Purification and characterization of a novel laccase from the mushroom Pleurotus nebrodensis

1Institute of Biotechnology and Germplasmic Resource, Yunnan Academy of Agricultural Science, Kunming, China; 2Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing, China; 3State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, China; 4Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China


INTRoDuCTIoN
Laccase (E.C.1.10.3.2, benzenediol:oxygen oxidoreductase) is a copper-protein belonging to the group of blue oxidases (Baldrian, 2006).Laccase catalyzes the oxidation of various phenolic compounds and aromatic amines with molecular oxygen as the electron acceptor (Palmieri et al., 1993).It is widely distributed among plants, insects, bacteria, and fungi, and laccase from fungi, especially from white rot fungi, plays an important role in the natural degradation of lignin (Baldrian, 2006).Fungal laccases demonstrate great applications in biodegradation of lignin, and recently they have also found potential applications in wood pulping, manufacturing of textile dyes, detoxification of polluted water, and as biosensors (Widsten & Kandelbauer, 2008).
Mushrooms have drawn attention of investigators since they constitute a source of important compounds comprising lectins, laccases, proteases, ribonucleases, ribosome inactivating proteins, antibacterial proteins, antifungal proteins, and polysaccharides (Ng, 2004).Many of them belong to white rot fungi, and a number of mushroom laccases have been reported in the literature (Hu et al., 2011;Li et al., 2010;Wang & Ng, 2006a).To date, there are only several publications on Pleurotus nebrodensis, most of which are on classification of the mushroom and the taxonomic relationship between P. nebrodensis and P. eryngii (Urbanelli et al., 2007;Zhang et al., 2006).One study showed that the mushroom is abundant in vitamin B12 and riboflavin, and has a low caloric content and a high gastronomic value (La Guardia et al., 2005).Only one protein, a hemolysin, has been isolated from P. nebrodensis (Lv et al., 2009).In view of the importance of laccases, the present study aimed to isolate and characterize a laccase from the mushroom P. nebrodensis, and to compare its characteristics with those of other laccases from the genus Pleurotus.

MATERIAlS AND METHoDS
Isolation of laccase.Fresh fruiting bodies (1500 g) of the mushroom P. nebrodensis were homogenized in distilled water (4500 ml) using a Waring blender.The homogenate was centrifuged at 12 000 rpm for 15 minutes.Tris/HCl buffer (1 M, pH 7.4) was added to the resulting supernatant to a final concentration of 10 mM.The supernatant was then loaded on 5 cm × 20 cm columns of DEAE-cellulose (Sigma, USA) in 10 mM Tris/HCl buffer (pH 7.4).Unadsorbed proteins were collected in the flowthrough fraction, and adsorbed proteins were eluted with the same buffer containing 0.8 M NaCl.Enzyme fraction was further purified on 2.5 cm × 20 cm columns of CM-cellulose (Sigma, USA) in 10 mM NH 4 OAc buffer (pH 4.5).After elution of unadsorbed proteins, the same NH 4 OAc buffer with 1 M NaCl was used for adsorbed ones.Subsequently, the active fraction was chromatographied on a 2.5 cm × 20 cm column of Q-Sepharose (GE Healthcare, USA).Following elution of unadsorbed proteins with 10 mM Tris/HCl buffer (pH 7.4), the adsorbed proteins were eluted with a linear concentration gradient of NaCl (0-1 M) in the same Tris/HCl buffer.Finally, the laccase fraction was dialyzed, lyophilized, and then chromatographied on a Superdex 75 HR 10/30 column (GE Healthcare) in 0.2 M NH 4 HCO 3 buffer (pH 8.5) using an AKTA Purifier (GE Healthcare, USA).
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 condition (Zhang et al., 2011).
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 was carried out using a Superdex 75 HR 10/30 column which had been calibrated with molecular mass standards (GE Healthcare, USA).
Analysis of N-terminal amino acid sequence.N-terminal amino acid sequence analysis was carried out using an HP G1000A Edman degradation unit and an HP1000 HPLC system (Zhang et al., 2011).
Substrate specificity of isolated laccase.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, catechol, hydroquinone, 2-methylcatechol, pyrogallol, and tyrosine.
The enzyme assay was performed as described above and the test buffer was sodium acetate buffer (pH 4.5).The substrate oxidation rate was followed by measuring the absorbance change with the molar extinction coefficients (ε) obtained from the literature (Eggert et al., 1996;Galhaup et al., 2002).
Assay for temperature and pH optima.To determine the optimal temperature, the standard laccase activity assay was ran at 20, 25, 30, 40, 50, 60, 70, 80, and 100°C.The assay buffer was the same sodium acetate buffer used in the standard assay described above.In the assay for optimal pH value, enzyme activities were determined using the standard laccase assay above, but in a series of assay buffers in a pH range of 3.0-9.0instead of the sodium acetate buffer (pH 4.5).The assay buffers were 50 mM NaOAc-HAc (pH 3.0-5.0),Mes (pH 5.0-7.0), and Hepes (pH 7.0-9.0).
Assay for bioactivities.HIV-1 reverse transcriptase inhibitory activity was carried out using an assay kit from Boehringer Mannhein (Germany) (Zhao et al., 2009).Antiproliferative activity towards tumor cell lines including human breast cancer (MCF-7) and hepatoma (HepG2) was assayed using a standard method described by Lam and Ng (2001).Antifungal activity towards Fusarium oxysporum, Rhizoctonia cerealis, Rhizoctonia solani, and Sclerotinia sclerotiorum was carried out using Li's method (Li et al., 2011).

Results and discussion
Purification of laccase.The fraction of P. nebrodensis fruiting body extract that was adsorbed on DEAEcellulose (fraction D2) and subsequently unadsorbed on CM-cellulose (fraction C1) contained the bulk of laccase activity (Table 1).Ion exchange chromatography of fraction C1 on Q-Sepharose yielded an unadsorbed peak (fraction Q1), a small adsorbed peak (fraction Q2) and a large adsorbed peak (fraction Q3) (Fig. 1).Laccase activity resided in fraction Q2 (Table 1).Q2 was resolved on Superdex 75 into two peaks of similar size (Fig. 2).Laccase activity was enriched in the first peak SU1 (Table 1).In brief, the laccase resided in adsorbed fraction D2 of DEAE-cellulose, then unadsorbed fraction C1, then adsorbed fraction Q2 of Q-Sepharose, and finally fraction SU1 of FPLC.
Most of the laccases reported in the literature were isolated from mycelia and not from fruiting bodies of mushroom.It is known that straw mushroom myce-  lia and fruiting bodies elaborate the same lectin (She et al., 1998).However, dissimilar lectins are extracted from Tricholoma mongolicum mycelia (Wang et al., 1996) and fruiting bodies (Wang et al., 1998).It is reported herein that a laccase can be purified from the fruiting bodies of the mushroom P. nebrodensis.The procedure employed in the present study was useful for isolating Pleurotus nebrodensis laccase.Proteins with little or no laccase activity were separated from the laccase-enriched fraction in each of the chromatographic steps on DEAE-cellulose, CM-cellulose, Q-Sepharose and Superdex 75.In contrast to some of the previously reported laccases, e.g., those from Coriolus hirsutus (Shin & Lee, 2000) and Rigidoporus lignosus (Cambria et al., 2000), which are adsorbed on cationic and anionic exchangers including DEAE-Sepharose, Q-Sepharose and Hitrap-SP, the P. nebrodensis laccase is adsorbed on DEAE-cellulose and Q-Sepharose but unadsorbed on CM-cellulose.
Molecular mass determination and N-terminal sequence analysis.SU1 appeared as a single band with a molecular mass of 64 kDa in SDS-PAGE (Fig. 3) and a single peak with the same molecular mass upon rechromatography on a Superdex 75 column (data not shown).It represented purified laccase.The N-terminal sequence of P. nebrodensis laccase showed only little similarity to other mushroom laccases (Table 2).Its molecular mass lies within the range of molecular masses found for mushroom laccases (Baldrian, 2006).Its N-terminal sequence does not, however, resemble previously isolated mushroom laccases to any great extent.

Mushroom laccase
N-terminal sequence Ref.
Temperature and pH dependence of laccase activity.The activity of the enzyme rose steadily when the temperature was raised from 20°C to 70°C.The activity dropped slightly when the temperature was further raised to 80°C (Fig. 4).Incubation of the enzyme at 100°C for 10 minutes brought about complete abolition of activity.The enzyme activity increased slowly when the pH was elevated from 3 to 5. A further rise in pH to 6 led to an abrupt drop in activity.Negligible laccase activity was detected at pH 7 while no activity was discerned at pH 8 and 9 (Fig. 5).
The P. nebrodensis laccase is unique in that its enzymatic activity undergoes a steady increase as the temperature is raised from 20°C to 70°C and that only a small decrement in activity ensues when the temperature is further elevated to 80°C.In contrast, R. lignosus laccase exhibits a temperature optimum at 45°C with only residual activity at 65°C (Cambria et al., 2000).However, the enzyme is denatured after treatment at 100°C for 10 minutes and all activity is lost.P. nebrodensis laccase requires a pH of 5 for it to manifest maximal activity.This finding is at variance with the observation of an optimum pH of 2-3 from several other mushroom laccases (Shin & Lee, 2000).It is noteworthy that a drastic decline in laccase activity results when the pH is elevated to 6 and that total loss of activity occurs at pH 8 and 9. Since the present laccase manifests a considerably high temperature optimum, it has good potential for industrial applications.
Assay for other biological activities.The laccase did not display antifungal activity, anti-proliferative activity on tumor cells or inhibitory activity toward HIV-1 reverse   transcriptase when tested at a concentration of 100 μM (not shown).Some mushroom products, e.g., lectins (Li et al., 2008), polysaccharopeptide (Wang & Ng, 2001), ribosome inactivating proteins (Lam & Ng, 2001) and ubiquitin-like proteins (Wang & Ng, 2000) manifest an inhibitory activity toward HIV-1 reverse transcriptase.Laccases from G. lucidum, H. erinaceum and T. giganteum also inhibit this retroviral enzyme.The P. nebrodensis laccase is devoid of this ability.The laccase isolated in this investigation lacks anti-proliferative and antifungal activities, in contrast to the observation that some mushroom lectins, antifungal proteins and ribonucleases show anti-proliferative activity (Wang & Ng, 2001), and mushroom antifungal proteins inhibit mycelial growth (Lam & Ng, 2001).

Table 1 . Yield and laccase activities of various chromatographic fractions (from 1.5 kg fresh fruiting bodies, 30°C)
Data corresponding to the most active fraction at each chromotographic step are shown in boldface.