 had minimal activity (Table 1).A 47.5-fold purification and activity recovery of 29.83% were achieved.

Molecular mass determination and analysis of the N-terminal amino acid sequence
The purified laccase appeared as a single band with a molecular mass of 59 kDa in SDS-PAGE (Fig. 3) and as a single peak with the same molecular mass upon rechromatography on Superdex 75 (not shown).The Nterminal amino acid sequence of T. matsutake laccase was ADPVGIVGD, which does not resemble previously isolated mushroom laccases to any appreciable extent.A comparison of the N-terminal amino acid sequence of the T. matsutake laccase with other fungal laccases is shown in Table 2.

Effect of temperature and pH on laccase activity
With ABTS as substrate, the activity of the enzyme rose slowly but steadily (about 30% increase) when the temperature was increased from 20 to 60°C.The activity fell when the temperature was further raised to 70 and 80°C (Fig. 3).Incubation of the enzyme at 100°C for 10 minutes brought about a total destruction of activity.The optimal pH value of the purified laccase was about 5.0, with a slow increase in activity as the pH value moved from 3 to 5. Negligible laccase activity was detected at pH 7 and no activity at pH 8 and 9 (Fig. 4, Table 4).

Effect of laccase on HIV-1 reverse transcriptase inhibitory activity
No inhibitory effect of the isolated laccase on the activity of HIV reverse transcriptase was observed at a laccase concentration of 10 mM (Table 4).

Dye decolorization by the purified laccase
The purified laccase decolorized structurally different dyes with a variable decolorizaiton rate from 10% to 40% (Table 5).

DISCUSSION
Previously most laccases were purified from culture broths and not from fruiting bodies of mushrooms.The laccase purified from the fruiting bodies of T. matsutake differs from the laccases isolated from other Tricholoma species.The protocol utilized in the present investigation was efficacious for purifying T. matsutake laccase.Pro-teins with little or no activity were removed from the laccase-enriched fraction in each of the chromatographic steps on DEAE-cellulose, Affi-gel blue gel, CM-cellulose, Con A-Sepharose and Superdex 75.Unlike some of the previously reported laccases, e.g., those from Tricholoma mongolicum (Li et al., 2010), Lentinus tigrinus (Xu et al., 2012) and Abortiporus biennis (Zhang et al., 2011), which were bound on cationic and anionic exchangers, the T. matsutake laccase did not bind to DEAE-or CMcellulose (Table 4).Its adsorption on Con A-Sepharose revealed that it is a glycoprotein like the previously reported Ganoderma lucidum laccase (Wang & Ng, 2006a).Its molecular mass was 59 kDa, within the range of molecular masses reported for fungal laccases (Garzillo et al., 2001;Giardina et al., 1999;Shin & Lee, 2000;Zhang et al., 2011).But was different from those of other Tricholoma sp.laccases: T. mongolicum (66 kDa) and T. giganteum (43 kDa) (Hyoung et al., 2004;Li et al., 2010).Its N-terminal sequence showed only a slight similarity to those of T. mongolicum and T. giganteum (Hyoung et al., 2004;Li et al., 2010).Similar to other fungal laccases, the best substrate for the enzyme was ABTS (Li et al., 2010;Zhang et al., 2010;2011) The optimum pH for the purified enzyme was pH 5, which was higher than that of laccases reported before, such as T. mongolicum (pH 23) (Li et al., 2010), T. giganteum (pH 4) (Wang & Ng, 2004a)

Ganoderma lucidum laccase GQNGDAVP
Identical corresponding amino acid residues were underlined.N-terminal sequence of laccases other than Tricholoma matsutake laccase were taken from Refs (Coll et al., 1993;Wang & Ng, 2004a;Zhang et al., 2010;Li et al., 2010;Wang & Ng, 2006a;Garzillo et al., 2001).ennis (pH 3.5) (Zhang et al., 2011).It is noteworthy that a dramatic decrement in laccase activity ensues when the ambient pH is raised to 6 and that there is complete loss of activity at pH 8 and 9.
Mushroom laccases, such as those from T. mongolicum (Li et al., 2010) and T. giganteum (Wang & Ng, 2004a), have shown HIV-1 reverse transcriptase inhibitory activity.Although T. matsutake has been widely used for medicinal or health functional purpose since ancient times in Asian countries (Tong et al., 2013), the laccase isolated in the present study was devoid of an HIV-1 reverse transcriptase inhibitory activity.
Compared with laccases from other sources (Telke et al., 2011;Forootanfar et al., 2011), the purified T. matsutake laccase manifested a considerably high decolorization activity towards Eriochrome black T but low activity towards other assayed dye.Notably, the laccase from Bacillus sp.ADR showed no decolorization activity (Telke et al., 2011).The T. matsutake laccase differs from its counterparts from T. giganteum and T. mongolicum in a number of aspects, such as molecular mass, pH and temperature optima, N-terminal amino acid and HIV-1 reverse transcriptase inhibitory activity (Table 4).The chromatographic behavior of T. matsutake laccase was quite different from other Tricholoma mushroom laccases.It was unbound on DEAE-cellulose, CM-cellulose or Affi-gel blue gel.In contrast, T. mongolicum laccase was bound on DEAE-and CM-cellulose, while T. giganteum laccase was bound on CM-cellulose and Affi-gel blue gel.Its  (Li et al., 2010), Tricholoma giganteum (Hyoung et al., 2004), Lentinus tigrinus (Xu et al., 2012), Lentinus edodes (Sun et al., 2011), Abortiporus biennis (Zhang et al., 2011), Pleurotus eryngii (Wang & Ng, 2006b), Ganoderma lucidum (Wang & Ng, 2006a).lack of inhibitory effect on the activity of the HIV enzyme is in contrast to the inhibitory action of T. giganteum and T. mongolicum laccases.It deserves attention that not all Tricholoma mushroom laccases exhibit an inhibitory effect towards HIV-1 reverse transcriptase.
In summary, we have reported the purification and characterization of a novel laccase from the fruiting body of T. matsutake.The purified enzyme manifested a molecular mass of 59 kDa, N-terminal amino-acid sequence of ADPVGIVGD, a pH optimum of 5 and temperature of 60°C.It showed activity in the decolorization of azo dyes without a mediator.The present enzyme was the first laccase purified from T. matsutake.It represents an addition to the growing list of mushroom laccases (Leonowicz & Malinowska, 1982;Shin & Lee, 2000;Dedeyan et al., 2000;Cambria et al., 2000;Fukuda et al., 2001;Baldrian, 2006;Tinoco et al., 2001).

Table 4 . Comparison of biochemical characteristics and activities of Tricholoma matsutake laccase with other mushroom laccases.
BD, Bound; UN, Unbound; -: not determined or not attempted.References: Tricholoma mongolicum