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Pleurotus ostreatus yellow laccase have been reported. However, this fungus deserves attention not only as one of the most efficient laccase producers but also ...
ISSN 0003-6838, Applied Biochemistry and Microbiology, 2006, Vol. 42, No. 1, pp. 56–61. © MAIK “Nauka /Interperiodica” (Russia), 2006. Original Russian Text © N.N. Pozdnyakova, O.V. Turkovskaya, E.N. Yudina, Ya. Rodakiewicz-Nowak, 2006, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2006, Vol. 42, No. 1, pp. 63–69.

Yellow Laccase from the Fungus Pleurotus ostreatus D1: Purification and Characterization N. N. Pozdnyakova*, O. V. Turkovskaya*, E. N. Yudina*, and Ya. Rodakiewicz-Nowak** *Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, pr. Entuziastov 13, Saratov, 410049 Russia e-mail: [email protected] **Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapominajek 8, Krakow, 30239 Poland e-mail: [email protected] Received May 5, 2004

Abstract—Yellow laccase was isolated from a solid-phase culture of the fungus Pleurotus ostreatus D1 and characterized. It is a copper-containing enzyme with a molecular weight of 64 kDa. Its lacks an absorption spectrum maximum at 610 nm, a result which is characteristic of fungal laccases and corresponds to the presence of type I copper atoms. The optimum pH values for the enzyme are determined. They prove to be 7.0 for syringaldazine, 8.0 for pyrocatechol, and 4.0 for 2,2'-azine-bis-(3-ethylbenzothiazoline-6-sulfonate and 2,6dimethoxyphenol. Kinetic parameters (Km and Vmax) for oxidation of these substrates are determined. The effect of inhibitors (SDS, 2-mercaptoethanol, and EDTA) on the activity of the enzyme is studied. It is shown that yellow laccase from Pleurotus ostreatus D1 in the absence of a mediator oxidizes anthracene to anthraquinone to 95%. DOI: 10.1134/S000368380601008X

Laccase (EC 1.10.3.2, p-diphenol: oxygen oxidoreductase) belongs to the blue copper-containing oxidases. It catalyzes the oxidation of ortho- and paradiphenols, aminophenols, polyphenols, arylamines, and some inorganic ions, while simultaneously reducing atmospheric oxygen to water. Laccase was first found in the varnish tree Rhus vernicifera; however, most laccases are found and studied in lignin-degrading basidiomycetes (white rot fungi) [1, 2]. The ability of white rot fungi to degrade not only lignin but also many aromatic xenobiotics is related to this enzyme. The increasing interest in fungal laccases is related to their potential use in organic synthesis, degradation of lignin and toxicants, production of biosensors, and other branches of biotechnology [3–5]. It has been long believed that the catalytic potential of laccases is limited to their ability to oxidize phenolics. Recent studies, however, point to a much broader range of substrates. On the one hand, the new substrates include benzyl alcohols and nonphenolic dimeric compounds of lignin and polyaromatic hydrocarbons (in the presence of mediators in the reaction mixture) [6–10]. On the other hand, it is suggested that solid-phase cultivation of the fungus on a natural lignin-containing substrate results in modification of the active center of laccase by lignin oxidation products, supposedly acting as mediators. This modification can also expand the substrate range of the enzyme to nonphenolic compounds [11–14].

However, this fungus deserves attention not only as one of the most efficient laccase producers but also as a degrader of many aromatic xenobiotics, including polyaromatic hydrocarbons (PAHs) [15]. The goal of the study was to isolate and characterize yellow laccase from a solid-phase culture of the fungus Pleurotus ostreatus D1. MATERIALS AND METHODS The fungus and its cultivation. The white rot agent Pleurotus ostreatus D1, used in this study1, is stored in the collection of the Laboratory of Microbiology and Mycology of the Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences. The culture was maintained on a rich agar medium for basidiomycetes (g/l): NH4NO3 (0.72), KH2PO4 (1.0), MgSO4 · 7H2O (1.0), KCl (0.5), FeSO4 · 7H2O (0.01), ZnSO4 · 7H2O (0.0028), CaCl2 · 2H2O (0.033), glucose (10.0), yeast extract (0.5), and peptone (10.0) [15]. For inoculation, the fungus was grown in 250 ml flasks with 100 ml of the same medium at 29°ë and 200 rpm for 7 days. To obtain a crude laccase preparation, the fungus was grown by solid-phase fermentation on sunflower-seed shells. Weighed amounts of shells (5 g) were placed into 200 ml flasks, wetted with 20 ml of tap water, sterilized at 1 atm for 30 min, inoc-

At present, neither isolation nor characterization of Pleurotus ostreatus yellow laccase have been reported.

1 Courtesy

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of Dr. V.E. Nikitina.

YELLOW LACCASE FROM THE FUNGUS PLEUROTUS OSTREATUS D1

ulated with 5 ml of homogenized mycelium, and incubated for 10–12 days. Assay of laccase activity. Laccase activity was determined spectrophotometrically by syringaldazine oxidation [16]. The specific activity was expressed in µmol/min/mg protein. Whenever the protein concentration could not be determined because of dark brown staining of the preparation with water-soluble lignin, the activity was expressed in arbitrary units, µmol/min/ml enzyme preparation. Enzyme purification included the following steps: (1) Isolation of the crude extracellular enzyme preparation. Fermented shells (100 g) were washed with three 200 ml portions of distilled water. The water extracts were combined, and the sediment was removed by centrifugation at 4000 rpm for 30 min. Approximately 600 ml of the crude enzyme solution was obtained from 20 flasks. (2) Anion-exchange chromatography in a 2.6 × 40 cm DEAE-Servacel column (Chemapol, Czech Republic) filled with a 50 mM potassium–sodium phosphate buffer at pH 6.0. The enzyme was eluted with a linear gradient of NaCl concentration (0–0.5 M) in the same buffer. (3) Precipitation with ammonium sulfate to 80% saturation. The preparation was allowed to stand for one hour, and the precipitate was collected by centrifugation at 4000 rpm for 40 min. (4) Gel filtration in a 1.8 × 100 cm Sephadex G-100 column (Aldrich, USA) filled with 50 mM pH-6.0 potassium–sodium phosphate–buffered saline containing 0.15 M NaCl. Protein concentration was determined by the Bradford method [17]. The purity of laccase preparations was tested by vertical disk electrophoresis (SDS-PAGE, 12% polyacrylamide) [18], followed by staining with Coomassie R-250 (Fluka, Switzerland) [17]. The molecular weight of laccase was determined with reference to standard proteins: L-lactalbumin (14.2 kDa), trypsin inhibitor (20.1 kDa), trypsinogen (24.0 kDa), carboanhydrase (29.0 kDa), glyceraldehyde-3-phosphate dehydrogenase (36.0 kDa), ovalbumin (45.0 kDa), and bovine serum albumin (66.0 kDa). Molecular features of the enzyme. The amino acid composition of the enzyme was determined with a 339 automated amino acid analyzer (Microtechna, Czech Republic) after hydrolysis of 0.3 mg of the enzyme with 6 N HCl for 24 h. Specific staining of the polyacrylamide gel for protein-bound copper was performed in the following solution: alizarin blue S in glacial acetic acid plus nine volumes of 70% acetic acid. The staining was carried out for 30 min, and the excess dye was removed by washing in 70% acetic acid [19]. Absorption spectra of laccase were recorded on a Specord M40 spectrometer (Karl Zeiss, Germany) in APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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the range 230–785 nm in 50 mM pH-6.0 potassium– sodium phosphate buffer at 22°ë. The protein concentration was 925 µg/ml. Catalytic properties of the enzyme. The effect of pH on the rates of enzymatic reactions was studied in the following systems: 50 mM pH-2.0 KCl–HCl, 25 mM citric acid in 50 mM pH-2.6–7.6 Na2HPO4, 50 mM pH-8.0 sodium– phosphate buffer, and 1.5 M pH-8.8 Tris–HCl [17]. The substrate specificity of laccase was determined spectrophotometrically at optimum pH values and 25°ë. The following substrates were tested: syringaldazine (Sigma, USA) [16], 2,2'-azine-bis-(3-ethylbenzothiazoline-6-sulfonate (ABTS) (Fluka, Switzerland) [20], pyrocatechol, hydroquinone (Sigma) [21], tyrosine [22], and 2,6-dimethoxyphenol (Sigma) [23]. The Vmax and Km values were calculated with the ENZFITER program (Biosoft, Cambridge). The effect of inhibitors on the enzyme activity was studied in a mixture containing 50 mM potassium– sodium phosphate buffer (pH 6.0), 1.1–1.7 µg enzyme, a substrate (syringaldazine), and an inhibitor. The reaction was performed at 25°ë. The following inhibitors were tested: 0.1–100 mM EDTA, 0.001–0.1 mM β-mercaptoethanol, and 0.1–4% SDS (Fluka, Switzerland). Oxidation of polycyclic aromatic hydrocarbons was carried out in the following mixture (5 ml): 50 mM potassium–sodium phosphate buffer; 20 µM anthracene (Fluka, Switzerland), 1% v/v; acetonitrile; 2 mM sodium bis-2-(ethylhexyl) sulfosuccinate (aerosol-OT (AOT); Fluka, Switzerland); and 1.3 µg enzyme. In the experiment with a mediator, the mixture was supplemented with 0.5 mM ABTS. Control experiments were performed with laccase inactivated by boiling for 10 min. The reaction mixtures were incubated at 29°ë for 24 h and acidified to pH 2.0. The remaining anthracene and reaction products were extracted with two 1 ml portions of chloroform, and the combined extracts were evaporated to dryness. The pellet obtained was redissolved in 100 µl of acetonitrile. Anthracene oxidation and reaction products were analyzed by HPLC on a Spectra Series P200 chromatograph equipped with a Spectra Series UV 100 detector in a Spherisorb S5 PAH column (Thermo Separation Products, USA). A 5 µl sample was loaded into the column, washed with 40% aqueous acetonitrile for 2 min, and eluted with a linear gradient of acetonitrile concentration in water (40–100%) at the rate 1.6 ml/min for 35 min. The results were statistically evaluated as in [24]. All inorganic chemicals were reagent or special purity grade (Reakhim, Russia). RESULTS AND DISCUSSION Time variation of laccase activity. To maximize laccase production, we optimized conditions of solidphase cultivation of P. ostreatus D1 on a natural lignincontaining substrate. We rested on the protocol formerly proposed for preparation of yellow laccase from

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Lacasse concentration, µmol/min per 1 ml 0.35

kDa 66.0

Lacasse

0.30 0.25

45.0

0.20 0.15 0.10 36.0

0.05

29.0 0

2

4

6

8

10

12

14

16

18

20 22 Days

Fig. 1. Production of laccase (µmol/min/ml) during solidphase cultivation of P. ostreatus D1.

the fungus Panus tigrinus 8/18 [11]. The natural substrate was sunflower-seed shells, most often used for growing P. ostreatus. Three activity maxima were observed during three weeks of cultivation, starting from day 4 (Fig. 1). The highest rate of enzyme production was observed on days 11–12 (0.27–0.33 µmol/min per 1 ml preparation). These data were used for obtaining a crude enzyme preparation. Twenty flasks with shells were cultivated until the maximum enzyme activity was attained. About 600 ml of the extract with a mean activity of 0.277 ± 0.01 µmol/min per 1 ml were obtained from 100 g of shells. The crude enzyme preparation was a dark brown opaque liquid. It was stored frozen without considerable loss of activity until the next steps of P. ostreatus D1 laccase isolation. Laccase purification. Ion-exchange chromatography is often the first step of laccase purification. In the purification of the corresponding enzyme from a solidphase culture of Panus tigrinus, this operation not only removes part of accompanying proteins but also frees the preparation from most of the contaminating lignin [11]. Laccase from P. ostreatus D1 was adsorbed in a DEAEServacel column and eluted as a single peak at 0.35 M NaCl. The enzyme-containing eluate was light yellow. Most of the lignin remained in the column to be eluted only at 2.0 M NaCl. The activity yield was 41.4%. Since the eluate volume was very large (300 ml), the enzyme was precipitated with ammonium sulfate before the next step of the purification. The pellet was dissolved in a minimum volume of the buffer and loaded into a Sephadex G-100 column for gel filtration. The purity of the enzyme was tested by PAGE. It necessary, gel filtration was repeated. This stage yielded an electrophoretically homogeneous laccase preparation (Fig. 2). The purification yielded 0.6 mg of P. ostreatus D1 laccase from 600 ml of the crude preparation.

24.0 20.1 14.2

Fig. 2. Electrophoresis of laccase in 12% polyacrylamide gel with SDS.

The specific activity of the pure preparation according to syringaldazine oxidation was 5.8 ± 0.04 µmol/min per 1 mg. The yield was 20.9%. Molecular properties of laccase. The molecular weight and subunit composition of the enzyme were determined by gel filtration using bovine serum albumin (BSA) as a reference (66 kDa). Gel filtration of BSA in a Sephadex G-100 column showed that the retention volume of this protein was 108.6 ml. The retention volume of laccase from the same column was 109.2 ml. Figure 3 shows superimposed elution profiles of the two proteins. Their matching (which was almost perfect) suggests that our laccase is a monomer with an Mr of ~66 kDa. More precise determination of the molecular weight of laccase was performed by denaturing PAGE (with proteins of Mr 14.2, 20.1, 24.0, 29.0, 36.0, 45.0, and 66.0 kDa as references). The molecular weight of P. ostreatus D1 laccase proved to be approximately 64 kDa (Fig. 2). As reported by Palmieri et al., the molecular weights of laccases from other strains of the same fungus vary from 64 to 70 kDa [25–27]. The amino acid composition of the enzyme was as follows (%): aspartate (18.2), threonine (9.2), serine (7.5), glutamate (9.9), proline (4.8), glycine (10.6), alanine (5.5), valine (7.2), isoleucine (3.1), leucine (4.1), tyrosine (1.4), phenylalanine (4.1), histidine (1.4), lysine (3.8), and arginine (4.1). Cysteine, methionine, and tryptophan were not detected.

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YELLOW LACCASE FROM THE FUNGUS PLEUROTUS OSTREATUS D1

The concentrated enzyme solution (0.925 mg/ml) was yellowish-brown. The presence of copper was confirmed by its characteristic dark blue color when stained with alizarin blue S in the polyacrylamide gel according to the Gaal method [19]. However, the absorption spectrum of laccase in the range 220–785 nm lacked the maximum at 610 nm characteristic of fungal laccases and corresponding to the presence of copper atom type I [2]. This combination of molecular features (unusual color and absence of the blue maximum in the absorption spectrum) was found previously in yellow laccases of the fungi Panus tigrinus, Phlebia radiata, and Phlebia tremellosa [11, 13, 14]. The Ä280/Ä610 ratio was 106.2, which falls within the range previously determined for yellow laccases (92.6–148.0), unlike typical blue laccases (15–20) [14]. Thus, we found that Pleurotus ostreatus D1 laccase is a copper-containing monomer with a molecular weight 64 kDa, like most fungal laccases, including laccases from other strains of the same fungus [1, 2, 25–27]. However, unlike laccases described before, the enzyme was light yellow and its absorption spectrum lacked the maximum at 610 nm characteristic of typical blue laccases [1, 2]. According to these features, we classify it with so-called yellow laccases, recently found in basidiomycetes [11, 13, 14]. Catalytic properties. We determined the optimum pH values for oxidation of four substrates in the range 2.0–8.8. The maxima for syringaldazine, pyrocatechol, and ABTS and 2,6-dimethoxyphenol were, respectively, 7.0, 8.0, and 4.0 (Fig. 4). These data are in good agreement with the literature. For example, it has been shown that optimum pH values for ABTS oxidation by laccases from other Pleurotus species are within 3.0–4.0, while those for syringaldazine oxidation are within 6.0–7.0 [25–27]. The data presented in Table 1 indicate that the enzyme oxidized syringaldazine, which is a conventional test substrate for determination of laccase activity [23]. Another piece of evidence for the laccase nature of our enzyme is that it has no effect on tyrosine. The Km values for oxidation of syringaldazine, 2,6-dimethoxyphenol, and ABTS are higher than the corresponding values for yellow laccase from Panus tigrinus 8/18 (0.011 mM for 2,6-dimethoxyphenol, 0.001 mM for syringaldazine, and 0.033 mM for ABTS) [11, 13, 28]. However, the values for our enzyme were close to values for the blue laccase obtained from another

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Concentration, µmol/min per 1 ml 1.6

A280 0.9

1.4

0.8

1.2

0.7 0.6

1.0

0.5

0.8

0.4

0.6

2

1

0.3

0.4

0.2

0.2

0.1

0

0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Fraction number Fig. 3. Superimposed elution profiles of BSA and laccase from a column with Sephadex G-100: (1) laccase and (2) BSA.

% 120 100 1

80 60

3

2 4

40 20 0

2.0 2.6 3.0 3.6 4.0 4.6 5.0 5.6 6.0 6.6 7.0 7.6 8.0 8.8 pH

Fig. 4. Optimum pH values of laccase (%): (1) syringaldazine, (2) ABTS, (3) pyrocatechol, and (4) 2,6dimethoxyphenol.

Pleurotus ostreatus strain (0.015 mM for syringaldazine and 0.28 mM for ABTS) [25]. The Km value for 2,6dimethoxyphenol was relatively high, comparable only with the value obtained for blue laccase of Phlebia radiata

Table 1. Kinetic constants of oxidation Substrate Syringaldazine ABTS 2,6-Dimethoxyphenol Pyrocatechol Hydroquinone Tyrosine

Km , mM

Vmax , µmol/min per 1 mg

kcat , min–1

0.0087 ± 0.0009 0.11 ± 0.01 0.43 ± 0.02 3.65 ± 0.53 – –

5.71 ± 0.17 11.00 ± 0.29 8.37 ± 0.16 20.61 ± 1.17 – –

365.44 704.00 536.50 1319.00 – –

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Table 2. Effect of inhibitors on the activity of P. ostreatus D1 yellow laccase Inhibitor concentration, mM

Inhibitor Control (no inhibitor) EDTA

– 0.1 1.0 10.0 100.0 3.5 35.0 70.0 140.0 0.001 0.005 0.01 0.05 0.1

SDS

β-Mercaptoethanol

Activity, % 100.0 99.0 ± 1.0 88.0 ± 4.7 93.0 ± 2.7 40.0 ± 2.0 100.0 ± 0.0 100.0 ± 0.0 20.0 ± 6.6 0.0 36.0 ± 6.6 10.0 ± 1.0 10.0 ± 1.0 4.5 ± 0.5 0.0

(0.25 mM) [29]. Unlike other fungal laccases, P. ostreatus D1 laccase did not oxidize hydroquinone [21]. We studied the effect of inhibitors on laccase activity. Another verification of the laccase nature of the enzyme is the inhibition of its activity by 140 mM SDS. It is known that SDS activates tyrosinase but either does not affect laccase at all or slightly reduces it [22]. We found that our enzyme was inhibited completely by 140 mM SDS, a result which also confirms the enzymes' laccase-like nature. The enzyme was inhibited by 0.1 mM β-mercaptoethanol by 100%. EDTA, a weak inhibitor of fungal laccases [30], reduced the activity of the enzyme by 60% at a concentration of 100 mM (Table 2). Although the patterns of inhibition were similar (the stronger inhibitor completely inactivated the enzyme, while the weaker one suppressed the A280 1.4 1.2 1.0 0.8 0.6

1

0.4 0.2 2 0 240 248 256 264 272 280 288 296 310 330 nm Fig. 5. Absorption spectra of the reaction mixture extracts after oxidation of anthracene with P. ostreatus D1 yellow laccase: (1) control and (2) laccase.

activity only by 60%), laccase from B D1, like yellow laccase from another white rot agent, Panus tigrinus [11], was more sensitive to inhibitors. For example, β-mercaptoethanol completely suppressed the activity of P. ostreatus D1 laccase at a concentration lower than that for known blue laccases by an order of magnitude. Thus, yellow laccase from P. ostreatus D1 is similar to the corresponding enzyme from Panus tigrinus in terms of catalytic properties as well [11, 13, 28]. Oxidation of polycyclic aromatic hydrocarbons by laccase. It is believed that the most important consequence of the appearance of yellow laccase species is their ability to oxidize nonphenolic lignin analogs [12]. Typical blue laccases do not oxidize such compounds without corresponding mediators [6, 31, 32]. We suggested that the yellow laccase isolated by us from P. ostreatus D1 also could oxidize nonphenolic species without mediators. To verify this suggestion, we studied the effect of this enzyme on a PAH with three condensed rings, anthracene, in both the presence and the absence of an exogenous mediator, ABTS. We found that P. ostreatus D1 yellow laccase oxidized anthracene by 95 ± 1.0% in the absence of a mediator. Addition of ABTS showed no notable effect on the efficiency of oxidation, which was 96 ± 0.5%. A peak corresponding to the reaction product was revealed by HPLC in a Spherisorb S5 PAH column. As reported previously, laccases generally oxidize anthracene to anthraquinone [9, 10, 33]. We prepared anthraquinone by chemical oxidation of anthracene with 35% nitric acid. Anthracene and anthraquinone were extracted with chloroform and analyzed in the same way as the enzymatic reaction mixture. The matching of the retention time of the product of enzymatic anthracene oxidation with the retention time of the synthetic anthraquinone suggested that yellow laccase also oxidized anthracene to anthraquinone. This suggestion was confirmed by an additional maximum in the absorption spectra of chloroform extracts of the reaction mixtures (Fig. 5). These results are comparable with those obtained for laccases from the fungi Trametes versicolor and Coriolopsis gallica [9, 10, 33]. Those studies demonstrated a significant increase in anthracene oxidation in the presence of ABTS, to 64–89%, whereas only traces of the oxidation product were noted without the exogenous mediator. To sum up, we isolated and characterized a new yellow laccase from the fungus P. ostreatus D1. This enzyme not only oxidized a nonphenolic aromatic substrate in the absence of a mediator but also catalyzed anthracene oxidation more efficiently than previously studied laccases. In recent years, chemical mediators of oxidation of nonphenolic substrates by laccases have been sought. The essential features required of such mediators are low cost, nontoxicity of both the mediator itself and the products of its oxidation, a high number of reversible redox cycles in the enzyme system, and the absence of colored absorbable oxidation products [34]. Yellow laccases do not need an exogenous

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mediator because, presumably, they contain an endogenous natural one. Thus, yellow lacasses meet the said requirement and are of great interest for biotechnology. Their further study is promising for understanding the mechanisms of xenobiotic degradation and application of white rot fungi, in particular, Pleurotus ostreatus, to bioremediation of PAH-polluted sites.

15. Bezalel, L., Hadar, Y., Fu, P.P., Freeman, J.P., and Cerniglia, C.E., Appl. Environ. Microbiol., 1996, vol. 62, no. 7, pp. 2554–2559.

ACKNOWLEDGMENTS

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The study was supported in part by the Mianowski Foundation for the Promotion of Science, Poland.

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18. Laemmli, U.K., Nature, 1970, vol. 227, pp. 680–685.

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