Comparison of physico-chemical characteristics of four laccases from different basidiomycetes

Biochimie 86 (2004) 693–703 www.elsevier.com/locate/biochi

Comparison of physico-chemical characteristics of four laccases from different basidiomycetes S.V. Shleev a,*, O.V. Morozova a, O.V. Nikitina a, E.S. Gorshina b, T.V. Rusinova b, V.A. Serezhenkov c, D.S. Burbaev c, I.G. Gazaryan d, A.I. Yaropolov a a

Laboratory of Chemical Enzymology, A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospekt 33, 119071 Moscow, Russia b FGUP “GosNIISintezbelok” B. Kommunisticheskaya 27, 109004 Moscow, Russia c N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Kosigina 4, 119977 Moscow, Russia d Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Lenin Hills, 119899, GSP-3 Moscow, Russia Received 7 March 2004; accepted 31 August 2004 Available online 21 September 2004

Abstract New strains of basidiomycetes producing extracellular laccases (Trametes ochracea 92-78, and Trametes hirsuta 56) have been found by screening of isolates of Trametes fungi. The laccases from T. hirsuta 56 and T. ochracea 92-78 as well as two laccases from previously found and described strains of basidiomycetes, namely Cerrena maxima and Coriolopsis fulvocinerea, were purified to homogeneity. The standard redox potentials of type 1 copper in the enzymes were determined and found to be 780, 790, 750, and 780 mV, respectively. The spectral and biochemical studies showed that the enzymes had no significant differences between the structures of their active sites (T1, T2, and T3). In spite of this fact, the basic biochemical properties as well as the redox potentials of the T1 sites of the enzymes were found to be different. The molecular weights of the laccases range from 64 to 70 kDa, and their pI values range from 3.5 to 4.7. The pH-optima are in the range 3.5–5.2. The temperature optimum for activity is about 50 °C. The thermal stabilities of the enzymes were studied. The catalytic and Michaelis constants for catechol, guaiacol, hydroquinone, sinapinic acid, and K4Fe(CN)6 were determined. Based on these results as well as results obtained by comparing with published properties of several laccases, it could be concluded that T. hirsuta and Cerrena maxima laccases have some superior characteristics such as high stability, high activity, and low carbohydrate content, making them attractive objects for further investigations as well as for application in different areas of biotechnology. © 2004 Elsevier SAS. All rights reserved. Keywords: Basidiomycete; Laccase; Redox potential; T1 site; Active site

1. Introduction Laccase (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) catalyses the oxidation of ortho- and paradiphenols, aminophenols, polyphenols, polyamines, lignins and aryl diamine as well as some inorganic ions coupled to the reduction of molecular dioxygen to water [1,2]. The enzyme exhibits a broad substrate specificity, which can be enhanced by addition of redox mediators. The efficiency of these mediators has been demonstrated in work presented in a number of publications [1,3]. Due to the broad variety of reactions catalysed by laccases, this enzyme holds great promise for many potential applications. * Corresponding author. Tel.: +7-95-954-44-77; fax: +7-95-954-40-07. E-mail address: [email protected] (S.V. Shleev). 0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.08.005

Laccases are classified into two groups in accordance with their source, i.e. plant and fungal. However, diphenol oxidases (laccases) have also been identified in bacteria [4,5] and insects [6]. The enzyme is a copper protein and contains four metal ions classified into three types, referred to as T1, T2, and T3 [1,2]. The T1 copper is responsible for the blue color of the enzyme and has a characteristic absorbance around 610 nm. The T2 copper cannot be detected spectrophotometrically; however, it generates a characteristic EPR signal [2,7,8]. The bi-nuclear T3 copper is diamagnetic. It displays a spectral absorbance shoulder in the region of 330 nm and also displays a characteristic fluorescence spectrum [9]. The key characteristic of laccase is the standard redox potential of the T1 site. The values of the redox potential of

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this site have been determined using potentiometric titrations with redox mediators for a great number of different laccases and they vary between 430 and 790 mV vs. NHE [10–15]. It has been shown for some laccases that T1 is the primary center at which electrons are accepted from reducing substrates [1,2,11]. Moreover, the catalytic efficiency (kcat/KM) for some reducing substrates depends on the redox potential of the T1 copper [11,16]. This is the reason why laccases with a high redox potential T1 site are of special interest in biotechnology, e.g., for different bleaching [17] and bioremediation processes [18]. The primary structures of laccase isolated from several organisms have been determined (for example, see the GenBank website: http://www.ncbi.nlm.nih.gov). It was shown that two multiple forms of laccase from one origin (for instance, basidiomycete T. hirsuta) with different biochemical properties had very similar primary structures (Ref. [19] and also GenBank). On the other hand, it has also been shown that while being similar biochemically, the isoenzymes of laccase from the same origin (T. hirsuta) can have different primary structures [9]. Currently, it is not absolutely clear whether basidiomycetes have several genes encoding laccases, or whether the diversity of laccase isoenzymes is due to postranslational modification and/or proteolysis during cultivation and purification. Laccase was first described by Yoshida as early as 1883 [20]. This makes it one of the first enzymes ever described. However, during the last few years of work on laccase applications has been extended. Reported applications include development of oxygen cathodes in biofuel cells [21], biosensors [22], green biodegradation of xenobiotics including pulp bleaching [3,17], and labelling in immunoassays [23]. Moreover, new interesting prospective directions for the application of the enzyme in biotechnology have been found: bioremediation [18], green organic synthesis [24], and even design of laccase fungicidal and bactericidal preparations [25]. These applications stimulate new waves of fundamental research concerning this enzyme. Activities of current interest include screening of laccase sources, studying new laccases [5,9,15,16,26,27], investigating the structure of the enzyme [28–31], elucidating the mechanism of the intraprotein electron transfer as well as the mechanism of oxygen reduction to water [32,33], investigating the electrochemical properties of laccases [34], and much more. There is no doubt that biochemical and physico-chemical properties of laccase (activity, stability, carbohydrate content, pH-optimum, etc.) comprise very important initial information for both fundamental studies and for application of laccases in biotechnology. The great differences in these properties even for different laccases from a single species motivate the importance of broad biochemical characterization of any enzyme which will be the focus of further work. Thus, the goals of the present paper were (i) to screen new strains of basidiomycetes for laccase secretion; (ii) to study and thoroughly compare biochemical, spectral, kinetic, and electrochemical properties of four laccases from strains se-

lected in the first step of our study; and (iii) to reveal the possible nature of the differences in their key characteristics in order to find new laccases as attractive objects for further investigations as well as for applications in different areas of biotechnology.

2. Materials and methods 2.1. Chemicals DEAE Toyopearl 650M and Toyopearl HW-55 were from Tosoh (Tokyo, Japan); ampholine was from Pharmacia LKB Biotechnology (Bromma, Sweden); Coomassie G-250 and R-250, and markers for electrophoresis and chromatography were from Serva (Heidelberg, Germany); 2-mercaptoethanol was from Ferak (Berlin, Germany); Tris and glycine were from ICN (Costa Mesa, CA, USA); Servacel DEAE 52, acrylamide, N,N-methylenebisacrylamide and ammonium persulfate were from Reanal (Budapest, Hungary); catechol, guaiacol, and sinapinic acid were from Sigma (St. Louis, MO, USA); hydroquinone, 2,2′-biquinoline, NaCl, Na2HPO4, KH2PO4, K3Fe(CN)6 and K4Fe(CN)6 were from Merck (Darmstadt, Germany); and glycerol, acetic acid, methanol, ethanol, MoO2, HCl, KCl, H3PO4, (NH4)2SO4 and NaOH were of the highest purity available from domestic sources. Buffers were prepared using water (18 MX) purified with a Milli-Q system (Millipore, Milford, CT, USA). The mediators (K4[Mo(CN)8] and K3[Mo(CN)8]) were synthesised and purified according to previously published methods [35]. 2.2. Material Basidiomycetes Cerrena maxima (Fr.) Ryvarden and Coriolopsis fulvocinerea (Murrill) Zmitr. were obtained from the Komarov Botanical Institute, Russian Academy of Sciences (St. Petersburg, Russia). Partially purified preparations of extracellular laccases from these basidiomycetes have been kindly provided by Drs. O.V. Koroleva and V.P. Gavrilova in the framework of the joint research performed with the support of INCO Copernicus Grant No. ICA2-CT2000-10050. Basidiomycetes Trametes hirsuta (Wulfen) Pilát and Trametes ochracea (Pers.) Gib. & Ryvarden were obtained from the laboratory collection of the State Research Institute of Protein Biosynthesis (Moscow, Russia). A list of strains which have been screened in the present study appears in Table 1 (top line). 2.3. Microbiological procedures The Cerrena maxima and Coriolopsis fulvocinerea basidiomycetes were grown as described in [8]. Basidiomycetes T. hirsuta and T. ochracea were grown by submerged cultivation using the following nutrient media

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Table 1 Dynamics of oxidase activity and biomass increase for different strains of Trametes fungi (T. hirsuta (Th) and T. ochracea (To)) Time 7 days 9 days 12 days 14 days

Parameters Oxidase activity (U/ml) Biomass (mg/ml) Oxidase activity (U/ml) Biomass (mg/ml) Oxidase activity (U/ml) Biomass (mg/ml) Oxidase activity (U/ml) Biomass (mg/ml)

Th NRB2 1.5 4.1 2.5 5.3 1.6 6.3 0.6 7.0

Th 86–43 0.3 8.2 0.8 8.5 a

7.6 0.5 6.7

Th 56 5.8 6.1 4.6 7.8 9,1 10.4 6.4 12.8

To 92-78 0.3 3.7 2.0 4.9 0.2 4.9 0.6 4.9

To 92-83 0.6 5.0 0.9 6.6 2,4 6.5 0.7 5.8

To 117 0.3 2,9 0.4 5.9 0.5 6.4 0.6 7.3

To M 103 0.5 5.3 0.9 8.3 0.8 8.1 0.8 7.79

To M 106 0.3 2.7 0.4 5.0 0.5 7.9 a

9.2

To 239 0.2 2.5 0.8 3.8 2.2 4.7 1.7 5.1

Notes: Strains selected for further study are indicated in bold. a indicates that only traces of activity were observed. Oxidase activity is presented in units per ml of culture fluid (U/ml). One unit of activity is defined as the amount of laccase oxidizing 1 µmol of catechol per minute under standard condition. The S.E.M. has been calculated from three independent experiments and did not exceed 10%.

(g/l): glucose, 10; peptone, 3; KH2PO4, 0.6; K2HPO4, 0.4; MgSO4, 0.5; ZnSO4, 0.001; FeSO4, 0.0005; MnSO4, 0.05; and CuSO4, 0.25. The cultivation was carried out in a fermenter “Marubishi” (Japan) with a volume of 30 l under the following conditions: pressure, 0.4 bar; temperature, 27 °C; aeration, 1.0 ppm; and agitation speed, 250 rpm. During cultivation, the pH of the solutions, as well as some biochemical parameters such as oxidase activity and biomass, were controlled. The nutrient media were inoculated with agar plugs (8 mm in diameter) of mycelium maintained on 2% malt extract agar. The oxidase activity test was performed on the agar media by Bavendamm’s reaction in accordance with a method presented in [36]. 2.4. Isolation and purification procedure The partly purified preparations of Cerrena maxima and Coriolopsis fulvocinerea laccases were received in the forms described in [8,26]. After cultivation, the culture fluids of T. hirsuta 56 and T. ochracea 92–83 basidiomycetes containing extracellular laccases were filtered to remove the mycelium. The used purification scheme included: precipitation of the enzymes from the cultural medium with saturated ammonium sulfate; FPLC ion-exchange chromatography on a Servacel DEAE 52; rechromatography on a DEAE-Toyopearl 650M performed on standard equipment purchased from Pharmacia LKB Biotechnology; isoelectric focusing in the pH range 3–5, then gel-filtration on Toyopearl HW-55 from Tohos. The final purification step for all laccases including partially purified enzymes from Cerrena maxima and Coriolopsis fulvocinerea was performed by means of HPLC on a TSK DEAE-2SW column from Pharmacia LKB Biotechnology using a “Stayer” system from Aquilon Ltd. (Moscow, Russia, web page: http://www.aquilab.ru/default_en.htm). The enzyme preparations were homogeneous as judged from SDS-PAGE [37]. The specific activity of each enzyme preparations was expressed in µM/min per mg of protein with catechol as substrate. Homogeneous preparations of laccase (ca. 10 mg/ml) were stored in 0.1 M phosphate buffer, pH 6.5, at –18 °C.

2.5. Biochemical characterization Preparative isoelectric focusing was performed in accordance with [38] on a 440 ml Pharmacia LKB Biotechnology column 8100 using a natural pH gradient 3–5 stabilized by a gradient of glycerol (10–60%) The applied power was 30 W at the beginning and 3 W at the end of the experiment. Fractions were collected at a flow rate of 4 ml/min. Determination of carbohydrate content involved the derivatization of the monosaccharides released after acid hydrolysis (incubation in 4 M CF3COOH for 3 h at 110 °C), and their subsequent analysis using a Biotronic LC 2000 analyzer (Maintal, Germany). The molecular mass of laccase was determined by gelfiltration on TSK G 2000 SWXL column from Tosoh, calibrated with a set of standard markers (15–92 kDa) from Serva. The assay of laccase-bound copper was performed with 2,2′-biquinoline [39]. The protein was determined in accordance with methodology presented in [40]. 2.6. Spectral investigation Absorbance spectra were recorded with a Hitachi557 spectrophotometer (Tokyo, Japan) with different scan rates and slit widths. Fluorescent spectra were recorded with a Shimadzu spectrofluorophotometer RF-5301 (Tokyo, Japan) with a scan rate of 120 nm/min and a bandwidth of 5 nm. EPR-spectra were registered with a Bruker EPRspectrometer ESC-106 (Ettlingen, Germany) in the X-band. CD-spectra were recorded using a Jasco spectropolarimeter J-715 (Tokyo, Japan) with a scan rate of 60 mm/min and a bandwidth of 5 nm. The parameters of the secondary structures were calculated using an in house written computer program “Belok” (Moscow, Russia). The program was designed by Dr. Vladimir V. Shubin (A.N. Bach Institute of Biochemistry, e-mail: [email protected]). 2.7. Electrochemical studies To determine the redox potentials of laccase T1 centers, the method of protein redox titration [41] was employed with potassium octocyanomolibdate (IV and V) mediators. The

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direct potential measurement on platinum electrodes was performed while simultaneously recording the absorbance spectra using a bioelectrochemical cell [42]. The potential was measured with an ionomer I-130.1 from APK Energoservis (St. Petersburg, Russia). Platinum wire with 1 mm diameter was used as a working electrode, and an Ag/AgCl electrode EVL-1M3 from BioEko (Moscow, Russia) with E =201 ±2 mV was used as a reference electrode. Laccases were placed anaerobically into a cell containing a high concentration of K3Mo(CN)8 in 0.1 M phosphate buffer, pH 6.0. Redox potentials were registered with platinum electrodes before and after the addition of enzymes. Further titration was performed with the reduced form of the mediator (K4Mo(CN)8) in 0.1 M phosphate buffer, pH 6.0. The absorbance spectra within the range from 550 to 800 nm were recorded after each addition after equilibrium was established. The calculations accounted for enzyme dilution, which never exceeded 10%. Anaerobic conditions were provided by argon purified through a system of solutions containing VCl2 [43].

3. Results As was mentioned in Section 1, one of the goals of present work was to study and thoroughly compare the properties of laccases from various basidiomycetes. As a start, two enzymes from different fungi, namely Cerrena maxima and Coriolopsis fulvocinerea, were kindly provided by our Russian colleagues. To broaden the scope of the investigations, the first task was to select additional strains for laccase production from our collection of Trametes fungi. Nine strains of Trametes basidiomycetes have been screened in order to find the most efficient producers of laccases (Table 1, upper line). The ability of the strains to produce extracellular oxidases was studied by Bavendamm’s reaction. All strains displayed positive Bavendamm’s reactions but significant variability in the reaction intensity among examined fungi was found (Fig. 1). The dynamics of oxidase activity during submerged cultivation have been investigated and significant variability of this parameter for examined strains was found (Table 1). However, the difference in biomass increase during cultivation was not so substantial as to correlate with corresponding level of oxidase activity (Table 1). The most productive Trametes strains (those with the highest oxidase activity), namely T. hirsuta 56 and T. ochracea 92-78, were selected for laccase purification. In particu-

Fig. 1. Color zone diameters of different strains of Trametes basidiomycetes (Bavendamm’s reaction). Two strains labeled in bold have been selected for further study. Black—T. hirsuta strains, gray—T. ochracea strains.

lar, T. hirsuta 56 possessed sufficiently higher oxidase activity compared to all other fungi screened (Table 1). Another basidiomycete in the group of T. ochracea strains was also selected for a high level of oxidase activity as well as for a relatively high growth rate. After selection of favorable properties, homogeneous laccases from basidiomycetes Cerrena maxima and Coriolopsis fulvocinerea as well as T. ochracea and T. hirsuta were prepared. The homogeneity of the preparations was confirmed without a doubt by SDS-PAGE and ion-exchange HPLC (data not shown). One clear line on the gel electrophoresis could be seen as well as one sharp peak in the HPLC chromatogram was received. The key biochemical properties of the selected laccases, e.g. molecular mass, pH-optimum, thermal stability, copper content, pI, and specific activity, are presented in Table 2. It was found that the preparations of each enzyme contained one form of the monomeric protein with a molecular weight of 64–70 kDa determined by SDS-PAGE and HPLC (data not shown). Laccases contain four copper ions per molecule and have a pH-optimum of 3.5–5.2 (Table 2). Like all known laccases, they are glycoproteins and the carbohydrate content

Table 2 Some biochemical properties of laccases Laccase T. ochracea Trametes hirsuta Cerrena maxima Coriolopsis fulvocinerea

MW (kD) 64 ± 2 70 ± 2 67 ± 2 65 ± 2

pI 4.7 ± 0.1 4.2 ± 0.1 3.5 ± 0.1 3.5 ± 0.1

pH-optimum 3.7–4.9 3.5–4.5 3.5–4.5 3.9–5.2

Carbohydrate content (%) 10 ± 1 12 ± 1 13 ± 1 32 ± 1

Copper content 4 4 4 4

Inactivation half-life at 50 °C 56 65 52 64

Note: If uncertainty values are not indicated, the S.E.M. has been calculated from results of three independent experiments, and was found not to exceed 10%.

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Table 3 Kinetic parameters of oxidation reactions Substrate

T. hirsuta KM (µM) 142

T. ochracea kcat (s–1) KM (µM) 80 110

Cerrena maxima kcat (s–1) KM (µM) 320 120

Coriolopsis fulvocinerea kcat (s–1) KM (µM) 90 85

Catechol

kcat (s–1) 390

Guaiacol

430

63

90

90

300

160

95

70

Hydroquinone

450

61

110

74

290

100

110

68

Sinapinic acid

580

24

170

11

330

24

140

21

K4[Fe(CN)6]

400

180

150

96

450

115

130

170

Note: The S.E.M. was calculated from five independent experiments and did not exceed 10%.

varies from 10% to 32%. It was found that the carbohydrate moieties of the enzymes consisted of mannose and N-acetylglucosamine (for T. hirsuta, Cerrena maxima, and Coriolopsis fulvocinerea laccases), or mannose, N-acetylglucosamine, and galactose (for T. ochracea laccase). The laccases are all highly stable enzymes retaining 50% of their initial activity after incubation at 50 °C for about 60 h. Afterwards kinetic investigations of the laccases were performed and the catalytic constants (kcat and KM) measured for some organic and inorganic compounds are presented in Table 3. As can be seen from Table 3 T. hirsuta laccase possessed the largest catalytic constants with respect to all organic substrates, but Cerrena maxima laccase showed the highest oxidation rate for the inorganic substance K4[Fe(CN)6]. Sinapinic acid is the best substrate for T. hirsuta, T. ochracea, and Coriolopsis fulvocinerea laccase, whereas K4[Fe(CN)6] is the best substrate for the enzyme from Cerrena maxima basidiomycete. In order to investigate the structure of the active sites of the enzymes, spectral studies have been performed. Absorbance spectra presented in Fig. 2 are typical for “blue” laccases and contain signals correlated with T1 and T3 cop-

per centers (about 610 and 330 nm, respectively). Fluorescence spectra, characteristic of the T3 site of the enzymes, are also not so different for the examined laccases (Fig. 3). The EPR spectra of the laccases under study are presented in Fig. 4. They are also typical for “blue” laccases and exhibit signals related to both copper centers (T1 and T2). Table 4 summarizes all spectral parameters measured for the T1, T2, and T3 sites of laccases. Secondary structures of laccases were investigated by circular dichroism (CD) spectroscopy. CD spectra are presented in Fig. 5. In the 190- to 250-nm wavelength range, the spectra were mathematically analyzed using the in-house developed computer program “Belok”. It was shown that the laccases contain about 10% a-helix, 30% b-sheet, 20% turns, along with 40% random coil. In the visible and near-infrared wavelength range, the CD spectra were similar to those of Trametes versicolor, Neurospora crassa, and Rhus vernicifera laccases [44–46]. The potential of the T1 copper was measured by direct redox titration using mediators. As an example, the redox titration of T. ochracea laccase is shown in Fig. 6. The reduction of the T1 copper was accompanied by the disap-

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Fig. 2. Absorbance spectra of extracellular fungal laccases from (a) T. ochracea, (b) T. hirsuta, (c) Coriolopsis fulvocinerea and (d) Cerrena maxima. The concentrations of laccases were about 1 mg/ml in 0.1 M phosphate buffer, PH 6.0.

Fig. 4. EPR-spectra of (a) T. ochracea, (b) T. hirsuta, (c) Coriolopsis fulvocinerea and (d) Cerrena maxima laccases in 0.1 M phosphate buffer PH 6.0. X-band, microwave power 20 mW. Modulation amplitude 0.5 mT; amplification 1 × 104 (a), 5 × 104 (b), 1 × 104 (c), 1 × 105 (d); four accumulations for (a), (b), (c), and eight accumulations for (d). Temperature 77°K. The concentrations of laccases were about 1 mg/ml.

T. ochracea laccase to 57 mV for Coriolopsis fulvocinerea laccase (Table 5).

4. Discussion

Fig. 3. Fluorescence spectra of laccases from (a) T. ochracea, (b) T. hirsuta, (c) Coriolopsis fulvocinerea and (d) Cerrena maxima in 0.1 M phosphate buffer, PH 6.0. (I) Emission spectra, excitation wavelength 330 nm; (II) excitation spectra, emission wavelength 420 nm. The concentrations of laccases were about 1 mg/ml.

pearance of the blue absorbance band at 610 nm. The redox potentials of the laccases were in the range from 750 to 800 mV vs. NHE. The slope of the Nernst dependence (Fig. 6, inset) during the course of laccases redox titrations in log[(A/(A0 – A)]/E coordinates varies from 32 mV for

The well known Bavendamm reaction shows, in general, the ability of basidiomycetes to produce extracellular oxidases such as laccase and tyrosinase [36]. During the oxidation of gallic acid, zones with black color are formed. The diameters of the color zones reflect the amount of oxidases which are produced by fungi during their cultivation. However, in our studies there was no direct positive correlation between Bavendamm’s reaction and the dynamics of oxidase activity during submerged cultivation of basidiomycetes. In particular, the most productive strains (those with the highest oxidase activities) showed the lowest levels of Bavendamm’s reaction. Our further investigations showed that the media as well as conditions of cultivation strongly affect the oxidase activity of the strains. The differences are probably connected with the totally different media and conditions of growth (submerged vs. agar plate medium cultivation procedures). Further investigations in this direction are necessary. The other properties of these laccases, such as molecular weight, activity, and carbohydrate composition, differ to a greater or lesser extent from the properties previously reported for laccases from the same fungi (see Table 1 and also Refs. [8,9,14,19,26,47,48]). The importance of carrying out the survey work reported here has been mentioned in Section 1. As an example, we would like to stress that different forms

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Table 4 Spectral characteristics of T1, T2, and T3 sites of laccases (UV-VIS, EPR, and fluorescence analyses) Laccase T. ochracea T. hirsuta Cerrena maxima Coriolopsis fulvocinerea

T1 site AbM (nm) 608 607 607 607

g兩 兩 2.20 2.19 2.18 2.18

A 兩 兩 (10–4 cm–1) 94 95 94 94

g兩 兩 2.24 2.26 2.24 2.21–2.23 (2.23)a

T2 site A 兩 兩 (10–4 cm–1) 194 186 193 190–210 (195)a

EmM (nm) 422 418 425 418

T3 site ExM (nm) 315 344 322 303

Notes: AbM, absorption maximum; EmM, emission maximum; ExM, excitation maximum. a indicates the most probable value for Coroilopsis fulvocinerea laccase.

of T. hirsuta laccase have differences in MW in the range from 55 to 70 kDa and in activity from 10 to 400 Units [8,9,14,19]. Knowledge about the actual properties of the particular purified enzymes is required before using them in further investigations. We can also emphasize an interesting observation that some biochemical properties of T. hirsuta laccase (for example, MW), which are described in this work, are close to the inducible form of the enzymes (in spite of the absence of the inducers during the cultivation procedure), while other characteristics (pI, pH-optimum, carbohydrate composition) have similarity with the constitutive form of the laccases from T. hirsuta [8,9]. Unfortunately, it is not possible to find these dependences for other laccases due to lack of information about the biochemical properties of their constitutive and inducible forms. However, in general (without consideration of the cultivation) the biochemical characteristics of T. ochracea, Cerrena maxima, and Coriolopsis fulvocinerea laccases are very different from those previously described [8,49]. All spectral values of laccases from T. hirsuta, T. ochracea, Cerrena maxima, and Coriolopsis fulvocinerea are in a good agreement with the published values for enzymes from

different sources [2,7–9,13,46] except for one EPR parameter (namely A 兩 兩 =210.10–4 Cm–1 for the T2 center) of Coriolopsis fulvocinerea laccase (Table 4). The explanation of this unusual parameter is the absence of a low-field shoulder for Coriolopsis fulvocinerea laccase, which made it difficult to calculate the correct value for the A 兩 兩 parameter. As for the secondary structure, it is typical for “blue” laccases [13,44,45] and does not differ from the studied laccases. Furthermore, the enzymes have similar b-barrel type architecture, related to the small blue copper proteins such as azurin or plastocyanin [30]. Thus, significant differences which could be correlated with possible differences between the structures of the active sites of the enzymes have not been found through the methodology employed in the present study. In a very recent publication a new classification scheme for laccases has been proposed [50]. From an electrochemical point of view as well as from analysis of the primary structures of the enzymes, all laccases can be divided into three groups in view of the potential of the T1 site: low,

Fig. 5. Circular dichroism spectra of laccases from (a) T. ochracea, (b) T. hirsuta, (c) Coriolopsis fulvocinerea and (d) Cerrena maxima in 0.1 M phosphate buffer, Ph 6.0. All samples have concentration 0.1 mg/ml except T. hirsuta laccase (0.2 mg/ml).

Fig. 6. Determination of the T1 copper redox potential of T. ochracea laccase in 0.1 M phosphate buffer, PH 6.0. N, native enzyme spectrum; 1–4, mixing the enzyme with K3Mo(CN)8 and K4Mo(CN)8 in various ratios ((1) 100:1, (2) 50:1, (3) 20:1, (4) 10:1).

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Table 5 Some electrochemical characteristics of the T1 site of laccases Laccase T. ochracea T. hirsuta Cerrena maxima Coriolopsis fulvocinerea

E0′, T1 (mV) 790 ± 10 780 ± 10 750 ± 5 780 ± 10

b (mV) 32 ± 2 49 ± 2 56 ± 1 57 ± 2

Ratio of concentrations (mediator/laccase) 50:1 100:1 120:1 100:1

Note: All potentials are given vs. NHE.

middle and high potential laccases. The low potential group includes enzymes from trees, e.g. R. vernicifera laccase with a T1 site potential of about 430 mV vs. NHE [10]. The middle group includes laccases from basidiomycetes like Myceliophthora thermophila [11], basidiomycete C30 [15], Rhizoctonia solany [11], and Coprinus cinereus [13]. These enzymes have T1 site potentials ranging from 470 to 710 mV vs. NHE. All the high potential laccases (e.g. those from T. hirsuta, T. versicolor, and Trametes villosa) have T1 site potentials of about 780 mV vs. NHE [10,11]. It was found that the redox potentials of examined laccases were in the range from 750 to 800 mV vs. NHE; thus, the isolated enzymes were seen to belong to a group of middle and high redox potential laccases (Table 5). It was previously suggested that the value of the redox potentials of the copper-containing oxidases depends on the ligands of the T1 copper and on the amino acids which form the T1 pocket [19,51]. Evidently, as can be seen from Table 6, all high redox potential laccases have absolutely similar amino acid sequences in the section of polypeptide chain containing the main ligands to the T1 copper and forming the T1 pocket. However, site-directed mutagenesis studies have shown only minor correlation between T1 Cu-ligating residues and E0 of the T1 site [12,27,51]. Based on the recently acquired structural data of the native high potential laccase (T. versicolor), a new mechanism was suggested by which laccase, and possibly other redox metalloenzymes, can in-

crease their redox potentials [30]. Such a mechanism assumes a reduction of electron density contribution at the metal and the ligating amino acid. In the case of the high potential laccase the distance between Cu and one of the histidines is longer compared with the distance between the same moieties in the middle potential group of the enzyme, for example Coprinus cinereus laccase [30,52]. The hydrogen bond between His-479 and Ser-113 (114 in the case of T. villosa laccase) seems to be responsible for this (Table 6, Fig. 7; see detailed explanation in [30]). It is obvious that the great differences between redox potentials for laccases from different sources are not yet fully understood, and further investigations in this direction are needed. However, our data show that the T1 site of T. hirsuta laccase has a redox potential of about 780 mV vs. NHE, which is reasonably justified by comparison to the sequences of other high potential laccases (Table 6). We can also suggest that knowledge about the redox potential of the T1 site can give an idea about the structure of the T1 site of laccases. Thus, analyzing the redox potentials of the T1 sites for the studied laccases, we can here speculate that the amino acid composition of polypeptide chain constituting the main ligands to the T1 coppers and forming the T1 pocket for T. ochracea and Coriolopsis fulvocinerea laccases are very close to that of the T. hirsuta and T. versicolor enzymes, while the Cerrena maxima laccase has a structure of the T1 site close to that of the enzyme from Marasmius querco-

Table 6 Comparison of the redox potentials, E0′, and the amino acid sequence alignment of the T1 site for oxidases from different sources Oxidase Trametes hirsuta

Sequence aliment G H S F L... H... H C H I D F H L E A G F

Trametes versicolor

G H S F L... H ... H C H I D F H L E A G F GenBank Accession number A35883 G N S F L... H ... H C H I D F H L E A G F GenBank Accession number JC5356 G H A F L... H ... H C H I D F H L E A G F GenBank Accession number AAM10738 G P A F V... H ... H C H I D F H L E A G L GenBank Accession number S68120 G H A F L... H ... H C H I E F H L M N G L GenBank Accession number AAD30964 G T N F I... H ... H C H F E R H T T E G M GenBank Accession number BAB63411 G E T F F... H ... H C H I E P H L H M G M GenBank Accession number A51027

Trametes villosa (pinsutus) Marasmius quercophilus C30 (I) Rhizoctonia solani Coprinus cinereus Rhus vernicifera Zucchini ascorbate oxidase

E0′, T1 (mV) 780a [19] 780 [10] 780 [11] 730 [15] 710 [11] 550 [13] 430 [10] 340 [12]

Notes: Underlined residues ligands to T1 copper; italicized—amino acids which might affect the electron density contribution at the metal and the ligating amino acid (in [30]); a —this paper.

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701

Fig. 7. Schematic drawing illustrating (A) the distance between significant amino acids for redox potentials of laccases, and (B) the movement of a helical segment in the enzymes (according to [30]).

philus basidiomycete (see the GenBank website: http://www.ncbi.nlm.nih.gov and also Ref. [10,15]). Comparing the properties of several laccases from different sources, also including multiple forms of laccases from the same species, it can be concluded that T. hirsuta and Cerrena maxima laccases have some superior characteristics such as high stability, high activity, and low carbohydrate content, making these laccases attractive objects for further investigations as well as applications. Moreover, we believe that detailed comparative studies of laccases from different sources can open up new opportunities for their use in different aspects of biotechnology as well as reveal the true mechanism of their function. Such studies (e.g., electrochemical investigation of the enzymes, creation of biosensors, studies of laccase mediator systems, achievement of laccase-based green organic synthesis) building on the survey results presented in this paper are being carried out in our laboratories [14,24,50]. Acknowledgements The authors thank Drs. O.V. Koroleva and V.P. Gavrilova for the preparation of Coriolopsis fulvocinerea and Cerrena maxima laccases. The authors also thank Dr. V.V. Shubin for the computer program for CD spectra calculation, and Dr. Curt T. Reimann for critical reading and helpful suggestions.

The work was supported by INCO-Copernicus Grant (contract No.ICA2-CT-2000-10050), the Russian Foundation for Basic Research (project No. 02-04-48885 and No. 03-04-48937), and by the FNTP of the Russian Ministry of Science and Technology (project No.43.073.1.1.2505).

References [1]

[2] [3]

[4]

[5]

[6]

A.I. Yaropolov, O.V. Skorobogat’ko, S.S. Vartanov, S.D. Varfolomeyev, Laccase. Properties, catalytic mechanism, and applicability, Appl. Biochem. Biotechnol. 49 (1994) 257–280. E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Multicopper oxidases and oxygenases, Chem. Rev. 96 (1996) 2563–2605. H.P. Call, I. Mucke, History, overview and applications of mediated lignolytic systems, especially laccase-mediator-systems (Lignozymprocess), J. Biotechnol. 53 (1997) 163–202. A. Givaudan, A. Effosse, D. Faure, P. Potier, M.L. Bouillant, R. Bally, Polyphenol oxidase in Azospirillum lipoferum isolated from rice rhizosphere: evidence for laccase activity in nonmotile strains of Azospirillum lipoferum, FEMS Microbiol. Lett. 108 (1993) 205–210. O. Martins Ligia, M. Soares Claudio, M. Pereira Manuela, M. Teixeira, T. Costa, H. Jones George, O. Henriques Adriano, Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat, J. Biol. Chem. 277 (2002) 18849–18859. F.M. Barrett, in: K. Binnigton, A. Retnakaram (Eds.), Physiology of the Insect Epidermis CISRO1991, pp. 194–196, East Melbourne, Australia.

702 [7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18] [19]

[20] [21]

[22]

[23]

[24]

[25]

S.V. Shleev et al. / Biochimie 86 (2004) 693–703 A.A. Leontievsky, T. Vares, P. Lankinen, J.K. Shergill, N.N. Pozdnyakova, N.M. Myasoedova et al., Blue and yellow laccases of ligninolytic fungi, FEMS Microbiol. Lett. 156 (1997) 9–14. O.V. Koroljova-Skorobogat’ko, E.V. Stepanova, V.P. Gavrilova, O.V. Morozova, N.V. Lubimova, A.N. Dzchafarova et al., Purification and characterization of the constitutive form of laccase from the basidiomycete Coriolus hirsutus and effect of inducers on laccase synthesis, Biotechnol. Appl. Biochem. 28 (1998) 47–54. K.-S. Shin, Y.-J. Lee, Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus, Arch. Biochem. Biophys. 384 (2000) 109–115. B.R.M. Reinhammar, Oxidation–reduction potentials of the electron acceptors in laccases and stellacyanin, Biochim. Biophys. Acta 275 (1972) 245–259. F. Xu, W. Shin, S.H. Brown, J.A. Wahleithner, U.M. Sundaram, E.I. Solomon, A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability, Biochim. Biophys. Acta 1292 (1996) 303–311. F. Xu, A.E. Palmer, D.S. Yaver, R.M. Berka, G.A. Gambetta, S.H. Brown et al., Targeted mutations in a Trametes villosa laccase, axial perturbations of the T1 copper, J. Biol. Chem. 274 (1999) 12372–12375. P. Schneider, M.B. Caspersen, K. Mondorf, T. Halkier, L.K. Skov, P.R. Ostergaard et al., Characterization of a Coprinus cinereus laccase, Enz. Microb. Technol. 25 (1999) 502–508. S.V. Shlev, E.A. Zaitseva, E.S. Gorshina, O.V. Morozova, V.A. Serezhenkov, D.S. Burbaev, B.A. Kuznetsov, A.I. Yaropolov, Spectral and electrochemical study of laccases from basidiomycetes, Moscow University Chem. Bull. 44 (2003) 35–39. A. Klonowska, C. Gaudin, A. Fournel, M. Asso, J. Le Petit, M. Giorgi, T. Tron, Characterization of a low redox potential laccase from the basidiomycete C30, Eur. J. Biochem. 269 (2002) 6119–6125. F. Xu, J.J. Kulys, K. Duke, K. Li, K. Krikstopaitis, H.J. Deussen et al., Redox chemistry in laccase-catalyzed oxidation of N-hydroxy compounds, Appl. Environ. Microbiol. 66 (2000) 2052–2056. M. Balakshin, C.-L. Chen, J.S. Gratzl, A.G. Kirkman, H. Jakob, Biobleaching of pulp with dioxygen in laccase-mediator system—effect of variables on the reaction kinetics, J. Mol. Catal. B: Enz. 16 (2001) 205–215. A.M. Mayer, R.C. Staples, Laccase: new functions for an old enzyme, Phytochemistry 60 (2002) 551–565. Y. Kojima, Y. Tsukuda, Y. Kawai, A. Tsukamoto, J. Sugiura, M. Sakaino et al., Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus, J. Biol. Chem. 265 (1990) 15224–15230. H. Yoshida, Chemistry of lacquer (urushi), J. Chem. Soc. 43 (1883) 472–486. S.C. Barton, H.-H. Kim, G. Binyamin, Y. Zhang, A. Heller, The “Wired” laccase cathode: high current density electroreduction of O2 to water at +0.7 V (NHE) at pH 5, J. Am. Chem. Soc. 123 (2001) 5802–5803. R.S. Freire, N. Duran, L.T. Kubota, Effects of fungal laccase immobilization procedures for the development of a biosensor for phenol compounds, Talanta 54 (2001) 681–686. B.A. Kuznetsov, G.P. Shumakovich, O.V. Koroleva, A.I. Yaropolov, On applicability of laccase as label in the mediated and mediatorless electroimmunoassay: effect of distance on the direct electron transfer between laccase and electrode, Biosens. Bioelectron. 16 (2001) 73–84. A.V. Karamyshev, S.V. Shleev, O.V. Koroleva, A.I. Yaropolov, I.Y. Sakharov, Laccase-catalyzed synthesis of conducting polyaniline, Enz. Microb. Technol. 33 (2003) 556–564. C. Johansen, PCT Int. Appl, Novo Nordisk A/s, Den, 1996, pp. 52, Wo.

[26] O.V. Koroleva, I.S. Yavmetdinov, S.V. Shleev, E.V. Stepanova, V.P. Gavrilova, Isolation and study of some properties of laccase from the basidiomycetes Cerrena maxima, Biochemistry (Moscow) 66 (2001) 618–622. [27] A.E. Palmer, R.K. Szilagyi, J.R. Cherry, A. Jones, F. Xu, E.I. Solomon, Spectroscopic characterization of the Leu513His variant of fungal laccase: effect of increased axial ligand interaction on the geometric and electronic structure of the type 1 Cu site, Inorg. Chem. 42 (2003) 4006–4017. [28] M. Antorini, I. Herpoel-Gimbert, T. Choinowski, J.-C. Sigoillot, M. Asther, K. Winterhalter, K. Piontek, Purification, crystallization and X-ray diffraction study of fully functional laccases from two ligninolytic fungi, Biochim. Biophys. Acta 1594 (2001) 109–114. [29] N. Hakulinen, L.-L. Kiiskinen, K. Kruus, M. Saloheimo, A. Paananen, A. Koivula, J. Rouvinen, Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site, Nat. Struct. Biol. 9 (2002) 601–605. [30] K. Piontek, M. Antorini, T. Choinowski, Crystal structure of a laccase from the fungus Trametes versicolor at 1.90 Å resolution containing a full complement of coppers, J. Biol. Chem. 277 (2002) 37663–37669. [31] F.J. Enguita, L.O. Martins, A.O. Henriques, M.A. Carrondo, Crystal structure of a bacterial endospore coat component: A laccase with enhanced thermostability properties, J. Biol. Chem. 278 (2003) 19416–19425. [32] S.-K. Lee, S. DeBeer George, W.E. Antholine, B. Hedman, K.O. Hodgson, E.I. Solomon, Nature of the intermediate formed in the reduction of O2 to H2O at the trinuclear copper cluster active site in native laccase, J. Am. Chem. Soc. 124 (2002) 6180–6193. [33] A.E. Palmer, L. Quintanar, S. Severance, T.-P. Wang, D.J. Kosman, E.I. Solomon, Spectroscopic characterization and O2 reactivity of the trinuclear Cu cluster of mutants of the multicopper oxidase Fet3p, Biochemistry 41 (2002) 6438–6448. [34] D.L. Johnson, J.L. Thompson, S.M. Brinkmann, K.A. Schuller, L.L. Martin, Electrochemical characterization of purified Rhus vernicifera laccase: voltammetric evidence for a sequential four-electron transfer, Biochemistry 42 (2003) 10229–10237. [35] C. Gercog, K. Gustav, I. Shtrele, Manual on Inorganic Synthesis, Mir, Moscow, 1985, pp. 386, (in Russian). [36] R. Roesch, Detection of phenoloxidases by Bavendamm’s reaction using the ring dish test, Zentralblatt fuer Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene 127 (1972) 555–563. [37] M.P. Tombs, D.P. Akroy, Acrilamide gel electrophoresis, Shandon Instr. Appl. 18 (1967) 1–16. [38] O. Vesterberg, H. Svensson, Isoelectric fractionation, analysis, and characterization of ampholytes in natural pH gradients. IV. Further studies on the resolving power in connection with separation of myoglobins, Acta Chem. Scand. A 20 (1966) 820–834. [39] G. Felsenfeld, The determination of cuprous ion in Cu proteins, Arch. Biochem. Biophys. 87 (1960) 247–251. [40] B. Ehresmann, P. Imbault, J.H. Weil, Spectrophotometric determination of protein concentration in cell extracts containing tRNA and rRNA, Anal. Biochem. 54 (1973) 454–463. [41] P.L. Dutton, Redox potentiometry: determination of midpoint potentials of oxidation–reduction components of biological electrontransfer systems, Methods Enzymol. 54 (1978) 411–435. [42] S.V. Shleev, S.V. Kuznetsov, A.F. Topunov, A cell for bioelectrochemical studies with parallel spectrophotometric monitoring of the studied substance, Appl. Biochem. Microbiol. (Moscow) 36 (2000) 354–358. [43] Z. Galus, Theoretical Bases of the Electrochemical Analysis, Mir, Moscow, 1974, pp. 189, (in Russian). [44] S.-P.W. Tang, J.E. Coleman, Y.P. Myer, Conformational studies of copper proteins. Pseudomonas blue protein and Polyporus laccase, J. Biol. Chem. 243 (1968) 4286–4297. [45] K.E. Falk, B. Reinhammar, Visible and near-infrared circular dichroism of some blue copper proteins, Biochim. Biophys. Acta 285 (1972) 84–90.

S.V. Shleev et al. / Biochimie 86 (2004) 693–703 [46] A. Messerschmidt, Multi-Copper Oxidases1997, pp. 465, Singapore. [47] A.L. Gindilis,Y.A. Baranov, E.O. Zhazhina, V.P. Gavrilova, V.V. Verzilov, A.I. Yaropolov, Laccase from the basidial fungus Cerrena maxima: some properties and kinetic mechanism of action, Biochemistry (Moscow) 55 (1990) 315–322. [48] A.L. Gindilis, E.O. Zhazhina, Y.A. Baranov, A.A. Karyakin, V.P. Gavrilova, A.I. Yaropolov, Isolation and properties of laccase from the basidial fungus Coriolus hirsutus (Fr.), Quel, Biochemistry (Moscow) 53 (1988) 735–739. [49] S.A. Smirnov, O.V. Koroleva, V.P. Gavrilova, A.B. Belova, N.L. Klyachko, Laccases from basidiomycetes: physicochemical characteristics and substrate specificity towards methoxyphenolic compounds, Biochemistry (Moscow) 66 (2001) 774–779.

703

[50] A. Christensson, N. Dimcheva, E. Ferapontova, L. Gorton, T. Ruzgas, L. Stoica, S. Shleev, Y.A.D. Haltrich, R. Thorneley, S. Aust, Direct electron transfer between ligninolytic redox enzymes and electrodes, Electroanalysis 6 (2004) 1074–1092. [51] F. Xu, R.M. Berka, J.A. Wahleithner, B.A. Nelson, J.R. Shuster, S.H. Brown et al., Site-directed mutations in fungal laccase: effect on redox potential, activity and pH profile, Biochem. J. 334 (1998) 63–70. [52] V. Ducros, A.M. Brzozowski, K.S. Wilson, S.H. Brown, P. Ostergaard, P. Schneider et al., Crystal structure of the type-2 Cu depleted laccase from Coprinus cinereus at 2.2 Å resolution, Nat. Struct. Biol. 5 (1998) 310–316.